Methods and compositions for endoplasmic reticulum ribosome regulation

Abstract

In general, the current disclosure relates to sterol regulatory element-binding protein cleavage-activating protein (SCAP) affecting endoplasmic reticulum ribosome translation.

Description

METHODS AND COMPOSITIONS FOR ENDOPLASMIC RETICULUM RIBOSOME REGULATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Application No. 63 / 719,869 filed November 13, 2024, which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 12, 2025, is named ARCDP0852WO.xml and is 24,635 bytes in size.STATEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under HG008935 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTIONI. Field of the Invention

[0004] This invention relates to the field of microbiology, cell biology, and medicine.II. Background

[0005] Among the ~40 modifications in mammalian tRNAs, queuosine (Q) is unique in that its installation requires an extracellular metabolite from the gut microbiome or diet. Queuosine is a 7-deaza-derivatived nucleoside in bacterial tRNA that is produced de novo by microbes using GTP in an eight-step biosynthesis pathway f Bacterial Q-modified tRNA is catabolized to queuine2'5, which is taken up by mammalian cells and becomes the substrate of the mammalian genome encoded enzyme complex (QTRT1 / QTRT2) to produce host Q-tRNA modification at the wobble anticodon position1’6'10. The unmodified G34-tRNA prefers decoding C-ending codons, whereas the Q34-tRNA reads the C and U-ending codons more equally and affects decoding speed and accuracyn'15.

[0006] An intermediate product of the biosynthesis pathway of Q-modified tRNA in bacteria is pre-queuosine 1 (preQi, Fig. 1A). In bacteria, preQi is the substrate of the modification enzyme tRNA-guanine transglycosylase (tgf) that incorporates preQi into the cognate tRNATyr / HlsMsn / Asp. The preQi -tRNA is further modified to Q-modified tRNA by 300397066.2 - 1 -additional enzymes. PreQi is constantly present in a bacterial cell and could become readily available upon bacterial turnover in the gut or elsewhere16'19. In the test tube, preQi can also be incorporated into the cognate tRNAs by the same human enzyme, QTRT1 / QTRT220,21. Even though two available metabolites from the same bacterial metabolic pathway are long known, however, the effect of preQi on mammalian cell biology and the associated molecular mechanism has not been explored.

[0007] A crucial question of tRNA quality control in a cell is to detect and remove tRNAs that are defective in translation. tRNAs are long-lived and tend to accumulate damage from many sources such as chemical reaction or radiation22'28. Cells have many ways in dealing with damaged RNAs including ribosome dependent and independent pathways26,29'38. The most well studied tRNA quality control pathways are ribosome independent26. For example, TRAMP complex is responsible for clearing defective pre-tRNAs in the nucleus39'42. Mature tRNAs undergo quality control called rapid tRNA decay (RTD) that is sensitive to tRNA modifications43'46. Hypomodified tRNAs are more readily degraded by ribonucleases. In addition, ribosome dependent RNA quality control has been well-characterized for mRNA-surveillance such as nonsense-mediated decay (NMD)32, no-go decay (NGD)34, and non-stop decay (NSD)36'38. NMD clears transcripts with a premature stop codon32. NGD clears transcripts that cause reduced translation speed or ribosome stalling34. NSD clears transcripts without stop codon36'38,47. These mRNA-surveillance pathways may also be used for the quality control of damaged RNAs through translation. For example, NGD can target 8-oxoG-containing RNA through translation48,49.

[0008] Since tRNAs are highly structured and carry many stabilizing modifications, damaged tRNAs can remain folded, possibly charged and still delivered to the ribosome. Therefore, defective tRNAs that are loaded on the ribosome can change translation speed, cause ribosome stalling or induce ribosome collision and may also undergo ribosome dependent RNA quality control. Human cells detect and relieve collided ribosome caused by stall-inducing transcripts in the cytosol through a process that involves E3 ubiquitin ligase ZNF598 and ASC-1 ribosome quality control complex50,51. ZNF598 catalyzes the ubiquitination of the collided 40S interface52'54. ASC-1 complex disassembles the collided ribosomes through the dissociation and splitting of uSIO polyubiquitinated ribosomes52>53>55>56. Defective mRNAs are degraded through NMD, NGD, or NSD in the process of detecting and rescuing stalling or collided ribosomes caused by stall-inducing transcripts32>34>36-38>47>51Nascent peptidyl-tRNAs remain complexed with the 60S ribosomal subunit after the ribosomal subunit dissociation51,57‘59. NEMF then binds to the 60S and tRNA interaction interface, blocking 40S ribosomal 300397066.2 - 2 -subunit binding and recruiting E3 ubiquitin ligase Listerin that catalyzes K48-linked polyubiquitination on the nascent peptide chain51,57'63. ANKZF1 cleaves the 3 ’-CCA nucleotides of the peptidyl-tRNA, releasing the polyubiquitinated nascent peptide for proteasomal degradation and generating intermediate tRNAs without trailing 3 ’-CCA64-67ELAC1 or ANGEL2 converts the 2’,3’-cyclic phosphate on the 3’ end of intermediate tRNA to 2’ and 3’-OH67-69. CCA tail was then added back by CCA-adding enzyme TRNT1 that also quality checks tRNAs and direct tRNAs with a destabilized acceptor stem for rapid degradation 45,67,69 Other components of stalled ribosomal complexes are processed or recycled by ribosome quality control processes such as the removal of defective rRNAs by nonfunctional rRNA decay70-74. However, whether ribosome collision caused by defective tRNAs triggers the same pathway and the fate of the tRNA that causes ribosome collision are unknown. One would expect that such tRNAs will be selectively degraded to prevent it from repeating the same unfavorable outcome on the ribosome.SUMMARY OF THE INVENTION

[0009] In general, the current disclosure relates to the discovery that a sterol regulatory element-binding protein cleavage-activating protein (SCAP) regulates ER translation in sensing low quality tRNAs that get on the ribosome and facilitating their degradation. Aspects relate to the discovery that SCAP directly interacts with the ribosome under all cellular conditions. SCAP can sense ribosome loaded with preQi -modified tRNA from the E-site through conformational change that activates the ER-resident IRE1 ribonuclease at the A-site to cleave the preQi -modified tRNA.

[0010] Disclosed herein are methods of treating a patient, methods of treating a disease associated with protein translation deregulation in a patient, methods of treating cancer, methods of treating a neurological disease, methods of treating a disease associated with SCAP, and / or methods of treating a disease. In some aspects, the method comprises one or more steps including administering a SCAP gene or gene product to the patient. The disease may comprise a disease associated with any of the biological mechanisms described herein. In some aspects, the disease is a neurological disease. In some aspects, the disease is a cancer. In some aspects, the disease is a cancer, a metabolic disease, an autoimmune disease, a fibrotic disease, a cardiovascular disease, a neurodegenerative disease, or an infection. In some aspects, the infection is a viral infection.300397066.2 - 3 -

[0011] In some aspects, the SCAP gene is a SCAP gene described herein. In some aspects, the SCAP or gene product an mRNA or a protein, including any of the SCAP mRNAs or proteins described herein. In some aspects, the SCAP gene or gene product is or is not mutated compared to a wild-type SCAP gene. In some aspects, the wild-type SCAP gene is a human SCAP gene. In some aspects, the SCAP gene or gene product comprises a loop 6 deletion and / or a WD domain deletion. In certain aspects, the SCAP gene comprises a polynucleotide of SEQ ID NOs: 9-11 or the SCAP gene product comprises a protein of SEQ ID NO: 7 or 8 or a polynucleotide of SEQ ID NOs: 9-11. In some aspects, the SCAP gene or gene product comprises a SCAP gene or gene product disclosed herein. In some aspects, the SCAP gene or gene product is administered at an amount sufficient to increase the SCAP gene product in a cell of interest in the patient. The increase may be over a baseline. The increase may be to a therapeutically relevant amount.

[0012] In some aspects, the method comprises administering a sterol regulatory elementbinding protein cleavage-activating protein (SCAP) inhibitor to the patient. In some aspects, the SCAP inhibitor is a nucleic acid capable of reducing a SCAP gene product. In some aspects, the SCAP inhibitor comprises a CRISPR-Cas9 system targeting a SCAP gene in a cell in the patient. In some aspects, the cell is a tumor cell. In some aspects, the cell is a neuron. In some aspects, the SCAP inhibitor is a SCAP inhibitor described herein, including an siRNA described herein.

[0013] Also disclosed are methods of regulating endoplasmic reticulum ribosome translation in a cell. In some aspects, the method comprises one or more steps including introducing a SCAP gene or gene product to the cell. In some aspects, the method comprises one or more steps including introducing a SCAP inhibitor to the cell. In some aspects, the SCAP gene or gene product is mutated compared to a wild-type SCAP gene. In some aspects, wild-type SCAP gene is a human SCAP gene. In some aspects, the SCAP gene or gene product comprises a loop 6 deletion and / or a WD domain deletion. In certain aspects, the SCAP gene comprises a polynucleotide of SEQ ID NOs: 9-11 or the SCAP gene product comprises a protein of SEQ ID NO: 7 or 8 or a polynucleotide of SEQ ID NOs: 9-11. In some aspects, the cell is a cancer cell. In some aspects, the cell is a neuron. In some aspects, the cell is a human cell.

[0014] In some aspects, the method comprises one or more steps including introducing a SCAP inhibitor to the cell. In some aspects, the cell is a cancer cell. In some aspects, the cell is a neuron. In some aspects, the cell is a human cell. In some aspects, the SCAP inhibitor is a nucleic acid capable of reducing a SCAP gene product. In some aspects, the SCAP inhibitor 300397066.2 - 4 -comprises a CRISPR-Cas9 system targeting a SCAP gene in the cell. In some aspects, the SCAP inhibitor is a SCAP inhibitor described herein, including an siRNA described herein.

[0015] Also disclosed are the following enumerated Aspects.Aspect 1. A method of treating a disease associated with protein translation deregulation in a patient, the method comprising administering a sterol regulatory element-binding protein cleavage-activating protein (SCAP) gene or gene product to the patient.Aspect 2. The method of Aspect 1, wherein the disease is a neurological disease.Aspect 3. The method of Aspect 1, wherein the disease is a cancer.Aspect 4. The method of any one of Aspects 1 to 3, wherein the SCAP gene or gene product is mutated compared to a wild-type SCAP gene.Aspect 5. The method of Aspect 4, wherein the wild-type SCAP gene is a human SCAP gene. Aspect 6. The method of Aspect 4 or 5, wherein the SCAP gene or gene product comprises a loop 6 deletion and / or a WD domain deletion.Aspect 7. The method of any one of Aspects 4 to 6, wherein the SCAP gene comprises a polynucleotide of SEQ ID NOs: 9-11 or the SCAP gene product comprises a protein of SEQ ID NO: 7 or 8 or a polynucleotide of SEQ ID NOs: 9-11.Aspect 8. A method of regulating endoplasmic reticulum ribosome translation in a cell, the method comprising introducing a sterol regulatory element-binding protein cleavage-activating protein (SCAP) gene or gene product to the cell.Aspect 9. The method of Aspect 8, wherein the SCAP gene or gene product is mutated compared to a wild-type SCAP gene.Aspect 10. The method of Aspect 9, wherein the wild-type SCAP gene is a human SCAP gene. Aspect 11. The method of Aspect 9 or 10, wherein the SCAP gene or gene product comprises a loop 6 deletion and / or a WD domain deletion.Aspect 12. The method of any one of Aspects 8 to 11, wherein the cell is a cancer cell.Aspect 13. The method of any one of Aspects 8 to 11, wherein the cell is a neuron.Aspect 14. The method of any one of Aspects 8 to 13, wherein the cell is a human cell.Aspect 15. A method of treating a disease associated with protein translation deregulation in a patient, the method comprising administering a sterol regulatory element-binding protein cleavage-activating protein (SCAP) inhibitor.Aspect 16. The method of Aspect 15, wherein the disease is a neurological disease.Aspect 17. The method of Aspect 15, wherein the disease is a cancer.Aspect 18. The method of any one of Aspects 15 to 17, wherein the SCAP inhibitor is a nucleic acid capable of reducing a SCAP gene product.300397066.2 - 5 -Aspect 19. The method of any one of Aspects 15 to 18, wherein the SCAP inhibitor comprises a CRISPR-Cas9 system targeting a SCAP gene in a cell in the patient.Aspect 20. The method of Aspect 19, wherein the cell is a tumor cell.Aspect 21. The method of Aspect 19, wherein the cell is a neuron.Aspect 22. A method of regulating endoplasmic reticulum ribosome translation in a cell, the method comprising introducing a sterol regulatory element-binding protein cleavage-activating protein (SCAP) inhibitor.Aspect 23. The method of Aspect 22, wherein the cell is a cancer cell.Aspect 24. The method of Aspect 22, wherein the cell is a neuron.Aspect 25. The method of any one of Aspects 22 to 24, wherein the cell is a human cell. Aspect 26. The method of any one of Aspects 22 to 25, wherein the SCAP inhibitor is a nucleic acid capable of reducing a SCAP gene product.Aspect 27. The method of any one of Aspects 22 to 26, wherein the SCAP inhibitor comprises a CRISPR-Cas9 system targeting a SCAP gene in the cell.

[0016] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0017] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprise plural form and vice versa.

[0018] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an aspect or aspect.

[0019] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0020] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The 300397066.2 - 6 -phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0021] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0022] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0023] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other aspects and embodiments are discussed throughout this application. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0024] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.

[0025] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.300397066.2 - 7 -BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0027] FIGs. 1A-1I: preQi represses cell proliferation and is counteracted by queuine. ns: not significant, * p<0.05, ** p<0.01, *** p<10'3, **** p<10'4. Mann-Whitney U test, two sided. FIG. 1A depicts bacterial biosynthesis pathway of preQi and queuine. The pathway starts with GTP. PreQi is made after 5 steps and is the substrate of the bacterial tgt enzyme for tRNA (G-tRNA = unmodified tRNA). Additional reactions produce queuosine (Q) modified tRNA. Queuine is produced after catabolic reactions of Q-modified tRNA (thick arrow). FIG. IB shows LC-MS / MS measurements of preQi, queuine, and queuosine (Q) in mouse plasma. The Y axis represents the ratio of the target area to that of the internal heavy standard (queuineN15) in the experiment, n = 3 individual mouse. FIG. 1C shows PAQS-seq of human stool showing presence of tRNA Q-modification in multiple bacterial classes in the same sample: 1: Bacteroidia, 2: Actinobacteria, 3: Bacilli, 4: Clostridia. FIG. ID shoes normalized cell count or proliferation of HEK293T cells under indicated preQi and queuine concentrations. All started as 0Q cells. All data normalized to the average of 0,0 condition at 24h. n = 8 biological replicates for each condition. FIG. IE depicts ability of preQi treated HEK293T cells to resume proliferation. 0Q cells were treated with 1 pM preQi in the medium for 24, 48 or 72h. Proliferation was measured upon replacing the medium with fresh media at different times, n = 3 biological replicates for each time point. FIG. IF shows normalized cell count or proliferation of HEK293T cells under varying queuine concentrations and 1 pM preQi. 0Q cells were treated with 1 pM preQi for 48h, queuine was then added to the medium at indicated concentration. All data normalized to the average of 1000,0 condition at 48h. n = 8 biological replicates for each condition. FIG. 1G shows normalized cell count or proliferation of HEK293T cells with different ratios of 0Q and 100Q starting cells at 1 pM preQi. All data normalized to the average of 0,0 condition at 24h. n = 8 biological replicates for each condition.FIG. 1H shows normalized cell count or proliferation of mouse embryonic fibroblast (MEF) cells under indicated preQi and queuine concentrations. All started as 0Q cells. All data normalized to the average of 0,0 condition at 24h. n = 8 biological replicates for each condition.FIG. II shows normalized cell count or proliferation of mouse BMDC cells under indicated preQi and queuine concentrations. All started as cells freshly isolated from mouse bone300397066.2 - 8 -marrow. All data normalized to the average of 0,0 condition at day 1. n = 5 replicates for each condition.

[0028] FIGs. 2A-2F: preQi effect is dependent on QTRT1 and QTRT2 and preQi is incorporated into tRNA. ns: not significant, * p<0.05, ** p<0.01, *** p<10'3, **** p<10'4. Mann-Whitney U test, two sided. FIG.2A shows an image of a western blot of HEK293T cells upon QTRT1 knockdown. GAPDH is the loading control. FIG. 2B shows normalized cell count or proliferation of QTRT1 knockdown cells under indicated preQi and queuine concentrations. All started as 0Q cells. All data normalized to the average of 0,0 condition at 24h. n = 8 biological replicates for each condition. FIG. 2C shows normalized cell count or proliferation of QTRT2 knockdown cells under indicated preQi and queuine concentrations. All started as 0Q cells. All data normalized to the average of 0,0 condition at 24h. n = 8 biological replicates for each condition. FIG.2D depicts LC-MS / MS of preQi sine calibration. The area of the MS signal (y-axis) as a function of preQi sine concentration (x-axis) shows a linear response. FIG. 2E shows LC-MS / MS measurements of queuosine (Q) and preQi sine (£) in queuine treated (left) or preQ 1 treated (right) HEK293T cells under indicated conditions. n = 3 biological replicates for each condition. FIG. 2F shows preQi sine detection of cognate tRNAs by chemical tagging (PEG-NHS) and Northern blots. The larger gel shifts in tRNAAsnand tRNATyrare derived from the known acp3U modification in those tRNAs that also reacts with the chemical tag.

[0029] FIGs 3A-3F: preQi is present in mouse tissues, can be incorporated into mouse tRNA, and reduces xenograft tumor growth, ns: not significant, * p<0.05, ** p<0.01, *** p<10'3, **** p<10'4. Mann-Whitney U test, two sided. FIG. 3 A shows LC-MS / MS measurements of preQi and queuine metabolites in mouse tissues. Fourteen tissues from C57BL / 6 mice were surgically collected immediately after sacrifice: bone marrow (BN), cerebellum (CB), colon (CL), cortex (CO), heart (HE), kidney (KI), liver (LI), lymph nodes (LN), lung (LU), mesenteric lymph nodes (MLN), small intestine (SI), spleen (SP), stomach (ST), thymus (TH). Metabolites were extracted and analyzed by LC-MS / MS for queuine and preQi . As in FIG. IB, data were normalized to the area of the internal heavy standard, queuineN15. n = 3 individual mouse. FIG. 3B shows LC-MS / MS measurements of queuosine (Q) and preQi sine (g) nucleosides in the same mouse tissues as (A). Data were normalized to the area of the internal heavy standard, queuineN15. n = 3 individual mouse. FIG. 3C shows LC-MS / MS measurements of Q (left) and preQi sine (right) nucleosides in mouse liver, kidney, heart, and lung with and without preQi injection, n = 4 mice for each group. FIG. 3D shows images of Northern blots of APB gels of total RNA samples from mouse liver and kidney with 300397066.2 - 9 -and without preQi injection using tRNAHlsor tRNAAsnprobes. 5S rRNAis the loading control, 0Q, 100Q HEK293T RNAs are positive controls, n = 4 mice for each group. FIG. 3E shows normalized cell count or proliferation of mouse B16 melanoma cells under indicated preQi and queuine concentrations. All started as 0Q cells. All data normalized to the average of 0,0 condition at 24h. n = 8 biological replicates for each condition. FIG. 3F shows tumor volume measurements of B16 cells 9 and 11 days after implantation and under mock or preQi treatment, n = 6 mice for control group and n = 9 mice for preQi group.

[0030] FIGs. 4A-4E: preQi selectively reduces the level of cognate tRNATyr / Hls / Asn / Asp. FIG. 4A shows all tRNA abundance change relative to 0Q samples without preQi treatment at the isoacceptor level measured by high throughput sequencing. Outliers are indicated and Q / preQl modifiable tRNAs are in red. Top: nuclear-encoded (cytosolic) tRNAs, bottom: mitochondrial-encoded tRNAs. FIG. 4B shows a heatmap of expression of individual tRNA isoacceptors showing selective decrease of cognate tRNA abundance indicated by arrows. All tRNA normalized to 0Q samples without preQi treatment. FIG. 4C shows Northern blot validation of nuclear-encoded tRNAHlsand tRNAAsplevel reduction. 5S rRNAis the loading control. FIG.4D shows a Northern blot of nuclear-encoded tRNATyrusing acid denaturing gels to measure charging levels. Deacylated samples are positive controls. FIG. 4E shows 5’ tRNATyrfragment levels relative to full-length tRNATyrmeasured by MSR-seq under indicated conditions.

[0031] FIGs. 5A-5H: preQi drastically reduces translation of ribosomal proteins and at A / T-ending codons. FIG.5A depicts sucrose gradient polysome profiles of HEK293T cells in mock and 1 pM treated preQi. mRNAs from disome and above (underlined) were collected for polysome mRNA sequencing. FIG. 5B shows preQi versus 0Q input mRNA (left) and polysome mRNA (right). Red, preQl / OQ >2. Blue, OQ / preQl > 2. The 117 ribosomal protein genes (named RPL / RPS) are highlighted in brown. FIG. 5C shoes translation efficiency (TE) of preQi treated and control samples, 0Q versus preQi. Highlighted are transcripts whose TE differs by >5-fold between mock and preQi -treated cells. FIG. 5D shows TE of preQi treated over untreated cells versus mRNA abundance of input (left) and polysome (right) under mock conditions. The 117 ribosomal protein genes are highlighted. FIG. 5E shows gene ontology (GO) terms of genes whose translation was drastically reduced (blue transcripts in panel C) or increased (red transcripts) upon preQi treatment. FIG. 5F shows comparison of codon usage of the 4 amino acids decoded by Q-modifiable tRNAs affected by preQi treatment. X-axis represents the codon usage (CU) of the blue transcripts in panel C, and y-axis the codon usage of the red transcripts. C-ending codons are in blue, U-ending codons are in green. FIG. 5G 300397066.2 - 10 -shows comparison of codon usage of all amino acids. X-axis represents the codon usage of the blue transcripts in panel C, and y-axis the codon usage of the red transcripts. Codon ends with A (red), C (blue), G (orange), U (green) indicated by colors. FIG. 5H shows difference in codon usage between genes with much higher TE in preQi over 0Q (red transcripts in panel C) and genes with much higher TE in 0Q over preQi (blue transcripts). Codons of the 4 amino acids decoded by Q-modifiable tRNAs are indicated by arrows. Codon ends with A (red), C (blue), G (orange), U (green) indicated by colors.

[0032] FIGs. 6A-6J: CRISPR screen identifies new cellular pathways in preQi effects -3 on proliferation and validation, ns: not significant, * p<0.05, ** p<0.01, *** p<10 , **** -4p<10 . Mann-Whitney U test, two sided. FIG. 6A shows integrated differential sgRNA expression by STARS. Genes with n value > 2.4503 and STARS p < 0.05 are highlighted in red; their depletion enhances proliferation compared to controls upon preQi treatment. Genes with p < 107are manually set to 7. QTRT1 and QTRT2 have TI scores of 66.99 and 104.58 respectively and are not shown here. FIG. 6B shows a heatmap of sgRNA enrichment of the 40 red genes in panel (A). Enrichment is normalized to the average of the four controls. FIG.6C shows gene ontology analysis of cellular component of the 40 red genes in FIG. 6A. FIG.6D shows clusters of protein-protein interaction analysis by STRING of the 40 red genes in FIG. 6A. Proteins in the same pathway are grouped and annotated with colored shadows. Filled colored dots indicate genes selected for validation. FIG. 6E shows enrichment of sgRNA of SCAP, MBTPS1 and MBTPS2 in the cholesterol metabolism pathway, relative to actin sgRNA as control. Each dot is a different sgRNA. FC: fold change. FIG. 6F shows enrichment of sgRNA of NBAS, USE1 and COG3 in the ER-Golgi trafficking pathway, relative to actin sgRNA as control. Each dot is a different sgRNA. FIG. 6G shows normalized cell count or proliferation of si-CTRL and si-SCAP cells without and with preQi treatment. All started as 0Q cells. All data normalized to the average of si-CTRL, no preQi condition at 24h. n = 8 biological replicates for each condition. FIG. 6H shows normalized cell count or proliferation under the same treatment as FIG. 6G, but for si-CTRL and si -MB TP 1 cells. FIG. 61 shows normalized cell count or proliferation under the same treatment as FIG. 6G, but for si-CTRL and si-NBAS cells. FIG. 6J depicts a CRISPR screen identifying SCAP (ER), MBTPS1 / MBTPS2 (Golgi), Cog3 / Cog4 / NBAS (COPI proteins for retrograde Golgi -ER trafficking), USO1 / USE1 / BNIP1 (SNARE proteins for reintegrating SCAP into ER) that rescue preQi -dependent cell proliferation, (i) SCAP is an ER resident protein that at low cholesterol levels traffics and chaperons SREBP transcription factors to Golgi, (ii) MBTPS1300397066.2 - 11 -and MBTPS2 proteases cleave SREBP and releasing SCAP to enter COPI. (iii) COPI retrograde transports SCAP back to ER. (iv) SCAP reintegrates into ER mediated by the SNARE complex.

[0033] FIGs. 7A-7L: preQl-dependent tRNA degradation requires active translation and SCAP domains. FIG. 7A shows an image of a Northern blot of si-CTRL and si-SCAP cells for tRNATyr. 5S rRNA is the loading control. FIG. 7B shows tRNATyr / 'Hls / Asn / Asplevel change of si-CTRL and si-SCAP ±preQl by Northern blot, normalized to 5S rRNA. All 4 tRNAs are represented at n = 4 biological replicates for each condition. FIG. 7C shows GO term of genes enriched in the polysome with preQi. FIG. 7D shows QQQ LC / MS results of queuosine (Q) and preQi sine (g) in the input and polysome samples. FIG. 7E shows Northern blot quantitation of tRNAHlsMsn / Tyr / AsP and control tRNALyslevels + / - the translation inhibitor emetine. FIG. 7F shows Northern blot quantitation of tRNAHls / Asplevels + / - the translation inhibitor cycloheximide. FIG. 7G shows an image of a Western blot of the SCAP protein in the ribosome fraction in sucrose cushion, ±preQl. FIG. 7H shows Western blot images of SCAP and RPL8 proteins in the monosome and across polysome fractions in sucrose gradient, ±preQl. FIG. 71 shows quantitation of SCAP / RPL8 ratio in monosome, disome, trisome, tetrasome, and larger polysomes, ±preQl. FIG. 7 J shows stable expression cell lines of Flag-SCAP, Flag-mutl, Flag-mut2 respond similarly to preQi treatment measured by tRNAHls / Asn / Tyr / AsP levels by tRNA-seq. FIG. 7K shows quantitation of SCAP / RPL8 ratio in monosome, disome, trisome, tetrasome, and larger polysomes without preQi for stable expression cell lines of Flag-SCAP, Flag-mutl, Flag-mut2. The ratio of SCAP detected using flag antibody (f) over SCAP antibody (s) is shown. FIG.7L shows quantitation of SCAP / RPL8 ratio in monosome, disome, trisome, tetrasome, and larger polysomes in the presence of preQi for stable expression cell lines of Flag-SCAP, Flag-mutl, Flag-mut2.

[0034] FIGs. 8A-8I: preQi represses cell proliferation and is counteracted by queuine. FIG. 8A shows queuine, preQi, queuosine (Q) andN15-labeled q (queuineN15) MS calibration curves with linear fits of the logio values. FIG. 8B shows short retrosynthetic chemical description of the different synthetic routes that led to the MS standard compounds used in examples described herein. FIG. 8C shows deletion rate of PAQS-seq of Q-modifiable tRNAs of genus roseburia in the human stool sample with and without periodate (IO) treatment. FIG.8D shows proliferation measurement by absorbance at 460 nm, HEK293T cells. Arrow indicates the time of addition of preQi and / or queuine. Error bar corresponds to the range of n = 8 biological replicates. FIG. 8E shows proliferation measurement by absorbance at 460 nm, HEK293T cells. Arrow indicates the time of addition of preQi and / or queuine. Error bar 300397066.2 - 12 -corresponds to the range of n = 8 biological replicates. preQi was added at t = 0 and queuine added at t = 48h. FIG. 8F shows proliferation measurement by absorbance at 460 nm, HEK293T cells. The starting cells were mixtures of 0Q and 100Q cells. Arrow indicates the time of addition of preQi. Error bar corresponds to the range of n = 8 biological replicates.FIG. 8G shows proliferation measurement by absorbance at 460 nm, MEF cells. Arrow indicates the time of addition of preQi and / or queuine. Error bar corresponds to the range of n = 8 biological replicates. FIG. 8H shows relative cell count of proliferation measurement, BMDC cells. Arrow indicates the time of addition of preQi and / or queuine. Cell counts are normalized to 0Q cells at t = 0. All started as cells freshly isolated from mouse bone marrow. n = 5 replicates for each condition. FIG. 81 depicts murine BMDCs that were stained with DAPI for flow cytometry and analyzed on the NovoCyte Penteon using Flow Jo software. From left to right: the myeloid cell population is identified by forward and side scatter, doublets are excluded, and live cells are quantified by selecting the DAPIlow population. Each figure is representative of one of five replicates.

[0035] FIGs. 9A-9D. preQi is incorporated into tRNA dependent on QTRT1 and QTRT2. FIG.9A shows proliferation measurement by absorbance at 460 nm, shRNA-QTRTl knockdown and control HEK293T cells. Arrow indicates the time of addition of preQi and / or queuine. Error bar corresponds to the range of n = 8 biological replicates. FIG. 9B shows an image of a Western blot showing shRNA knockdown of the QTRT2 protein. CycB is the loading control. FIG. 9C shows proliferation measurement by absorbance at 460 nm, shRNA-QTRT2 knockdown and control cells. FIG.9D shows a reaction scheme of the preQi -modified tRNA for Northern blot analysis.

[0036] FIGs. 10A-10C. preQi metabolite is present in mouse tissues, can be incorporated into tRNA, and preQi treatment reduces xenograft tumor growth. FIG. 10A shows LC-MS / MS results of mouse feces showing queuine and preQi metabolites and queuosine and preQi sine nucleosides. FIG. 10B shows quantitation of Q-modification levels from Northern blot results of preQi injected liver and kidney tRNAHlsand tRNAAsn. FIG. 10C shows proliferation measurement by absorbance at 460 nm, B 16 cells. Arrow indicates the time of addition of preQi and / or queuine. Error bar corresponds to the range of n = 8 biological replicates.

[0037] FIGs. 11A-11B. preQi selectively reduced the expression of Q-modified tRNAs. FIG. 11A shows a comparison of the fraction tRNA reads for the biological replicates under different preQi and queuine treatments. Pearson’s r = 0.9981 (0Q), 0.9868 (preQi), 0.9986300397066.2 - 13 -(preQl lnM Q), 0.9952 (preQl_10nM_Q), 0.9963 (100Q). FIG. 11B shows Northern blots for Q-modifiable tRNAs in MEF cells. 5S rRNAis the loading control.

[0038] FIGs. 12A-12J: SCAP directly interacts with ER ribosome to coordinate tRNA degradation through IRE1, and a model of tRNA quality control on the ER ribosome. FIG. 12A shows SCAP-CLiP-seq of rRNA shows three 28S rRNA regions directly interacting with SCAP without preQi. FIG. 12B depicts the regions in FIG. 12A superimposed on the cryo-EM 80S structures (PDB: 4V6X,106). The E-site tRNA is in orange. The uL24 and uL29 proteins lining the end of the exit tunnel are in blue. Residues in the three clusters with mutation rate of >1% are shown in red. Region 3090-3180 residues are located in an rRNA region not visible in the cryo-EM structure, the beginning and the end of this missing region are shown in magenta. FIG. 12C shows SCAP-CLiP-seq of rRNA shows four 28 S rRNA regions change their interaction with SCAP with preQi. FIG. 12D depicts the regions in FIG. 12C superimposed on the cryo-EM 80S structures (PDB: 4V6X). The E-site tRNA is in orange. The uL24 and uL29 proteins lining the end of the exit tunnel are in blue. Residues in the four clusters with mutation rate differences of >1% are shown in red (increase with preQi) or cyan (decrease with preQi). Region 3090-3180 changes are positive with preQi, magenta residues at the beginning and the end indicate this missing region in the cryo-EM structure. FIG. 12E shows SCAP-N-Tad8-seq results of 28S rRNA, ±preQl . Residues with >0.4% A-to-G mutation rate differences are numbered. FIG. 12F depicts residues of SCAP-Tad8-seq superimposed on the cryo-EM 80S structures (PDB:4V6X). The E-site tRNA is in orange. The uL24 and uL29 proteins lining the end of the exit tunnel are in blue. Changes of A-to-G mutations with and without preQi. Residues with >0.4% A-to-G mutation rate differences are shown in red (increase in preQi) or cyan (decrease in preQi), residues in the missing rRNA regions in the cryo-EM structure are indicated by magenta (increase in preQi) or green (decrease in preQi) residues at the beginning and the end. FIG. 12G shows tRNA-seq results showing the addition of the IREl ribonuclease inhibitor 4p8C selectively rescues the abundance of tRNAHls / Asp / Tyrin the presence of preQi. FIG. 12H shows addition of the IREl ribonuclease inhibitor 4p8C alleviates the preQi effect of cell proliferation. FIG. 121 shows correlation of tRNA species that interact with IREl (data from110) and tRNA fragments in cells without preQi treatment.FIG. 12J depicts tRNA quality control occurs on the ER translating ribosome. SCAP senses stalled ER ribosomes and enables IREl -mediated cleavage of translation-deficient tRNAs. Ribosome is in gray (normal) or magenta (stalled), Sec61 is the central channel of the translocon. (Left) Under normal conditions without preQi, SCAP interacts with the ER translation machinery near the E site of the ribosome, IREl interacts with the ribosome near 300397066.2 - 14 -the A site. (Right) preQi -modified tRNA stalls ribosome, triggering a chain of events, (i) PreQl-tRNA stalls ribosome at the A site, (ii) SCAP changes conformation and activates IRE1 ribonuclease, (iii) IRE1 cleaves tRNA. (iv) Cleaved tRNA leaves, and new tRNA comes to A site, thus rescuing the stalled ribosome.

[0039] FIGs. 13A-13J: Additional data for FIGS. 6A-6F. FIG. 13A depicts an experimental setup. FIG. 13B shows differential expression of individual sgRNAs of the “red’ genes in FIG. 6 A. FIG. 13C shows enrichment of sgRNA of QTRT1 and QTRT2, relative to actin sgRNA as control. Each dot is a different sgRNA. FC: fold change. FIG. 13D shows gene ontology (GO) analysis for biological process of the “red” genes in FIG. 5A. FIG. 13E shows GO analysis of molecular function of the “red” genes in FIG. 6A. FIG. 13F shows an image of a Western blot of si-CTRL and si-SCAP protein. GAPDH was the loading control. FIG. 13G shows a proliferation measurement by absorbance at 460 nm, si-SCAP and si-Ctrl cells. Arrow indicates the time of addition of preQi and / or queuine. Error bar corresponds to the range of n = 8 biological replicates. FIG. 13H shows proliferation measurement by absorbance at 460 nm, si-MBTPSl and si-Ctrl cells. Arrow indicates the time of addition of preQi and / or queuine. Error bar corresponds to the range of n = 8 biological replicates. FIG. 131 shows normalized cell number of si-MBTPS2 and si-Ctrl cells under conditions of indicated preQi concentration. All started as 0Q cells. All data normalized to the average of 0,0 condition at 24h. n = 8 biological replicates for each condition. (Lower right inset) Proliferation measurement by absorbance at 460 nm, si-MBTPSl and si-Ctrl cells. Arrow indicates the time of addition of preQi and / or queuine. Error bar corresponds to the range of n = 8 biological replicates. FIG. 13J shows proliferation measurement by absorbance at 460 nm, si-NBAS and si-Ctrl cells. Arrow indicates the time of addition of preQi and / or queuine. Error bar corresponds to the range of n = 8 biological replicates. NBAS mRNAKD is also shown.

[0040] FIGs. 14A-14F: additional data for FIGS. 6G-6J, 7G-7L. FIG. 14A shows Si-NBAS rescue of tRNA levels. FIG. 14B shows tRNA levels by Northern blot with and without emetine. FIG. 14C shows tRNA levels and preQi -modification by Northern blot with and without cycloheximide. FIG. 14D shows a diagram showing SCAP domain structure, and the constructs of mutl (loop 6 deletion) and mut2 (WD domain deletion). FIG. 14E shows an image of a Western blot of wild-type SCAP, mutl, and mut2 expression using SCAP (top) or Flag (middle) antibodies. FIG. 14F shows proliferation of the stable expression WT-OE, mutl and mut2 cell with (1000 nM) and without preQi.

[0041] FIGs. 15A-15I: additional data for FIGS. 12A-F, 12J. FIG. 15A depicts an experimental scheme for SCAP-CLiP. FIG. 15B shows an image of a PCR gel showing the 300397066.2 - 15 -MSR-seq libraries for SCAP-CLiP. PCR products between 150-300 bp were cut out and eluted for sequencing. FIG. 15C shows SCAP-CLiP-seq replicate mapping of mutation rates, 28S rRNA. FIG. 15D depicts an experimental scheme for SCAP-Tad8 deaminase. FIG. 15E shows an image of a Western blot showing stable expression of SCAP-Tad8 protein. FIG. 15F shows an image of a Western blot of preQi treated cells ±4p8C, the inhibitor of IRE1 ribonuclease activity. FIG. 15G shows eIF2a phosphorylation with and without preQi. FIG. 15H shows XBP1 mRNA splicing with and without preQi. FIG. 151 shows conservation of SCAP and IRE1 protein from Pfam.DETAILED DESCRIPTION OF THE INVENTION

[0042] The mammalian gut microbiome produces two intermediary metabolites, queuine and pre-queuosine (preQi) in the same tRNA modification pathway. Queuine is incorporated into the mammalian host tRNAs that fine-tunes translation, but how preQi affects the host cell biology has not been explored. Aspects herein show that preQi is present in mouse plasma and tissues, incorporated into mammalian tRNAs by the tRNA Q-modification writer, and strongly represses cell proliferation, but this effect is suppressed by queuine. PreQi treatment reduces tumor growth in a cancer model. Mechanistically, preQi reduces cognate tRNA levels specifically and translation of house-keeping genes. Genomic-wide CRISPR screen and validation identify pathways in cholesterol biosynthesis regulation and Golgi-ER trafficking that mitigate the preQi proliferation effects through modulation of cognate tRNA levels.

[0043] Aspects herein identify the sterol regulatory element binding protein cleavageactivating protein (SCAP) as the sensor of preQi -modified tRNA on the endoplasmic reticulum (ER) ribosome through direct SCAP-ribosome interaction, and the ER localized inositol-requiring enzyme 1 (IREl) ribonuclease as the enzyme responsible for the selective degradation of preQi -modified tRNAs on the translating ribosomes. Aspects herein link lipid metabolism, ER translation, and tRNA quality control by two integral ER resident proteins.

[0044] Aspects herein show the effect of preQi on mammalian cell physiology and the underlying molecular mechanism. Aspects herein show that preQi is present in the plasma and mouse tissues. PreQi drastically reduces cell proliferation, but this effect is attenuated or reversed by queuine and depends on the same host enzyme that catalyzes Q-modification, QTRT1 / QTRT2. PreQi is incorporated into the cognate Q-modified tRNAs, and preQi treatment slows the growth of xenograft tumors in mice. Mechanistically, preQi specifically reduces the abundance of their cognate tRNAs and alters translation of house-keeping genes in300397066.2 - 16 -a highly codon dependent manner. Applying genome wide CRISPR screen and validation, the inventors identify genes from multiple cellular pathways in lipid metabolism that alleviate the preQi effect on cell proliferation through the modulation of cognate tRNA levels.

[0045] Genomic-wide CRISPR screen and validation identify pathways in cholesterol biosynthesis regulation and Golgi-ER trafficking that mitigate the preQi proliferation effects through modulation of cognate tRNA levels. Certain aspects herein identify sterol regulatory element binding protein cleavage-activating protein (SCAP) as a sensor of preQi -modified tRNA on the endoplasmic reticulum (ER) ribosome, including through direct SCAP-ribosome interaction. In certain aspects, SCAP coordination with the IRE1 ribonuclease can selectively degrade the preQi -modified tRNA. Accordingly, certain aspects herein link lipid metabolism, ER translation, and tRNA quality control by an integral ER resident protein, SCAP.

[0046] Certain aspects herein therefore are related to SCAP, a SCAP gene, or a SCAP gene product, compositions comprising thereof, and methods of use thereof. SCAP or a SCAP gene product may refer to a SCAP protein, including those described in the GenBank database. In certain aspects, SCAP or a SCAP gene product comprises or consists of a protein having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) sequence identity with a SCAP protein described in the GenBank database. In certain aspects, SCAP or a SCAP gene product comprises or consists of a protein having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) sequence identity to SEQ ID NO: 7 or 8, or a homolog or variant thereof.

[0047] MLLHPSPNCSLRAESLVHVHFKEEIGVAELIPLVTTYIILFAYIYFSTRKIDM VKSKWGLALAAVVTVLSSLLMSVGLCTLFGLTPTLNGGEIFPYLVVVIGLENVLVLT KSVVSTPVDLEVKLRIAQGLSSESWSIMKNMATELGIILIGYFTLVPAIQEFCLFAVVG LVSDFFLQMLFFTTVLSIDIRRMELADLNKRLPPEACLPSAKPVGQPTRYERQLAVRP STPHTITLQPSSFRNLRLPKRLRVVYFLARTRLAQRLIMAGTVVWIGILVYTDPAGLR NYLAAQVTEQSPLGEGALAPMPVPSGMLPPSHPDPAFSIFPPDAPKLPENQTSPGESPE RGGPAEVVHDSPVPEVTWGPEDEELWRKLSFRHWPTLFSYYNITLAKRYISLLPVIPV TLRLNPREALEGRHPQDGRSAWPPPGPIPAGHWEAGPKGPGGVQAHGDVTLYKVA ALGLATGIVLVLLLLCLYRVLCPRNYGQLGGGPGRRRRGELPCDDYGYAPPETEIVP LVLRGHLMDIECLASDGMLLVSCCLAGHVCVWDAQTGDCLTRIPRPGQRRDSGVGS GLEAQESWERLSDGGKAGPEEPGDSPPLRHRPRGPPPPSLFGDQPDLTCLIDTNFSAQ PRSSQPTQPEPRHRAVCGRSRDSPGYDFSCLVQRVYQEEGLAAVCTPALRPPSPGPVL300397066.2 - 17 -SQAPEDEGGSPEKGSPSLAWAPSAEGSIWSLELQGNLIVVGRSSGRLEVWDAIEGVL CCSSEEVSSGITALVFLDKRIVAARLNGSLDFFSLETHTALSPLQFRGTPGRGSSPASP VYSSSDTVACHLTHTVPCAHQKPITALKAAAGRLVTGSQDHTLRVFRLEDSCCLFTL QGHSGAITTVYIDQTMVLASGGQDGAICLWDVLTGSRVSHVFAHRGDVTSLTCTTS CVISSGLDDLISIWDRSTGIKFYSIQQDLGCGASLGVISDNLLVTGGQGCVSFWDLNY GDLLQTVYLGKNSEAQPARQILVLDNAAIVCNFGSELSLVYVPSVLEKLD (SEQ ID NO: 7).

[0048] MTLTERLREKISRAFYNHGLLCASYPIPIILFTGFCILACCYPLLKLPLPGTG PVEFTTPVKDYSPPPVDSDRKQGEPTEQPEWYVGAPVAYVQQIFVKSSVFPWHKNLL AVDVFRSPLSRAFQLVEEIRNHVLRDSSGIRSLEELCLQVTDLLPGLRKLRNLLPEHG CLLLSPGNFWQNDWERFHADPDIIGTIHQHEPKTLQTSATLKDLLFGVPGKYSGVSL YTRKRMVSYTITLVFQHYHAKFLGSLRARLMLLHPSPNCSLRAESLVHVHFKEEIGV AELIPL VTTYIILF AYIYF STRKIDMVKSKWGLAL AAVVTVLS SLLMS VGLCTLFGLTP TLNGGEIFPYLVVVIGLENVLVLTKSVVSTPVDLEVKLRIAQGLSSESWSIMKNMATE LGIILIGYFTLVPAIQEFCLFAVVGLVSDFFLQMLFFTTVLSIDIRRMELADLNKRLPPE ACLPSAKPVGQPTRYERQLAVRPSTPHTITLQPSSFRNLRLPKRLRVVYFLARTRLAQ RLIMAGTVVWIGILVYTDPAGLRNYLAAQVTEQSPLGEGALAPMPVPSGMLPPSHPD PAFSIFPPDAPKLPENQTSPGESPERGGPAEVVHDSPVPEVTWGPEDEELWRKLSFRH WPTLFSYYNITLAKRYISLLPVIPVTLRLNPREALEGRHPQDGRSAWPPPGPIPAGHW EAGPKGPGGVQAHGDVTLYKVAALGLATGIVLVLLLLCLYRVLCPRNYGQLGGGP GRRRRGELPCDDYGYAPPETEIVPLVLRGHLMDIECLASDGMLLVSCCLAGHVCVW DAQTGDCLTRIPRPGRQRRDSGVGSGLEAQESWERLSDGGKAGPEEPGDSPPLRHRP RGPPPPSLFGDQPDLTCLIDTNFSAQPRSSQPTQPEPRHRAVCGRSRDSPGYDFSCLVQ RVYQEEGLAAVCTPALRPPSPGPVLSQAPEDEGGSPEKGSPSLAWAPSAEGSIWSLEL QGNLIVVGRS SGRLEVWD AIEGVLCCS SEEVS SGITALVFLDKRIVAARLNGSLDFF S LETHTAL SPLQFRGTPGRGS SP ASP VYS S SDT VACHLTHT VPC AHQKPIT ALK AA AGR LVTGSQDHTLRVFRLEDSCCLFTLQGHSGAITTVYIDQTMVLASGGQDGAICLWDVL TGSRVSHVFAHRGDVTSLTCTTSCVISSGLDDLISIWDRSTGIKFYSIQQDLGCGASLG VISDNLLVTGGQGCVSFWDLNYGDLLQTVYLGKNSEAQPARQILVLDNAAIVCNFG SELSLVYVPSVLEKLD (SEQ ID NO: 8).

[0049] In certain aspects, a SCAP protein described herein is mutated. In certain aspects, the SCAP has one or more deletions, additions, or substitutions relative to a wild-type SCAP or relative to SEQ ID NO: 7 or 8. In certain aspects, the SCAP or the SCAP gene product comprises a loop 6 deletion. In certain aspects, the SCAP or the SCAP gene product comprises 300397066.2 - 18 -a deletion of amino acids 452-512 in SEQ ID NO: 8 or the corresponding amino acids in SEQ ID NO: 7. In certain aspects, the SCAP or the SCAP gene product comprises a WD domain deletion. In certain aspects, the SCAP or the SCAP gene product comprises a deletion of amino acids 741-1279 in SEQ ID NO: 8 or the corresponding amino acids in SEQ ID NO: 7. In certain aspects, the SCAP protein is a fusion protein. In some aspects, the fusion protein comprises the SCAP protein and a base editor. In some aspects, the SCAP protein is fused, at its N terminus or its C terminus, a base editor. In some aspects, the base editor is a Tad8e protein.

[0050] In certain aspects, the SCAP gene or SCAP gene product comprises or consist of a nucleic acid encoding a SCAP protein, including any SCAP described herein. In some aspects, the SCAP gene or SCAP gene product is described in the GenBank database. In certain aspects, the SCAP gene or SCAP gene product comprises or consists of a polynucleotide having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) sequence identity to SEQ IDN0:9-ll

[0051] ATGACCCTGACTGAAAGGCTGCGTGAGAAGATATCTCGGGCCTTCTAC AACCATGGGCTCCTCTGTGCATCCTATCCCATCCCCATCATCCTCTTCACAGGGTT CTGCATCTTAGCCTGCTGCTACCCACTGCTGAAACTCCCCTTGCCAGGAACAGGA CCTGTGGAATTCACCACCCCTGTGAAGGATTACTCGCCCCCACCTGTGGACTCTG ACCGCAAACAAGGAGAGCCTACTGAGCAGCCTGAGTGGTATGTGGGTGCCCCGG TGGCTTATGTCCAGCAGATATTTGTGAAGTCCTCAGTGTTTCCCTGGCACAAGAA CCTCCTGGCAGTAGATGTATTTCGTTCACCTTTGTCCCGGGCATTCCAACTGGTGG AGGAGATCCGGAACCACGTGCTGAGAGACAGCTCTGGGATCAGGAGCTTGGAGG AGTTGTGTCTGCAAGTGACCGACCTGCTGCCAGGCCTTAGGAAGCTCAGGAACCT ACTCCCTGAGCATGGATGCCTGCTGCTGTCCCCTGGGAACTTCTGGCAGAATGAC TGGGAACGCTTCCATGCTGATCCTGACATCATTGGGACCATCCACCAGCACGAGC CTAAAACCCTGCAGACTTCAGCCACACTCAAAGACTTGTTATTTGGTGTTCCTGG GAAGTACAGCGGGGTGAGCCTCTACACCAGGAAGAGGATGGTCTCCTACACCAT CACCCTGGTCTTCCAGCACTACCATGCCAAGTTCCTGGGCAGCCTGCGTGCCCGC CTGATGCTTCTGCACCCCAGCCCCAACTGCAGCCTTCGGGCGGAGAGCCTGGTCC ACGTGCACTTCAAGGAGGAGATTGGTGTCGCTGAGCTCATCCCCCTTGTGACCAC CTACATCATCTTGTTTGCCTACATCTACTTCTCCACGCGGAAGATCGACATGGTC AAGTCCAAGTGGGGGCTGGCCCTGGCTGCCGTGGTCACAGTGCTCAGCTCGCTGC TCATGTCTGTGGGACTCTGCACACTCTTCGGCCTGACGCCCACCCTCAATGGCGG CGAGATTTTCCCCTACCTTGTGGTGGTTATTGGGTTAGAGAATGTGTTGGTGCTCA300397066.2 - 19 -CCAAGTCTGTGGTCTCAACCCCGGTAGACCTGGAGGTGAAGCTGCGGATCGCCC AAGGCCTAAGCAGCGAGAGCTGGTCCATCATGAAGAACATGGCCACGGAGCTGG GCATCATCCTCATCGGCTACTTCACCCTAGTGCCCGCCATCCAGGAGTTCTGTCTC TTTGCTGTCGTGGGGCTGGTGTCTGACTTCTTCCTTCAGATGCTGTTTTTCACCAC TGTCCTGTCCATTGACATTCGCCGGATGGAGCTAGCAGACCTGAACAAGCGACTG CCCCCTGAGGCCTGCCTGCCCTCAGCCAAGCCAGTGGGACAGCCAACGCGCTAC GAGCGGCAGCTGGCTGTGAGGCCGTCCACACCCCACACCATCACGTTGCAGCCG TCTTCCTTCCGAAACCTGCGGCTCCCCAAGAGGCTGCGTGTTGTCTACTTCCTGGC CCGCACCCGCCTGGCACAGCGCCTCATCATGGCTGGCACCGTTGTCTGGATTGGC ATCCTGGTATACACAGACCCAGCAGGGCTGCGCAACTACCTCGCTGCCCAGGTG ACGGAACAGAGCCCATTGGGTGAGGGAGCCCTGGCTCCCATGCCCGTGCCTAGT GGCATGCTGCCCCCCAGCCACCCGGACCCTGCCTTCTCCATCTTCCCACCTGATG CCCCTAAGCTACCTGAGAACCAGACGTCGCCAGGCGAGTCACCTGAGCGTGGAG GTCCAGCAGAGGTTGTCCATGACAGCCCAGTCCCAGAGGTAACCTGGGGGCCTG AGGATGAGGAACTTTGGAGGAAATTGTCCTTCCGCCACTGGCCGACGCTCTTCAG CTATTACAACATCACACTGGCCAAGAGGTACATCAGCCTGCTGCCCGTCATCCCA GTCACGCTCCGCCTGAACCCGAGGGAGGCTCTGGAGGGCCGGCACCCTCAGGAC GGCCGCAGTGCCTGGCCCCCACCGGGGCCCATACCTGCTGGGCACTGGGAAGCA GGACCCAAGGGCCCAGGTGGGGTGCAGGCCCATGGAGACGTCACGCTGTACAAG GTGGCGGCGCTGGGCCTGGCCACCGGCATCGTCTTGGTGCTGCTGCTGCTCTGCC TCTACCGCGTGCTATGCCCGCGCAACTACGGGCAGCTGGGTGGTGGGCCCGGGC GGCGGAGGCGCGGGGAGCTGCCCTGCGACGACTACGGCTATGCGCCACCCGAGA CGGAGATCGTGCCGCTTGTGCTGCGCGGCCACCTCATGGACATCGAGTGCCTGGC CAGCGACGGCATGCTGCTGGTGAGCTGCTGCCTGGCAGGCCACGTCTGCGTGTG GGACGCGCAGACCGGGGATTGCCTAACGCGCATTCCGCGCCCAGGCAGGCAGCG CCGGGACAGTGGCGTGGGCAGCGGGCTTGAGGCTCAGGAGAGCTGGGAACGACT TTCAGATGGTGGGAAGGCTGGTCCAGAGGAGCCTGGGGACAGCCCTCCCCTGAG ACACCGCCCCCGGGGCCCTCCGCCGCCTTCCCTCTTCGGGGACCAGCCTGACCTC ACCTGCTTAATTGACACCAACTTTTCAGCGCAGCCTCGGTCCTCACAGCCCACTC AGCCCGAGCCCCGGCACCGGGCGGTCTGTGGCCGCTCTCGGGACTCCCCAGGCT ATGACTTCAGCTGCCTGGTGCAGCGGGTGTACCAGGAGGAGGGGCTGGCGGCCG TCTGCACACCAGCCCTGCGCCCACCCTCGCCTGGGCCGGTGCTGTCCCAGGCCCC TGAGGACGAGGGTGGCTCCCCCGAGAAAGGCTCCCCTTCCCTCGCCTGGGCCCCC AGTGCCGAGGGTTCCATCTGGAGCTTGGAGCTGCAGGGCAACCTCATCGTGGTG300397066.2 - 20 -GGGCGGAGCAGCGGCCGGCTGGAGGTGTGGGACGCCATTGAAGGGGTGCTGTGC TGCAGCAGCGAGGAGGTCTCCTCAGGCATTACCGCTCTGGTGTTCTTGGACAAAA GGATTGTGGCTGCACGGCTCAACGGTTCCCTTGATTTCTTCTCCTTGGAGACCCA CACTGCCCTCAGCCCCCTGCAGTTTAGAGGGACCCCAGGGCGGGGCAGTTCCCCT GCCTCTCCAGTGTACAGCAGCAGCGACACAGTGGCCTGTCACCTGACCCACACA GTGCCCTGTGCACACCAAAAACCCATCACAGCCCTGAAAGCCGCTGCTGGGCGC TTGGTGACTGGGAGCCAAGACCACACACTGAGAGTGTTCCGTCTGGAGGACTCG TGCTGCCTCTTCACCCTTCAGGGCCACTCAGGGGCCATCACGACCGTGTACATTG ACCAGACCATGGTGCTGGCCAGTGGAGGACAAGATGGGGCCATCTGCCTGTGGG ATGTACTGACTGGCAGCCGGGTCAGCCATGTGTTTGCTCACCGTGGGGATGTCAC CTCCCTTACCTGTACCACCTCCTGTGTCATCAGCAGTGGCCTGGATGACCTCATC AGCATCTGGGACCGCAGCACAGGCATCAAGTTCTACTCCATTCAGCAGGACCTG GGCTGTGGTGCAAGCTTGGGTGTCATCTCAGACAACCTGCTGGTGACTGGCGGCC AGGGCTGTGTCTCCTTTTGGGACCTAAACTACGGGGACCTGTTACAGACAGTCTA CCTGGGGAAGAACAGTGAGGCCCAGCCTGCCCGCCAGATCCTGGTGCTGGACAA CGCTGCCATTGTCTGCAACTTTGGCAGTGAGCTCAGCCTGGTGTATGTGCCCTCT GTGCTGGAGAAGCTGGAC (SEQ ID NO: 9).

[0052] GGGCACCCGGCGGCCAGGAGAGAGAGGGAGGGCGCCACGCACCGGA CTGCGGGCCGAGAGCGCGCACGCCGCGCTCCGCCCCTGCTGCCGCCCCCGTCGCC GCCGCCGCCGCCGCCGCAGCTTGGGAGGTGCTGCCACCACAGGTACCTGCACAT GTTGTTCTTTGTCAGTGCTGTCAAGTGTGTGCCAGGGTGATCCATGGTCACTTTCC GGGATGGCAGCAAGGTGACTTCGGCTGAGGATGACCCTGACTGAAAGGCTGCGT GAGAAGATATCTCGGGCCTTCTACAACCATGGGCTCCTCTGTGCATCCTATCCCA TCCCCATCATCCTCTTCACAGGGTTCTGCATCTTAGCCTGCTGCTACCCACTGCTG AAACTCCCCTTGCCAGGAACAGGACCTGTGGAATTCACCACCCCTGTGAAGGATT ACTCGCCCCCACCTGTGGACTCTGACCGCAAACAAGGAGAGCCTACTGAGCAGC CTGAGTGGGTTCCTGGGCAGCCTGCGTGCCCGCCTGATGCTTCTGCACCCCAGCC CCAACTGCAGCCTTCGGGCGGAGAGCCTGGTCCACGTGCACTTCAAGGAGGAGA TTGGTGTCGCTGAGCTCATCCCCCTTGTGACCACCTACATCATCTTGTTTGCCTAC ATCTACTTCTCCACGCGGAAGATCGACATGGTCAAGTCCAAGTGGGGGCTGGCC CTGGCTGCCGTGGTCACAGTGCTCAGCTCGCTGCTCATGTCTGTGGGACTCTGCA CACTCTTCGGCCTGACGCCCACCCTCAATGGCGGCGAGATTTTCCCCTACCTTGT GGTGGTTATTGGGTTAGAGAATGTGTTGGTGCTCACCAAGTCTGTGGTCTCAACC CCGGTAGACCTGGAGGTGAAGCTGCGGATCGCCCAAGGCCTAAGCAGCGAGAGC300397066.2 - 21 -TGGTCCATCATGAAGAACATGGCCACGGAGCTGGGCATCATCCTCATCGGCTACT TCACCCTAGTGCCCGCCATCCAGGAGTTCTGTCTCTTTGCTGTCGTGGGGCTGGT GTCTGACTTCTTCCTTCAGATGCTGTTTTTCACCACTGTCCTGTCCATTGACATTC GCCGGATGGAGCTAGCAGACCTGAACAAGCGACTGCCCCCTGAGGCCTGCCTGC CCTCAGCCAAGCCAGTGGGACAGCCAACGCGCTACGAGCGGCAGCTGGCTGTGA GGCCGTCCACACCCCACACCATCACGTTGCAGCCGTCTTCCTTCCGAAACCTGCG GCTCCCCAAGAGGCTGCGTGTTGTCTACTTCCTGGCCCGCACCCGCCTGGCACAG CGCCTCATCATGGCTGGCACCGTTGTCTGGATTGGCATCCTGGTATACACAGACC CAGCAGGGCTGCGCAACTACCTCGCTGCCCAGGTGACGGAACAGAGCCCATTGG GTGAGGGAGCCCTGGCTCCCATGCCCGTGCCTAGTGGCATGCTGCCCCCCAGCCA CCCGGACCCTGCCTTCTCCATCTTCCCACCTGATGCCCCTAAGCTACCTGAGAAC CAGACGTCGCCAGGCGAGTCACCTGAGCGTGGAGGTCCAGCAGAGGTTGTCCAT GACAGCCCAGTCCCAGAGGTAACCTGGGGGCCTGAGGATGAGGAACTTTGGAGG AAATTGTCCTTCCGCCACTGGCCGACGCTCTTCAGCTATTACAACATCACACTGG CCAAGAGGTACATCAGCCTGCTGCCCGTCATCCCAGTCACGCTCCGCCTGAACCC GAGGGAGGCTCTGGAGGGCCGGCACCCTCAGGACGGCCGCAGTGCCTGGCCCCC ACCGGGGCCCATACCTGCTGGGCACTGGGAAGCAGGACCCAAGGGCCCAGGTGG GGTGCAGGCCCATGGAGACGTCACGCTGTACAAGGTGGCGGCGCTGGGCCTGGC CACCGGCATCGTCTTGGTGCTGCTGCTGCTCTGCCTCTACCGCGTGCTATGCCCGC GCAACTACGGGCAGCTGGGTGGTGGGCCCGGGCGGCGGAGGCGCGGGGAGCTG CCCTGCGACGACTACGGCTATGCGCCACCCGAGACGGAGATCGTGCCGCTTGTG CTGCGCGGCCACCTCATGGACATCGAGTGCCTGGCCAGCGACGGCATGCTGCTG GTGAGCTGCTGCCTGGCAGGCCACGTCTGCGTGTGGGACGCGCAGACCGGGGAT TGCCTAACGCGCATTCCGCGCCCAGGGCAGCGCCGGGACAGTGGCGTGGGCAGC GGGCTTGAGGCTCAGGAGAGCTGGGAACGACTTTCAGATGGTGGGAAGGCTGGT CCAGAGGAGCCTGGGGACAGCCCTCCCCTGAGACACCGCCCCCGGGGCCCTCCG CCGCCTTCCCTCTTCGGGGACCAGCCTGACCTCACCTGCTTAATTGACACCAACT TTTCAGCGCAGCCTCGGTCCTCACAGCCCACTCAGCCCGAGCCCCGGCACCGGGC GGTCTGTGGCCGCTCTCGGGACTCCCCAGGCTATGACTTCAGCTGCCTGGTGCAG CGGGTGTACCAGGAGGAGGGGCTGGCGGCCGTCTGCACACCAGCCCTGCGCCCA CCCTCGCCTGGGCCGGTGCTGTCCCAGGCCCCTGAGGACGAGGGTGGCTCCCCCG AGAAAGGCTCCCCTTCCCTCGCCTGGGCCCCCAGTGCCGAGGGTTCCATCTGGAG CTTGGAGCTGCAGGGCAACCTCATCGTGGTGGGGCGGAGCAGCGGCCGGCTGGA GGTGTGGGACGCCATTGAAGGGGTGCTGTGCTGCAGCAGCGAGGAGGTCTCCTC300397066.2 - 22 -AGGCATTACCGCTCTGGTGTTCTTGGACAAAAGGATTGTGGCTGCACGGCTCAAC GGTTCCCTTGATTTCTTCTCCTTGGAGACCCACACTGCCCTCAGCCCCCTGCAGTT TAGAGGGACCCCAGGGCGGGGCAGTTCCCCTGCCTCTCCAGTGTACAGCAGCAG CGACACAGTGGCCTGTCACCTGACCCACACAGTGCCCTGTGCACACCAAAAACC CATCACAGCCCTGAAAGCCGCTGCTGGGCGCTTGGTGACTGGGAGCCAAGACCA CACACTGAGAGTGTTCCGTCTGGAGGACTCGTGCTGCCTCTTCACCCTTCAGGGC CACTCAGGGGCCATCACGACCGTGTACATTGACCAGACCATGGTGCTGGCCAGT GGAGGACAAGATGGGGCCATCTGCCTGTGGGATGTACTGACTGGCAGCCGGGTC AGCCATGTGTTTGCTCACCGTGGGGATGTCACCTCCCTTACCTGTACCACCTCCTG TGTCATCAGCAGTGGCCTGGATGACCTCATCAGCATCTGGGACCGCAGCACAGG CATCAAGTTCTACTCCATTCAGCAGGACCTGGGCTGTGGTGCAAGCTTGGGTGTC ATCTCAGACAACCTGCTGGTGACTGGCGGCCAGGGCTGTGTCTCCTTTTGGGACC TAAACTACGGGGACCTGTTACAGACAGTCTACCTGGGGAAGAACAGTGAGGCCC AGCCTGCCCGCCAGATCCTGGTGCTGGACAACGCTGCCATTGTCTGCAACTTTGG CAGTGAGCTCAGCCTGGTGTATGTGCCCTCTGTGCTGGAGAAGCTGGACTGAGCG CAGGGCCTCCTTGCCCAGGCAGGAGGCTGGGGTGCTGTGTGGGGGCCAATGCAC TGAACCTGGACTTGGGGGAAAGAGCCGAGTATCTTCCAGCCGCTGCCTCCTGACT GTAATAATATTAAACTTTTTTAAAAAACCATATCATCATCTGTCAGGCACTTTGG GAGCTA (SEQ ID NO: 10)

[0053] GGGCACCCGGCGGCCAGGAGAGAGAGGGAGGGCGCCACGCACCGGA CTGCGGGCCGAGAGCGCGCACGCCGCGCTCCGCCCCTGCTGCCGCCCCCGTCGCC GCCGCCGCCGCCGCCGCAGCTTGGGAGGTGCTGCCACCACAGGTACCTGCACAT GTTGTTCTTTGTCAGTGCTGTCAAGTGTGTGCCAGGGTGATCCATGGTCACTTTCC GGGATGGCAGCAAGGTGACTTCGGCTGAGGATGACCCTGACTGAAAGGCTGCGT GAGAAGATATCTCGGGCCTTCTACAACCATGGGCTCCTCTGTGCATCCTATCCCA TCCCCATCATCCTCTTCACAGGGTTCTGCATCTTAGCCTGCTGCTACCCACTGCTG AAACTCCCCTTGCCAGGAACAGGACCTGTGGAATTCACCACCCCTGTGAAGGATT ACTCGCCCCCACCTGTGGACTCTGACCGCAAACAAGGAGAGCCTACTGAGCAGC CTGAGTGGTATGTGGGTGCCCCGGTGGCTTATGTCCAGCAGATATTTGTGAAGTC CTCAGTGTTTCCCTGGCACAAGAACCTCCTGGCAGTAGATGTATTTCGTTCACCTT TGTCCCGGGCATTCCAACTGGTGGAGGAGATCCGGAACCACGTGCTGAGAGACA GCTCTGGGATCAGGAGCTTGGAGGAGTTGTGTCTGCAAGTGACCGACCTGCTGCC AGGCCTTAGGAAGCTCAGGAACCTACTCCCTGAGCATGGATGCCTGCTGCTGTCC CCTGGGAACTTCTGGCAGAATGACTGGGAACGCTTCCATGCTGATCCTGACATCA300397066.2 - 23 -TTGGGACCATCCACCAGCACGAGCCTAAAACCCTGCAGACTTCAGCCACACTCA AAGACTTGTTATTTGGTGTTCCTGGGAAGTACAGCGGGGTGAGCCTCTACACCAG GAAGAGGATGGTCTCCTACACCATCACCCTGGTCTTCCAGCACTACCATGCCAAG TTCCTGGGCAGCCTGCGTGCCCGCCTGATGCTTCTGCACCCCAGCCCCAACTGCA GCCTTCGGGCGGAGAGCCTGGTCCACGTGCACTTCAAGGAGGAGATTGGTGTCG CTGAGCTCATCCCCCTTGTGACCACCTACATCATCTTGTTTGCCTACATCTACTTC TCCACGCGGAAGATCGACATGGTCAAGTCCAAGTGGGGGCTGGCCCTGGCTGCC GTGGTCACAGTGCTCAGCTCGCTGCTCATGTCTGTGGGACTCTGCACACTCTTCG GCCTGACGCCCACCCTCAATGGCGGCGAGATTTTCCCCTACCTTGTGGTGGTTAT TGGGTTAGAGAATGTGTTGGTGCTCACCAAGTCTGTGGTCTCAACCCCGGTAGAC CTGGAGGTGAAGCTGCGGATCGCCCAAGGCCTAAGCAGCGAGAGCTGGTCCATC ATGAAGAACATGGCCACGGAGCTGGGCATCATCCTCATCGGCTACTTCACCCTAG TGCCCGCCATCCAGGAGTTCTGTCTCTTTGCTGTCGTGGGGCTGGTGTCTGACTTC TTCCTTCAGATGCTGTTTTTCACCACTGTCCTGTCCATTGACATTCGCCGGATGGA GCTAGCAGACCTGAACAAGCGACTGCCCCCTGAGGCCTGCCTGCCCTCAGCCAA GCCAGTGGGACAGCCAACGCGCTACGAGCGGCAGCTGGCTGTGAGGCCGTCCAC ACCCCACACCATCACGTTGCAGCCGTCTTCCTTCCGAAACCTGCGGCTCCCCAAG AGGCTGCGTGTTGTCTACTTCCTGGCCCGCACCCGCCTGGCACAGCGCCTCATCA TGGCTGGCACCGTTGTCTGGATTGGCATCCTGGTATACACAGACCCAGCAGGGCT GCGCAACTACCTCGCTGCCCAGGTGACGGAACAGAGCCCATTGGGTGAGGGAGC CCTGGCTCCCATGCCCGTGCCTAGTGGCATGCTGCCCCCCAGCCACCCGGACCCT GCCTTCTCCATCTTCCCACCTGATGCCCCTAAGCTACCTGAGAACCAGACGTCGC CAGGCGAGTCACCTGAGCGTGGAGGTCCAGCAGAGGTTGTCCATGACAGCCCAG TCCCAGAGGTAACCTGGGGGCCTGAGGATGAGGAACTTTGGAGGAAATTGTCCT TCCGCCACTGGCCGACGCTCTTCAGCTATTACAACATCACACTGGCCAAGAGGTA CATCAGCCTGCTGCCCGTCATCCCAGTCACGCTCCGCCTGAACCCGAGGGAGGCT CTGGAGGGCCGGCACCCTCAGGACGGCCGCAGTGCCTGGCCCCCACCGGGGCCC ATACCTGCTGGGCACTGGGAAGCAGGACCCAAGGGCCCAGGTGGGGTGCAGGCC CATGGAGACGTCACGCTGTACAAGGTGGCGGCGCTGGGCCTGGCCACCGGCATC GTCTTGGTGCTGCTGCTGCTCTGCCTCTACCGCGTGCTATGCCCGCGCAACTACG GGCAGCTGGGTGGTGGGCCCGGGCGGCGGAGGCGCGGGGAGCTGCCCTGCGAC GACTACGGCTATGCGCCACCCGAGACGGAGATCGTGCCGCTTGTGCTGCGCGGC CACCTCATGGACATCGAGTGCCTGGCCAGCGACGGCATGCTGCTGGTGAGCTGCT GCCTGGCAGGCCACGTCTGCGTGTGGGACGCGCAGACCGGGGATTGCCTAACGC300397066.2 - 24 -GCATTCCGCGCCCAGGCAGGCAGCGCCGGGACAGTGGCGTGGGCAGCGGGCTTG AGGCTCAGGAGAGCTGGGAACGACTTTCAGATGGTGGGAAGGCTGGTCCAGAGG AGCCTGGGGACAGCCCTCCCCTGAGACACCGCCCCCGGGGCCCTCCGCCGCCTTC CCTCTTCGGGGACCAGCCTGACCTCACCTGCTTAATTGACACCAACTTTTCAGCG CAGCCTCGGTCCTCACAGCCCACTCAGCCCGAGCCCCGGCACCGGGCGGTCTGTG GCCGCTCTCGGGACTCCCCAGGCTATGACTTCAGCTGCCTGGTGCAGCGGGTGTA CCAGGAGGAGGGGCTGGCGGCCGTCTGCACACCAGCCCTGCGCCCACCCTCGCC TGGGCCGGTGCTGTCCCAGGCCCCTGAGGACGAGGGTGGCTCCCCCGAGAAAGG CTCCCCTTCCCTCGCCTGGGCCCCCAGTGCCGAGGGTTCCATCTGGAGCTTGGAG CTGCAGGGCAACCTCATCGTGGTGGGGCGGAGCAGCGGCCGGCTGGAGGTGTGG GACGCCATTGAAGGGGTGCTGTGCTGCAGCAGCGAGGAGGTCTCCTCAGGCATT ACCGCTCTGGTGTTCTTGGACAAAAGGATTGTGGCTGCACGGCTCAACGGTTCCC TTGATTTCTTCTCCTTGGAGACCCACACTGCCCTCAGCCCCCTGCAGTTTAGAGG GACCCCAGGGCGGGGCAGTTCCCCTGCCTCTCCAGTGTACAGCAGCAGCGACAC AGTGGCCTGTCACCTGACCCACACAGTGCCCTGTGCACACCAAAAACCCATCAC AGCCCTGAAAGCCGCTGCTGGGCGCTTGGTGACTGGGAGCCAAGACCACACACT GAGAGTGTTCCGTCTGGAGGACTCGTGCTGCCTCTTCACCCTTCAGGGCCACTCA GGGGCCATCACGACCGTGTACATTGACCAGACCATGGTGCTGGCCAGTGGAGGA CAAGATGGGGCCATCTGCCTGTGGGATGTACTGACTGGCAGCCGGGTCAGCCAT GTGTTTGCTCACCGTGGGGATGTCACCTCCCTTACCTGTACCACCTCCTGTGTCAT CAGCAGTGGCCTGGATGACCTCATCAGCATCTGGGACCGCAGCACAGGCATCAA GTTCTACTCCATTCAGCAGGACCTGGGCTGTGGTGCAAGCTTGGGTGTCATCTCA GACAACCTGCTGGTGACTGGCGGCCAGGGCTGTGTCTCCTTTTGGGACCTAAACT ACGGGGACCTGTTACAGACAGTCTACCTGGGGAAGAACAGTGAGGCCCAGCCTG CCCGCCAGATCCTGGTGCTGGACAACGCTGCCATTGTCTGCAACTTTGGCAGTGA GCTCAGCCTGGTGTATGTGCCCTCTGTGCTGGAGAAGCTGGACTGAGCGCAGGG CCTCCTTGCCCAGGCAGGAGGCTGGGGTGCTGTGTGGGGGCCAATGCACTGAAC CTGGACTTGGGGGAAAGAGCCGAGTATCTTCCAGCCGCTGCCTCCTGACTGTAAT AATATTAAACTTTTTTAAAAAACCATATCATCATCTGTCAGGCACTTTGGGAGCTA (SEQ IDNO: 11)300397066.2 - 25 -I. Therapeutic Compositions

[0054] Disclosed herein are therapeutic compositions comprising SCAP, a SCAP gene, a SCAP gene product, or a SCAP inhibitor. The therapeutic composition may be specifically formulated to effectively deliver SCAP, a SCAP gene, a SCAP gene product, or a SCAP inhibitor to a patient, a specific tissue in the patient, and / or a specific cell type in the patient.

[0055] In certain aspects, the compositions or agents for use in the methods are suitably contained in a pharmaceutically acceptable carrier, making a therapeutic composition. The carrier can be non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as a tumor or other tissue) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices and the like.

[0056] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0057] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0058] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner300397066.2 - 26 -responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0059] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0060] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0061] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0062] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0063] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers,300397066.2 - 27 -buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol may be between about 0.01 ml and 0.5 ml, for example.

[0064] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0065] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.

[0066] It is contemplated that other agents may be used in combination with certain aspects of the present aspects to improve the therapeutic efficacy of treatment.II. Administration of Therapeutic Compositions

[0067] Certain therapies provided herein may comprise administration of one or a combination of therapeutic agents. The therapeutic agent can comprise SCAP, a SCAP gene, a SCAP gene product, or a SCAP inhibitor. In certain aspects, the therapy comprises a first therapy, which may be SCAP, a SCAP gene, a SCAP gene product, or a SCAP inhibitor, and a second therapy, such as any additional anti-cancer agent and / or anti-cancer intervention. The therapies may be administered in any suitable manner known in the art. For example, the first and second therapy may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, the first and second therapy are administered in a separate composition. In some aspects, the first and second therapy are in the same composition.

[0068] In some aspects, the first and second therapy are administered substantially simultaneously. In some aspects, the first and second therapy are administered sequentially

[0069] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.300397066.2 - 28 -

[0070] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some aspects, the therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some aspects, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0071] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0072] In some aspects, the first therapy, which may be SCAP, a SCAP gene, a SCAP gene product, or a SCAP inhibitor, is administered at a dose of between 1 mg / kg and 5000 mg / kg. In some aspects, the first therapy is administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 300397066.2 - 29 -314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg / kg.

[0073] In some aspects, a single dose of the second therapy is administered. In some aspects, multiple doses of the second therapy are administered. In some aspects, the second therapy is administered at a dose of between 1 mg / kg and 5000 mg / kg. In some aspects, the second therapy is administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 300397066.2 - 30 -279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg / kg.

[0074] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain aspects, it is contemplated that doses in the range from 10 mg / kg to 200 mg / kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg / kg, mg / kg, pg / day, or mg / day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0075] In certain aspects, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another aspect, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM 300397066.2 - 31 -to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other aspects, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pM or any range derivable therein. In certain aspects, the therapeutic agent that is administered to a subj ect is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0076] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0077] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels). It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0078] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.

[0079] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the 300397066.2 - 32 -like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0080] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0081] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0082] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0083] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about300397066.2 - 33 -by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0084] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0085] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0086] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.A. Cancer Therapy

[0087] In some aspects, the method further comprises administering a cancer therapy to the patient. The cancer therapy may be chosen based on the expression level measurements, alone or in combination with the clinical risk score calculated for the patient. In some aspects, the cancer therapy comprises a local cancer therapy. In some aspects, the cancer therapy excludes a systemic cancer therapy. In some aspects, the cancer therapy excludes a local therapy. In some aspects, the cancer therapy comprises a local cancer therapy without the administration of a system cancer therapy. In some aspects, the cancer therapy comprises an immunotherapy, which may be an immune checkpoint therapy. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered.300397066.2 - 34 -

[0088] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain aspects, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In some aspects, the cancer is recurrent cancer. In some aspects, the cancer is Stage I cancer. In some aspects, the cancer is Stage II cancer. In some aspects, the cancer is Stage III cancer. In some aspects, the cancer is Stage IV cancer.

[0089] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar 300397066.2 - 35 -rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin’s disease; hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.B. Surgery

[0090] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).300397066.2 - 36 -

[0091] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.III. Kits

[0092] Certain aspects of the present disclosure also concern kits containing compositions of the disclosure or compositions to implement methods disclosed herein. Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[0093] Individual components may also be provided in a kit in concentrated amounts; in some aspects, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more.

[0094] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different aspects may be combined. The claims originally filed are contemplated to cover claims that are multiply dependent on any filed claim or combination of filed claims.Examples

[0095] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1PreQi strongly reduces cell proliferation but is attenuated and reversed by queuine

[0096] Queuine and preQi are both derived from the same bacterial metabolic pathway and may affect host cell properties. The inventors measured the presence of both queuine and300397066.2 - 37 -preQi in the plasma of specific pathogen-free (SPF) mice by mass spectrometry and found appreciable amounts of both, suggesting that mouse tissues could indeed be exposed to both metabolites (FIGs. IB, 8A, 8B). The inventors performed PAQS-seq75of human stools and identified the presence of Q-tRNA modification in multiple bacterial classes (FIGs. 1C, 8C), indicating that queuine and preQi can come from many taxa of gut bacteria.

[0097] To test the effect of preQi on cell proliferation, the inventors generated HEK293T cells depleted of Q-tRNA modification (0Q cells)76,77and added queuine, preQi, or both together to the culture medium (FIGs. ID, 8D). The addition of queuine alone slightly increased cell proliferation. However, the addition of preQi alone strongly reduced cell proliferation. Strikingly, the reduction of cell proliferation with preQi was fully reversed when queuine was also present.

[0098] To further evaluate the preQi effects on proliferation the inventors performed cell proliferation experiments with preQi and queuine added at different times and with cell mixtures with different starting Q-tRNA modification levels. The inventors first treated cells with preQi for up to 72 hours, then measured cell proliferation upon addition of queuine at different times and concentrations after preQi pretreatment. Cells pre-exposed to preQi up to 72 hours proliferated equally well (Fig. IE). The addition of 10 nM to 1 pM queuine, but not 1 nM queuine to cells pre-exposed 48h to preQi fully rescued proliferation (FIGs. IF, 8E).The above experiments were carried out with 0Q cells before preQl / queuine addition. To test whether the Q-tRNA modification status of the starting cells mattered in preQi -dependent reduction in proliferation, the inventors added preQi to cell mixtures at varying proportions of 0Q and fully Q-modified tRNA (100Q cells)76,77. PreQi had the same effect on proliferation regardless of the Q-tRNA modification status of the starting mixture FIGs. 1G, 8F).

[0099] The inventors further tested whether the preQi effect on cell proliferation went beyond transformed human cells. For mouse embryonic fibroblast (MEF) cells (FIGs. 1H, 8G), the addition of queuine had no effect on proliferation. The addition of preQi strongly reduced proliferation, and the addition of queuine in the presence of preQi partially restored proliferation. The inventors also measured queuine and preQi effect on the proliferation of primary mouse bone marrow-derived dendritic cells (BMDCs, FIGs. II, 8H, 81). The addition of queuine alone had only a small effect on proliferation, but preQi strongly inhibited proliferation. For BMDCs, up to 100 nM queuine was needed to fully restore proliferation in the presence of 100 nM preQi.

[0100] Taken together, these results indicate that preQi strongly represses cell proliferation of transformed cells, non-transformed cells, and primary immune cells, but 300397066.2 - 38 -queuine can overcome the preQi effect. PreQi does not reduce the ability of cells to proliferate, rather, cells are poised to uptake queuine to resume growth, and preQi effect is independent on the Q-tRNA modification level of the starting cells. The sensitivity of the preQ 1 and queuine effects on proliferation depends on the cell type: preQi effect varies from 100 nM to 1 pM, and queuine rescue varies from 10 nM to 100 nM in the medium.PreQi effect is dependent on QTRT1 / 2 and is incorporated into tRNA in cells

[0101] Mammalian Q-tRNA modification is installed by the QTRT1 / QTRT2 complex in which QTRT1 is the catalytic subunit. The recombinant QTRT1 / QTRT2 complex can use both queuine and preQi to modify tRNA in the test tube20,21. The inventors knocked down QTRT1 (FIG. 2A) and measured the effect of preQi on cell proliferation (FIGs.2B, 9A). The addition of queuine had little effect on the proliferation of the QTRT1-KD cells. However, the addition of preQi reduced the proliferation of QTRT1-KD cells to a much smaller extent than that of the wild-type control cells. Knocking down QTRT2 also alleviated the preQi effect on proliferation FIGs. 2C, 9B, 9C).

[0102] The inventors applied two independent methods to show that preQi was incorporated into human tRNA in cells. First, the inventors established a mass spectrometry method to measure the preQi nucleoside (preQi sine, £) by LC-MS / MS (FIG. 2D, FIG. 8B).The inventors employed Multiple Reaction Monitoring (MRM) mode, a specific and sensitive mass spectrometry technique for quantifying selective compounds within complex mixtures. In MRM, preQi sine was selected, then fragmented in the collision cell, and specific products of fragmentation were detected. The inventors employed custom made preQi sine dichlorohydrate as a standard to calibrate the method. The inventors measured the queuosine and preQi sine levels after digestion of total RNA from queuine or preQi treated cells (FIG.2E). As expected, a high level of queuosine was present in the queuine, but not in preQi treated cells. Conversely, a high level of preQi sine was present in the preQi, but not in queuine treated cells. Second, the inventors took advantage of the chemical structure of preQi sine containing a primary amine group absent in queuosine. The inventors devised a chemical tagging strategy that required primary amine reaction with a large chemical group which would result in a gel shift (FIG. 9D). Q-modified tRNAs could be detected by N-acryloyl-3-aminophenylboronic acid (APB) or acid gel electrophoresis77, but the inventors did not observe a gel shift from preQi -modified tRNA on these gels without chemical tagging, likely due to the smaller size of the preQi sine moiety compared to queuosine (FIG. 2F). After reacting with the chemical tag, tRNATyr / Hls / Asn / Aspfrom preQi treated cells showed significantly retarded migration (FIG.2F).300397066.2 - 39 -Unexpectedly, the addition of just 1 nM queuine in the presence of preQi increased the abundance and preQi -tRNA fraction of tRNAAsn / Tyr, even though this low queuine concentration only very moderately rescued proliferation (FIG. IF).

[0103] Taken together, these results indicate that the preQi effect is mediated by QTRT1 and QTRT2, the same enzyme complex that installs Q-tRNA modification. PreQi is incorporated into tRNAs in cells, however, the addition of a low level of queuine can increase preQi levels in the low affinity Q-modification substrates of tRNATyr / Asn 77, suggesting a complex interplay between preQi and queuine in the installation of preQl / Q-tRNA modifications.PreQi incorporation in tRNA in mouse tissues and effect on xenograft tumor growth

[0104] To further explore the relationship of queuine and preQi and their tRNA modifications, the inventors measured the presence of queuine and preQi metabolites in SPF mouse tissues (FIG. 3A). Queuine was readily detectable at varying amounts in all tissues examined. PreQi was detectable in cerebellum, cortex, colon, kidney, liver, lymph nodes, mesenteric lymph nodes, spleen, thymus, sometimes in stomach and small intestine, but not in bone marrow, heart, lung. The inventors also measured the presence of queuosine (Q) and preQi sine (g) nucleotides in these tissues (FIG. 3B). Q was present at varying levels in all tissues, and the variation could be derived from the tRNA Q-modification and / or tissuedependent tRNA expression levels. £ was not detected in any tissue. As a control, queuine, preQi, Q and £ were all detected in feces (FIG. 10A) which contained mostly bacteria.

[0105] The inventors injected preQi into SPF mice to determine whether preQi sine could be detected upon a sudden influx of preQi metabolite. Using mass spectrometry, the inventors readily detected a reduction of queuosine levels and simultaneously a substantial level of preQi sine nucleotide in the total RNA of liver, kidney, heart, and lung, the four tissues examined in this experiment harvested after three days of inj ecting preQ 1 every 24 hours (FIG.3C). Using APB gel electrophoresis which could measure the Q-modification levels in tRNAHls / Asn, the inventors validated that preQi injection corresponded to a substantial reduction of Q-modification levels in these tRNAs, consistent with preQi sine replacing queuosine in these tRNAs (FIGs. 3D, 10B).

[0106] To identify a physiological effect of preQi treatment in mice, the inventors used ovalbumin-expressing mouse melanoma B16 cells (B16-OVA) as xenograft tumors. The inventors determined first that preQi also strongly reduced the proliferation of B16-OVA cells, and queuine addition restored proliferation in vitro (FIGs. 3E, 10C). Both control and preQi-300397066.2 - 40 -treated cells were implanted in mouse skin and tumor growth was measured over time. PreQi treatment significantly reduced the tumor volume at 9- and 11 -days post-implantation of Bl 6-OVA cells (FIG. 3F).

[0107] Taken together, the results indicate that queuine and preQi metabolites are present in many mouse tissues. Mouse tissues are poised to take up preQi to incorporate into their cognate tRNAs upon an influx of preQi. Furthermore, preQi treatment can result in long-term consequences in a cancer model.PreQi reduces cognate tRNA levels and translation in a pathway and codon dependent manner

[0108] To determine the effect of preQi treatment on tRNA, the inventors performed multiplex small RNA sequencing78to measure tRNA abundance change. For the nuclear-encoded tRNAs, preQi alone significantly reduced the levels only of the 4 cognate tRNATyr / Hls / Asn / Aspthat could be Q- or preQi -modified (FIG. 4A). In the presence of preQi, the level of these tRNAs started to recover upon the addition of 1 nM of queuine, and recovery was complete for tRNATyr / Asp / Asnat 10 nM queuine (FIG. 4B). The inventors validated the preQi -dependent depletion of cognate tRNAs by Northern blots (FIG.4C). The inventors also observed depletions of cognate tRNAs in MEF cells (FIG. 11 A). The human mitochondrial tRNATyr / Hls / Asn / Aspare also Q-modified79,8°. PreQi alone also depleted the cognate mitochondrial tRNAs, but the recovery of the mitochondrial tRNATyr / Hls / Asn / Asplevels required more than 10 nM queuine (FIGs. 4 A, 4B).

[0109] PreQi -dependent cognate tRNA depletion could be related to deficiency in tRNA charging and / or elevated cleavage by cellular ribonucleases. For nuclear-encoded tRNATyr, preQi treatment resulted in decreased charging levels (FIG. 4D). At the same time, more 5’ tRNATyrfragments (tRFTyr) were present, consistent with increased degradation of tRNATyr(FIG. 4E). The addition of queuine decreased the tRFTyrlevel, consistent with the restoration of full-length tRNATyrlevel under these conditions.

[0110] To determine the preQi effect on translation, the inventors performed polysome profiling of preQi treated and control cells. PreQi substantially reduced global translation, consistent with its severe reduction of cell proliferation (FIG. 5A). In the input and polysome samples, 1098 and 590 transcripts were increased, 1307 and 491 transcripts decreased by 2-fold in preQi treated compared to untreated cells, respectively (FIG. 5B). Analysis of translation efficiency (TE, FIG. 5C) of transcripts changed by >5-fold showed that many highly expressed transcripts corresponding to house-keeping genes such as the ribosomal 300397066.2 - 41 -proteins were depleted in the polysome (FIG. 5D). Gene ontology analysis (FIGs. 5E, 12B) showed that the major cellular pathways reduced in translation were structural components of cytosolic ribosomes, which corresponded to the highly expressed transcripts (FIG. 12C).[OHl] The inventors analyzed the preQi effect on the codon context in translation. For the amino acid codons read by the cognate tRNATyr / Hls / Asn / Asp, preQi treatment substantially reduced the translation of the U-ending, but not the C-ending codons (FIG. 5F). Furthermore, preQi exerted a clear difference on translation based on the third codon sequence (FIGs. 5G, 12E). Translation on all A / U-ending codons were reduced and comparably translation on nearly all C / G ending codons were enhanced in the presence of preQi. The only major exception to this third codon sequence trend was lysine AAG which was however accompanied by an even stronger reduction in translation of lysine AAA codon. Hence, for all codon pairs that end with A / G or C / U, preQi reduced translation of A-ending over G-ending codons and U-ending over C-ending codons in every instance (FIG. 5H). Decoding of the four cognate amino acids by Q / preQlsine modified tRNAs followed this trend, but their effect was not among the strongest.

[0112] Taken together, the results show that preQi strongly and specifically reduces the levels of cognate tRNATyr / Hls / Asn / Asp, and queuine counteracts this reduction. PreQi -dependent tRNA reduction may be derived from their lower charging levels and preQi -modified tRNAs could be more prone for degradation. PreQi also specifically reduces the level of cognate mitochondrial tRNAs, although their queuine rescue effect is complex. PreQi strongly and selectively reduces the translation of highly abundant mRNAs such as those of ribosomal proteins, consistent with its detrimental effect on proliferation. Finally, preQi -dependent reduction on translation is not restricted to the codons read directly by the cognate tRNAs, rather, it is highly selective for A / U-ending codons, suggesting a global consequence on decoding of all amino acids.

[0113] The inventors summarize the findings in a model linking preQi and queuine effects on cell proliferation. PreQi and queuine compete for the modification of tRNATyr / Hls / Asn / Aspby the same enzyme. Q-modification blocks preQi -modification of the same tRNA. PreQi modified tRNA is defective in charging, decoding, and undergoes more rapid degradation. These tRNA property changes cumulatively decrease cell proliferation by impacting the translation of ribosomal proteins required for proliferation.300397066.2 - 42 -Genome-wide CRISPR screen identifies new cellular pathways in preQl-dependent cell proliferation

[0114] To identify additional cellular pathways involved in preQl-dependent cell proliferation, the inventors performed genome-wide CRISPR screen. The screen used Brunello genome-wide CRISPR library that contains four sgRNAs for each annotated human gene for a total of 77,441 sgRNAs in the library81. The inventors collected lentivirus infected cells in the absence and presence of preQi after 5 days and sequenced their sgRNA libraries that were integrated into the genomic DNA (FIG. 13A). The inventors first generated single clonal HEK293T cells that stably expressed Cas9 protein (Cas9 stable cells). Two hundred million Cas9 stable cells were infected with lentivirus packaged sgRNA library to ensure average coverage of over 500 copies of each sgRNA. Infected cells after puromycin selection were separated into two groups for mock or preQi treatment. The inventors amplified sgRNAs in genomic DNA from over 40 million cells for each condition to ensure appropriate coverage and performed next-generation sequencing.

[0115] The inventors analyzed the enriched sgRNAs in preQi treated versus untreated cells using STARS81and found 40 genes enhancing (right of right dashed line) and 11 genes reducing (left of left dashed line) proliferation (FIG. 6A). For the 40 enhancing genes, at least 2, and for many all 4 sgRNAs (FIG. 13B) as well as the average fold change showed significant enrichment (FIG. 6B) The sgRNAs for the Q-modification writer complex QTRT1 and QTRT2 were the most enriched (FIG. 13C). This positive control corresponded to the QTRT1 and QTRT2 knockdown results (FIG. 2) and validated the CRISPR approach for the positive identification of new genes and pathways involved in preQi effect on proliferation.

[0116] The inventors carried out gene ontology analysis of the genes whose depletion alleviated the preQi effect on proliferation (FIG. 6C, 13C, 13D). Top hits were proteins localized in cellular organelles and trans-organelle transport between endoplasmic reticulum (ER) and Golgi82'85, and subunits of vacuolar ATPases (V-ATPase) that acidifies intracellular organelles, vesicles, and endosome using ATP hydrolysis86'89. The inventors performed clusters of protein-protein interaction enrichment analysis using STRING90and found several major complexes or multiple components involved in the same cellular pathway (FIG. 6D).Aside from the obvious QTRT1 / QTRT2 complex, new pathways with significant enrichment in corresponding sgRNAs included regulation of cholesterol biosynthesis (FIG. 6E, SCAP, MBTPS1, MBTPS2)91’95and Golgi-ER trafficking (FIG. 6F, NBAS, USE1, COG3)82’85.300397066.2 - 43 -

[0117] The inventors followed up the CRISPR screen results by knocking down individual gene products and testing for effects on cell proliferation and tRNA levels. Focusing on the 4 genes in the cellular pathways of cholesterol biosynthesis (SCAP, MBTPS1, MBTPS2) and Golgi-ER trafficking (NBAS), knocking down SCAP (FIG. 13F) resulted in a small reduction in proliferation in mock treatment, but the reduction in proliferation upon preQi treatment was significantly diminished compared to si-CTRL (FIG.6G, 13G). Knocking down MBTPS1 had little effect on proliferation without preQi, and preQi treatment resulted in much smaller reduction (FIG. 6H, 13H). Knocking down MBTPS2 had a large effect on proliferation without preQi, and the reduction of preQi treatment was less severe compared to si-CTRL (FIG. 131). Knocking down NBAS (FIG. 13 J) had little effect on proliferation without preQi, whereas the differential proliferation effect was smaller with preQi (FIG. 61, 13 J). These results were consistent with the expectations of the CRISPR screen for these individual genes.

[0118] To understand how these genes were involved in mediating preQi -dependent proliferation, we performed Northern blots for the four cognate tRNAs upon knock-down of SCAP or NBAS. As exemplar, tRNATyrwas significantly reduced upon preQi treatment in si-CTRL, whereas its level was largely maintained upon SCAP knock-down (Fig. 2D). Similar findings could be found for all four cognate tRNAs (Fig. 2E) which exhibited varying magnitudes of reduction for individual tRNAs with preQi in control cells. The same type of analysis showed that NBAS knock-down (Fig. S2G) also desensitized the preQi -dependent reduction of cognate tRNA levels compared to si-CTRL. These results indicate that these proteins in this new pathway facilitated preQi -dependent cognate tRNA reduction in the wildtype cells.

[0119] The inventors summarize the CRISPR screen findings in a model (FIG. 6J). The inventors identify ER and other membrane proteins in controlling preQi -mediated inhibition of cell proliferation including SCAP (ER), MBTPS1 / MBTPS2 (Golgi), Cog3 / Cog4 / NBAS (COPI proteins for retrograde Golgi-ER trafficking), USO1 / USE1 / BNIP1 (SNARE proteins for reintegrating SCAP into ER) that rescue preQi -dependent cell proliferation. SCAP may play a central role in the regulation of preQi -dependent cell proliferation through the following steps: (i) SCAP is an ER resident protein that at low cholesterol levels traffics and chaperons SREBP transcription factors to Golgi, (ii) MBTPS1 and MBTPS2 proteases cleave SREBP, and releasing SCAP to enter COPI. (iii) COPI retrograde transports SCAP back to ER. (iv) SCAP reintegrates into ER mediated by the SNARE complex.The SCAP protein is required for preQl-modified tRNA reduction in active translation300397066.2 - 44 -

[0120] To understand how these genes were involved in mediating preQi -dependent proliferation, the inventors performed Northern blots for the four cognate tRNAs upon knockdown of SCAP or NBAS. As exemplar, tRNATyrwas significantly reduced upon preQi treatment in si-CTRL, whereas its level was largely maintained upon SCAP knock-down (FIG.7A). Similar findings could be found for all four cognate tRNAs (FIG. 7B) which exhibited varying magnitudes of reduction for individual tRNAs with preQi in control cells. The same type of analysis showed that NBAS knock-down (FIG. 14A) also desensitized the preQi -dependent reduction of cognate tRNA levels compared to si-CTRL. These results indicate that these proteins in this new pathway facilitated preQi -dependent cognate tRNA reduction in the wild-type cells.

[0121] Is the preQi -dependent tRNA reduction dependent on their participation in translation? In the polysome profiling (FIG. 4), major mRNAs depleted in preQi treatment code for ribosomal proteins which require cytosolic translation and are a consequence of reduced tRNATyr / Hls / Asn / Asplevels. In contrast, mRNAs enriched in preQi treatment code for ER lumen and membrane proteins (FIG. 7C) which require translation on the ER. The inventors measured both low level of Q-modified and high level of preQi -modified tRNA in the input and the polysome by mass spectrometry and found that preQi -modified tRNAs can indeed be present on the polysome (FIG. 7D). However, comparing to the input, the Q-modified tRNAs are enriched, whereas preQi -modified tRNAs are reduced on the polysome, consistent with preQ 1 -modified tRNA being a naturally occurring defective tRNA in translation. The inventors then added translation inhibitors such as emetine (FIG. 7E, 14B) or cycloheximide (FIG. 7F, S6C) to cells and found that translation inhibition fully restored the cognate tRNATyr / Hls / Asn / Asplevels in the presence of preQi, even though tRNAs remained preQi -modified (FIG. 14C).These results indicate that preQi -modified tRNA reduction requires active translation in the cell, which enables the loading of the preQi -modified tRNAs onto the ribosome.

[0122] What are the sensor proteins that detect ribosomes containing preQi -modified tRNAs? The increased ER-translated mRNAs in the polysome of preQi -treated cells suggest that this sensory protein is localized on the ER. The inventors focused on the SCAP protein, which is a known ER resident transmembrane protein91,96. The inventors treated the cells with mild detergents and found that SCAP co-localized with ribosomes in sucrose cushion (FIG.7G). The inventors performed Western blots of SCAP in the sucrose gradient without or with preQi treatment (FIG. 7H). Intriguingly, SCAP already associates with the 80S monosome and the disome at appreciable levels in the absence of preQi, i.e. under normal, control conditions. The presence of preQi further shifted SCAP to trisome or higher polysome 300397066.2 - 45 -fractions; furthermore, the total SCAP / RPL8 ratio increased by 1.9-fold with preQi, consistent with increased engagement of SCAP with the ribosome (FIG. 71).

[0123] Which domain of SCAP is involved in ribosome interaction? The human SCAP protein is composed of 8 transmembrane helices, plus a 74 amino acid loop 6 domain and a large, 549 amino acid C-terminal WD domain in the cytosol that may interact with the ribosome (FIG. 14D) To determine whether either or both cytosolic domains are involved in SCAP-ribosome effects, the inventors constructed three stable cell lines (FIG. 14D) that express the flag-tagged wild-type SCAP (OE), a loop 6 deletion mutant (mutl) and a WD domain deletion mutant (mut2). The inventors did not knockout the endogenous SCAP protein as it may be essential for cell viability97‘". All three constructs generated stable proteins (FIG. 14E) and responded similarly to preQi -dependent reduction in cell proliferation (FIG. 14F).Furthermore, all three cell lines showed the same depletion of the cognate tRN ATvr'Hls / Asn / Aspinthe presence of preQi (FIG.7 J). However, the behavior of the SCAP mutants on the polysome is distinct among these constructs. The inventors used both SCAP and Flag antibodies to distinguish the ribosome interaction of the endogenous / OE SCAP and the mutants. Without preQi, all three constructs are present at similar or above wild-type SCAP levels in the ribosome (FIG. 7K). This result is consistent with SCAP oligomer interaction with the ribosome, as SCAP oligomerization is mediated by its membrane domain10°, and all three constructs can increase SCAP oligomerization. However, compared to the endogenous SCAP upon preQi treatment, only the wild-type Flag-SCAP protein maintained its interaction levels with the ribosome (FIG. 7L). The loop 6 deletion mutant reduced its polysome presence by about half, whereas the WD domain deletion mutant almost completely lost its interaction with the ribosome. This result indicates that the WD domain of SCAP plays a major role in the ribosome response to the presence of preQi, with the assistance of loop 6 residues.SCAP directly binds ribosome and coordinates tRNA reduction with IRE1

[0124] The inventors employed two strategies to determine where SCAP interacts with the ribosome in cells. The first approach used UV crosslinking followed by SCAP immunoprecipitation and RNA sequencing (SCAP-CLiP-seq), a common method used to map direct protein-mRNA binding sites101’102. The inventors modified this method to identify direct SCAP-rRNA crosslinks using mutational signatures in the sequencing data (103, FIG. 15A).Since no SCAP antibody suitable for immunoprecipitation is available, the inventors used the stably expressing SCAP cell lines characterized above for this mapping experiment (FIG.15B). The inventors obtained good mutation rate correlation among biological replicates (FIG.300397066.2 - 46 -15C). To maximize signal to noise, the inventors applied a differential mutation rate between the SCAP-IP and control samples of >1% present in >10 residues within 100 nucleotides. Using this criterion, the inventors identified three clusters in the 28S rRNA of 570-670, 3090-3190, and 4100-4200 (FIG. 12A), but none in the 18S, 5.8S or 5S rRNA. The 570-670 and 3090-3190 regions are in the mammalian 28S rRNA expansion regions, whereas the 4100-4200 region is conserved in all eukaryotic rRNA104’105. Superimposing these clusters on the human 80S cryo-EM structure shows that they are located close to each other and project away from the ER-located ribosomes engaged with the Sec61 translocon106'108, exactly within the expected topology of ribosome interaction by an ER resident protein (FIG. 12B). Furthermore, these SCAP interaction regions are near the tRNA exit site (E-site), thus positioning SCAP to the E-site region under normal conditions. In the presence of preQi, the inventors observed a dramatic change in the SCAP-ribosome interaction in all three clusters, plus the appearance of a new region of interaction (FIG. 12C). All three clusters gain or lose mutation signatures, indicating SCAP movement on the ribosome (FIG. 12D). Furthermore, the inventors identified a gain of SCAP interaction in the 3900-4000 region which is located from the E-site tRNA only 5.4 A at the closest point in the 80S structure. This result suggests that the loading of a preQi -modified tRNA triggers the movement of SCAP to a location where it may temporarily block the exit of the E-site tRNA.

[0125] The second approach used the expression of a SCAP fused adenosine deaminase protein (SCAP-N-Tad8, FIG. 14E) in cells and the identification of A-to-I signatures in the rRNA109. This method provides an orthogonal strategy to inform on SCAP-ribosome interaction by simply mapping the A-to-I signatures (i.e. A-to-G mutation in the sequencing data) from samples under any condition. A disadvantage is that the deaminase fusion protein only reports rRNA in the proximity of SCAP and on the ribosome surface. To maximize signal to noise, the inventors applied a differential A-to-G mutation rate between the samples with or without preQi treatment of >0.4%. Using this criterion, the inventors identified ten residues in the 28 S rRNA (FIG. 12E), but none in the 18 S, 5.8 S or 5 S rRNA. Several of these residues are located in multiple regions near the ribosome E-site (FIG. 12F), consistent with the SCAP-CLiP-seq result of SCAP location with preQi. Several residues are located opposite to the E site of the ribosome which is consistent with SCAP interaction with the disome in the polysome data (FIG. 71).

[0126] The inventors next asked which ribonuclease cleaves these tRNAs on the ribosome. A seminal study by Walter and colleagues110showed that the ER resident ribonuclease inositol-requiring enzyme type 1 (IRE1) can directly bind tRNAs in the A-site of the ER 300397066.2 - 47 -ribosome. IRE1 ribonuclease is required for the unfolded protein response (UPR) by cleaving the XBP1 pre-mRNA111 13However, the reported IRE1 interaction with tRNA is present with or without the UPR stress. To test the hypothesis that IRE1 is the ribonuclease responsible for cleaving preQi -modified tRNA, the inventors treated cells with 4p8C, a well-characterized inhibitor specific for the IRE1 ribonuclease activity114Inhibition of IRE1 ribonuclease fully rescued the preQi -dependent tRNA reduction even though tRNAs remained preQi -modified (FIG. 12G, 15F). Furthermore, the addition of 4p8C can partially alleviate the preQi -mediated reduction in cell proliferation (FIG. 12H). The inventors also found that the preQi treatment did not trigger the integrated stress response measured by eIF2a phosphorylation (FIG. 15G) or XBP1 pre-mRNA splicing (FIG. 15H). The result is thus consistent with IRE1 cleaving the preQi -modified tRNA without requiring the activation of the general stress response.

[0127] Does IRE1 cleave other cellular tRNAs? One intriguing observation under normal condition is that the tRNAs interacting with IRE1110correlates with the tRNA fragments that the inventors measure in the tRNA-seq data (FIG. 121). While the inventors cannot rule out that other ribonuclease(s) are responsible to degrade translation-defective tRNAs, this result suggests that IRE1 cleavage of tRNAs occurs at any time and is a source of cellular tRNA fragments under steady-state conditions.

[0128] The inventors propose a model of tRNA quality control occurring on the ER translating ribosome (FIG. 12J). Under normal conditions, SCAP interacts with the ER translation machinery near the E site and IRE1 interacts with the ribosome near the A site. SCAP senses stalled ER ribosomes and enables IRE 1 -mediated cleavage of translationdeficient tRNAs by going through these steps: (i) preQl-tRNA stalls ribosome at the A site, (ii) SCAP changes conformation and activates IREl ribonuclease, (iii) IRE1 cleaves tRNA. (iv) Cleaved tRNA leaves, and new tRNA comes to A site, thus rescuing the stalled ribosome. In the context of preQi, tRNATyr / Hls / Asn / Aspis selectively cleaved and depleted which results in the inhibition of translation of abundant proteins such as ribosomal proteins, which amplifies and sustains protein synthesis inhibition.Discussion

[0129] In aspects herein, the inventors expand the microbiome-mammalian host interaction through tRNA and translation into two metabolites that act on cell proliferation in opposing directions. Queuine is derived from the catabolic product of bacterial queuosine tRNA modification and is taken up by mammalian cells to generate their own Q-tRNA modification. The mammalian Q-tRNA modification has been studied for decades, and yet, this tRNA300397066.2 - 48 -modification seems to only confer subtle phenotypes in cells and whole organismsn-15>76>115-126. PreQi is an upstream metabolite in the same pathway that produces bacterial Q-tRNA modification. The inventors find here that preQi has a profound effect on mammalian cells, it very severely reduces cell proliferation, but maintains the ability of cells to proliferate. The preQi -dependent proliferation effect is blocked or rescued by queuine, but the amount of queuine needed to fully overcome the preQi effect is cell type dependent, ranging from 1 : 100 in HEK293T cells to 1:1 in BMDC cells. The demonstration that both preQi and queuine circulate in the mouse blood and are detectable in many mouse tissues indicate that proliferating mammalian cells are poised to respond to the fluctuating preQi and queuine levels derived from microbiome components and activity or diet.

[0130] The combinatory action of preQi and queuine provides the opportunity to examine the host cell and tissue response to microbiome dynamics at the organismal level. Upon bacterial turnover in the gut, preQi would be immediately available, whereas the availability of queuine requires additional enzymatic reactions and likely takes longer to enter the blood stream. This differential timing may prime certain cells, such as immune cells that must respond to these circulating metabolites and others to undergo rapid proliferation. Intriguingly, the strongest preQi effect the inventors observe here is for BMDC cells that respond fully at 100 nM preQi and require an equal molar queuine to fully restore its proliferation. This is in line with the tremendous effects of gut microbiome on the cellular immunity127'131, and preQi and queuine may make a significant contribution to tuning the immune response.

[0131] PreQi specifically reduces tRNA levels for the cognate tRNAs, and this reduction in specific tRNA levels results in translational repression of ribosomal protein genes. Since preQi is incorporated directly into tRNAs, the preQi modified tRNAs seem to be the target of more rapid degradation. Unexpectedly, the translation effect of preQi -dependent tRNA depletion is not restricted to the decoding of just the codons of the four amino acids read by the cognate tRNAs, rather, preQi exerts a global reduction of translation of all A / T-ending codons. Ribosome takes longer to read A / T-ending codons132,133. PreQi -dependent tRNA depletion may exacerbate this and disproportionally affect translation of highly abundant mRNAs such as ribosomal proteins that must be translated at high levels for proliferation.

[0132] The CRISPR screen and validation identify multiple genes in several cellular pathways that can partially rescue the preQi effects on proliferation upon knockdown. These new genes and pathways encompass the V-ATPase complex that acidifies intracellular vesicles, organelles, and endosomes86'89, enzymes required for fatty acid synthesis134'137. The CRISPR hits also include ER and Golgi-localized proteases and accessory factor that regulate 300397066.2 - 49 -cholesterol biosynthesis91-95and genes involved in Golgi-ER trafficking82-85. The inventors focused on SCAP, an ER resident protein that has been well characterized for its role in the regulation of cholesterol biosynthesis. In response to changing cholesterol levels, SCAP traffics from ER and Golgi and back, so that the SCAP levels on the ER membrane is subjected to the control of specific proteins that reside in Golgi such as the MBTPS1 / S2 proteases and those in the ER-Golgi trafficking vesicles such as NBAS. Therefore, the microbiome-host interaction study on tRNA modification has identified connections of RNA biology to new cellular pathways that center around proteins required in the regulation of cholesterol biosynthesis and intracellular vesicle trafficking.

[0133] Many crucial components and mechanism of Q and preQi biology remain unknown. These include the queuine and preQi transporters in eukaryotes1>5, the biological reason for localizing the QTRT1 / QTRT2 enzyme on the mitochondrial membrane138, the function of mitochondrial Q-tRNA modifications79,8°, to name a few. The CRISPR screen with preQi opens the possibility of studying new genes to address these questions and enabling more in-depth mechanistic studies.

[0134] The most profound discovery here is how SCAP regulates ER translation in sensing low quality tRNAs that get on the ribosome and facilitating their degradation. Translation in a polysome needs to be evenly paced to minimize ribosome collision which triggers cellular stress response52,139. The elaborate mechanism used to relieve cytosolic colliding ribosomes has been recently elucidated52,53’55. However, an unanswered question is how ribosome senses the loading of a deficient tRNA that may slow-down or pause the ribosome. Left unchecked, such translation deficient tRNA, exemplified here by the naturally occurring preQi -modified tRNA may cause both cytosolic and ER ribosome collision. The polysome profiling results showed the enrichment of ER translated transcripts (FIG. 6C), suggesting an ER localized mechanism for relieving collided ER ribosomes and rescuing of ER translation. The inventors found that SCAP directly interacts with the ribosome under all cellular conditions. SCAP senses ribosome loaded with preQi -modified tRNA from the E-site through conformational change that activates the ER-resident IRE1 ribonuclease at the A-site to cleave the preQi -modified tRNA. This SCAP-IRE1 coordinated action results in both the degradation of the preQi -modified tRNA and the release of the paused ribosome for a new round of A-site tRNA loading before the ribosomes may collide. This mechanism could be active for the sensing of any translation deficient tRNA. Both SCAP and IRE1 proteins are conserved among many branches of eukaryotes (FIG. S7I), suggesting that this tRNA quality control mechanism is widespread among the eukaryotes.300397066.2 - 50 -

[0135] In summary, the inventors show here that two metabolites from the same pathway in the gut bacteria act on mammalian host cell proliferation in opposing ways. PreQi incorporation in cognate tRNAs leads to their depletion with the consequence of reducing translation of house-keeping genes required for proliferation. Genome-wide CRISPR screen identifies genes in multiple new pathways that have not been associated previously with RNA biology. The inventors identify a new mechanism of ER ribosome sensing and tRNA quality control through the ER resident proteins of SCAP and IRE1 that are highly conserved in eukaryotes. Future work will define how these pathways and in-depth mechanisms are involved in the regulation of cell proliferation, tRNA quality control, and ribosome sensing and their effects on physiology in different mammalian cell types and tissues.Example 2: Materials and Methods

[0136] Cells: HEK293T cells were cultured in 37 °C incubator with 5% CO2 using complete DMEM medium without pyruvate (Cytiva) containing 10% FBS (ThermoFisher) and 1% Pen / Strep (ThermoFisher). HEK293T cells depleted from queuosine tRNA modification (0Q) were obtained by culturing cells using complete DMEM medium containing 10% dialyzed FBS (ThermoFisher) and 1% Pen / Strep for 2-3 weeks. Queuosine modification levels were measured by Acrylamidophenylboronic acid (APB, Frontier Scientific)-gel based Northern blot77.

[0137] MEF cells were cultured in 37 °C incubator with 5% CO2 using complete DMEM medium with pyruvate (ATCC) containing 10% FBS and 1% Pen / Strep. 0Q MEF cells were obtained in the same way as HEK293T cells by culturing MEF cells with dialyzed medium for 2-3 weeks and verified as above.

[0138] B16-OVA cells were cultured in 37 °C incubator with 5% CO2 using DMEM medium with pyruvate (ThermoFisher). 0Q B16-OVA cells were obtained and verified as described for HEK293T cells.

[0139] Bone marrow-derived dendritic cells (BMDCs) were generated from 6-8 week old female C57BL / 6 mice. Bone marrow cells were collected from femora and tibiae and plated at IxlO5cells per well in a flat-bottom, non-tissue culture treated 96-well plate (GenClone) in 150 pL of complete RPMI-1640 (ThermoFisher) supplemented with 10% volume / volume (v / v) dialyzed fetal bovine serum (ThermoFisher), L-glutamine (2 mM ThermoFisher), penicillinstreptomycin (ThermoFisher), MEM non-essential amino acids (Corning), HEPES (10 mM; ThermoFisher), sodium pyruvate (1 mM; Corning), and P-mercaptoethanol (55 pM300397066.2 - 51 -ThermoFisher). Recombinant murine GM-CSF (15 ng / mL Peprotech) was added to the complete RPMI medium.

[0140] Mice: Female C57BL / 6J mice (wild-type) were obtained from the Jackson Laboratories. Animals were housed in specific pathogen-free conditions at The University of Chicago, and all experiments were performed in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by The University of Chicago Institutional Animal Care and Use Committee.

[0141] LC / MS / MS measurements of preQL queuine, queuosine and preQlsine: Queuine, Queuine N15, queuosine, PreQlsine standards'. Queuine dihydrochloride was synthesized by Synthenova SAS using the protocol from Brooks et al.140. Its purity was determined at 99% by HPLC analysis. PreQi dihydrochloride was purchased from Sigma-Aldrich (SML0807).

[0142] Synthesis scheme shown in FIG. SIB: preQi was synthesized by catalytic reduction of the nitrile function141of compound 1140in presence of ammonia under hydrogen pressure before being deprotected in an acid medium. Nitrile 1 was formed in two stages: before being tritylated in position 2, the pyrrolopyrimidinone bicycle was performed by reacting methyl 2-cyano-2-formyl acetate 2 and 2,6-diaminopyrimidin-4-one 3 according to the work of Migawa et al.142. The Queuine itself was obtained by deprotection in an acid medium of compound 4 resulting from a reductive amination reaction between amine 5 and aldehyde 6140. Aldehyde 6 was produced by reduction of nitrile 1 with Dibal-H. The modified nucleosides Queuosine and PreQisine were obtained after several deprotection steps following the reductive amination reactions of the respective amines 5 and benzylamine with aldehyde 8. This later was synthesized by glycosylation of aldehyde 6 using l-acetyl-2,3,5-tribenzoyl-ribose. Finally, the inventors synthesized "heavy queuine" by introducing three15N isotopes into its formula. The heavy queuine was isolated after a final deprotection step that followed a type 2 nucleophilic substitution of brominated compound 9143by the heavy amine PreQi144Heavy PreQi was formed in several steps from15N3-2,6-diaminopyrimidin-4-one 10 by an intermolecular cyclisation reaction between methyl 2-cyanoacetate 11 and commercial15N3-guanidinium chloride 12.

[0143] Metabolites extraction: Each mouse tissue was crushed with 1* PBS. 100 pL of homogenized mixture were taken to process to extraction of metabolites. 450 pL of methanol / water buffer (ratio 8 / 1) previously cooled to -20°C and 1 pL of 10 pM queuine-N15 were added. The samples were incubated with orbital agitation at 4°C for 20 minutes, then centrifuged at 16000* g at 4°C for 5 minutes. The supernatants were filtered with Captiva300397066.2 - 52 -EMR-Lipid plate (Agilent) to remove phospholipids. The samples were dried in vacuum and resuspended in 5 mM ammonium acetate pH 5.3 before LC-MS / MS analysis.

[0144] Nucleoside mass-spectrometry analysis: (LC-method) 5 pL samples were separated by reverse phase ultra-performance liquid chromatography (Nexera LC-40 system, Shimadzu) on a C18 column (Synergi™ Fusion-RP; 4 pm particle size, 250 mm x 2 mm, 80 A, Phenomenex). The mobile phases consisted of 5 mM ammonium acetate pH 5.3 (solvent A) and pure acetonitrile (solvent B). The 30-minute elution gradient started with 100% phase A followed by a linear gradient to 8% solvent B at 13 min. Solvent B was increased further to 40% over 10 minutes. After 2 minutes, solvent B was decreased back to 0% at 25.5 minutes. Initial conditions were regenerated by rinsing with 100% solvent A for additional 4.5 minutes. The flow rate was 0.4 mL / min and the column temperature was 35 °C.

[0145] (MRM method) Nucleoside detection was performed using a Shimadzu TripleQuad NX8060 in positive ion mode. MS was operated in dynamic MRM mode with a retention time window of 3 min and a maximum cycle time set at 297 ms.

[0146] Extraction of total RNA from gut microbiome and RNA sequencing: RNA was extracted from human stool samples and prepared for MSR-seq as previously described 78,145,146 Briefly, samples were deacylated without periodate. After the first ligation reaction, samples were split into two parts. One aliquot was treated with AlkB demethylase and the other was treated with same solutions without AlkB (mock). Both samples were again split into two equal parts, one set of aliquots were treated with sodium periodate as previously described for PAQS-seq75, the other set of aliquots was untreated. These 4 samples were then used in reverse transcription following the usual MSR-seq protocol.

[0147] Cell proliferation assay with queuine / preQl treatment: 0Q HEK293 T, MEF, or B 16- OVA cells in 10-cm / 15-cm plates were grown to 80% confluency and collected using 0.25% trypsin (Trypsin). Cells were counted using trypan blue stain (ThermoFisher) and cell counter (ThermoFisher Countess II). Equal amount of trypan blue and cell mixture was mixed. 10 pl mixture was loaded onto each side of the chamber slide and counted using cell counter. Live cell concentration and percentage were recorded. For each queuine (Toronto Research Chemicals) and / or preQi (Cayman Chemical) treatment, six milliliters of 5 *104cells / ml mixture in 15-ml conical tubes were prepared. Queuine and / or preQi were added to final concentrations as indicated. 8 replicates of 100 pl cell mixture from each condition were then transferred into 96-well plates. Control (ctrl) wells with only medium were also prepared. 6 plates of cells were prepared for 6-day measurements (day 0 to day 5). All cells were incubated at 37 °C for 2 hours to allow cells to adhere to the bottom. 10 pl CCK-8 reagent (Dojindo) was 300397066.2 - 53 -then added to each well of the “day 0” plates using multichannel pipets followed by incubation at 37 °C for 2 hours. Absorbance at 450 nm were measured using Synergy Neo microplate reader (BioTek). On day 1 to day 5, absorbance at 450nm was measured at the same time of each day.

[0148] For cell’s ability to proliferate-measurement after preQi treatment, _HEK293T cells depleted from queuosine modification (0Q cells) were cultured in 37 °C incubator with 5% CO2 using complete DMEM medium (10% dialyzed FBS and 1% Pen / Strep). PreQi was added to the medium 72 h, 48 h, 24 h, and 0 h to a final concentration of 1 pM before collection of cells. Cells were then collected at the same time using 0.25% trypsin and counted using trypan blue stain and cell counter. Cell concentration and percentage of live cells were recorded.

[0149] For proliferation rescue experiment with variable queuine concentrations, 0Q HEK293T cells were cultured to 60-70% confluency and then treated with 1 pM preQi for 24 h. Cell mixture at 5 * 104cells / ml with 1 pM preQi were prepared and transferred into 96-well plates as above. Absorbance at 450 nm was measured as above on day 0 to day 2. Queuine was added to the day 3 to day 5 96-well plates on day 2 to final concentrations of 0 nM, 1 nM, 10 nM, 100 nM, and 1000 nM. Absorbance at 450 nm was then measured as above on day 3 to day 5.

[0150] For proliferation with different percentage of fully Q-modified cells, two plates of 0Q HEK293T cells were cultured to 60-70% confluency followed by treatment with sterile H2O or 1 pM queuine for 24 hours to get 0Q cells or fully Q-modified (100Q) cells. 0Q and 100Q cells were collected using 0.25% trypsin and counted using Trypan blue stain. 0Q cells and 100Q cells were then mixed to get 0%, 25%, 50%, 75%, and 100% 100Q cells. The mixed cells were then used to perform cell proliferation assay as above under 1 pM preQi. 0Q cells without any treatment were used as control.

[0151] For proliferation with treatment of different concentrations of IRE 1 ribonuclease inhibitor 4p8C, 0Q HEK293T cells were cultured to 80% confluency. Cells were then collected using 0.25% trypsin and counted using Trypan blue stain. Sterile H2O or 4p8C was added to each set of the 15-ml conical tubes to 0 pM, 10 pM, or 50 pM final. Sterile FEO or preQi were then added to 0 nM or 1000 nM final. Proliferation assay was then performed as above.

[0152] BMDC Cell proliferation with preQl / queuine treatment: BMDC cells were obtained and cultured as described above. All treatment conditions were plated in quintuplicate in 96-well plate. Cells were fed at days 2 and 4 with 50 pL of complete RPMI medium (ThermoFisher) containing GM-CSF (Peprotech). Queuine and / or preQi were added as required to maintain the initial treatment concentration.300397066.2 - 54 -

[0153] Live Cells were counted each day at the same time. Cells were resuspended and transferred into a 96-well V-bottom plate (Sarstedt). To loosen adherent cells, 100 pM of 10 mM EDTA (ThermoFisher) in lx dPBS (ThermoFisher) was added to the flat-bottom plate, which was then warmed at 37°C for 10 minutes. Following incubation, cells were resuspended and transferred to the corresponding well of the V-bottom plate. A final wash using 50 pL 1 x dPBS was used to transfer any remaining cells from the flat-bottom plate to the corresponding wells in the V-bottom plate. Immediately before data acquisition, the V-bottom plate was centrifuged at 1500 rpm (4°C) for 5 minutes, supernatant was removed, and pellets were resuspended in 130 pL MACS buffer (Miltenyi Biotec) with 0.1 mg / mL DAPI (Biotium). Flow cytometry was completed using the NovoCyte Penteon (Agilent) and data analysis was completed using FlowJo. Flow cytometry was performed at the Cytometry and Antibody Technology Facility at University of Chicago.

[0154] Proliferation measurements of QTRT1 and QTRT2 knockdown cells: QTRT 1 , QTRT2, and Control shRNAlentiviral particles were obtained from Santa Cruz Biotechnology. The manufacturer’s manual was followed to obtain QTRT1 and QTRT2 stable knockdown cell lines. Briefly, lx 105HEK29T 0Q cells were plated into each well of 12-well plate 24 hours prior to viral infection. After 24 hours of incubation, the medium was replaced with complete medium containing 8 pg / ml Polybrene (Santa Cruz). Lentiviral particles were thawed at room temperature and used promptly. Corresponding amount of QTRT1 or QTRT2 lentiviral particles were added to each well to infect the cells at Multiplicity of infection (MOI) 1, 5, and 10. The infected cells were continued incubated for 48-72 hours. Then, the culture medium was replaced with complete DMEM containing 5 pg / ml puromycin to select stable clones expressing the shRNAs. The transduced cells were continuously incubated with puromycin (Santa Cruz) containing medium for a few days and split 1 :3 to 1 :5 when it was necessary until the cells without virus transduction were completely dead. Stable knockdown cells were stocked and harvested for protein expression test by Western blot after the cells had been sufficiently expanded. Proliferation assays with QTRT1 and QTRT2 stable knockdown cells were performed as above using 0Q QTRT1 or QTRT2 and shRNA control knockdown cells. Proliferation was done under queuine and preQi concentrations indicated in Fig. 2.

[0155] Western blot: Western blot was performed as previously reported147,148. Briefly, equal amounts of total protein lysate from each sample were boiled and reduced at 95 °C for 5 mins and loaded onto SDS-PAGE gel (ThermoFisher). The samples were then transferred to PVDF membrane (Millipore) using Bio-Rad semi-dry transfer system (Trans-Blot Turbo Transfer System) or wet transfer cell (Mini Trans-Blot Electrophoretic Transfer Cell). The 300397066.2 - 55 -membrane was then blocked using 5% (w / v) non-fat dry milk (Bio-Rad) in 1 * TBST (National Diagnostics) overnight at 4 °C. The membrane was then washed three times with lx TBST for 10 min each. The membrane was then sliced into several strips to blot target proteins at different molecular weights. The membrane strips were incubated with control or target antibodies in 1 x TBST with 5% (w / v) non-fat dry milk at 4 °C overnight. After primary antibody incubation, each membrane strip was incubated with corresponding secondary antibodies in 1 x TBST with 5% (w / v) non-fat dry milk at room temperature for 30 min. The membrane strips were washed three times for 10 min each and detected using ECL substrate (Bio-Rad) and Bio-Rad ChemiDoc. Blot stripping and reprobing were performed using low pH protocol as previously reported149. Briefly, the membrane strips were incubated with stripping buffer (0.2 M glycine-HC1, 0.1% SDS, 1% Tween 20, pH 2.2) for 30 min at room temperature. The membrane strips were then washed three times with agitation for 10 minutes each in lx TBST buffer. The membrane strips were blocked with 5% (w / v) non-fat dry milk in lx TBST buffer prior to reprobing.

[0156] PreQlsine detection of cognate tRNAs by chemical tagging and Northern blots:PreQlsine has a primary amine that can react with NHS esters. 0Q HEK293T cells were cultured to -80% confluency and treated with preQi and / or queuine at indicated concentrations for 24 hours. Cells were then collected, and total RNA were extracted using TRIzol reagent (ThermoFisher) according to manufacturer’s manual. Northern blot was performed as previously described77. Briefly, 6 pg of total RNA from each sample was deacylated in 20 pl 100 mM NaHCO3 / Na2CC>3 buffer (pH 9) at 37 °C for 30 min. Deacylated RNA was purified using Zymo RNA Clean & Concentrator Kit (Zymo) and eluted in 8 pl sterile H2O. Each deacylated sample was then split into two equal parts (4 pl each). 1 pl 1 M NaHCO3 / Na2CC>3 (pH 9) buffer and 1 pl 250 mM NHS ester, m(dPEG)24-NHS (ThermoFisher) or 1 pl DMSO (untreated control) was added to each sample, respectively. The reaction mixture was then incubated at room temperature for 30 minutes. Equal amount (6 pl) of 2x acidic denaturing RNA loading buffer (7M urea, 0.03% (w / v) Bromophenol blue, 0.03% (w / v) Xylene cyanol, 12% (w / v) Ficoll, 2x TBE, 0.1M NaOAc / HOAc, pH 4.8) was added to each sample. All samples were loaded onto a pre-run 10% acidic urea-denaturing PAGE gel containing 100 mM NaOAc / HOAc, pH 4.8 for electrophoresis separation. The gel was run at constant power until the Xylene cyanol band was -4 cm to the bottom of the gel. The gel was then stained with 1 x SYBR gold (ThermoFisher) in lx TBE buffer to check RNA quality. The RNA was then transferred to positively charged Nylon membrane (Cytiva Hybond-XL membrane) using gel dryer (Bio-Rad) under vacuum or using semi-dry transfer system (Bio-Rad). The gel- 300397066.2 - 56 -membrane assembly was then soaked in deionized water to separate the gel and membrane. The RNA was crosslinked to membrane under 254 nm UV twice with 120 mJ / cm2each time. The membrane was then blocked with hybridization buffer (20 mM phosphate, pH 7, 300 mM NaCl, 1% SDS) at room temperature for 30 min followed by incubation with 200 pmol biotinylated DNA probe (Table S3) against Q-modified tRNAin 50 ml hybridization buffer in 60 °C hybridization oven (UVP) with rotation overnight. The membrane was washed with washing buffer solution (20 mM phosphate (pH 7.2), 300 mM NaCl, 2 mM EDTA, and 0.1% SDS) twice for 30 min each in 60 °C hybridization oven. The membrane was incubated with streptavidin-HRP (Genscript, 1:5000 - 1:1000 dilution) in 30 mL hybridization buffer for 30 min at room temperature. The membrane was washed three times for 10 min each in 30 ml washing buffer. The signal was detected using ECL substrate (Bio-Rad) and Bio-Rad ChemiDoc. The membrane was then stripped by incubation with boiling 1% SDS solution for 30 second followed by incubation at room temperature for 15 min twice. The stripped membrane was then incubated with Northern blot probes of other tRNA.

[0157] Detection of queuosine modification levels using APB gel based Northern blot: The APB gel based Northern blots were performed as previously reported77. Briefly, 3 pg of cellular or mouse tissue total RNA with different preQl / queuine treatment was deacylated in 10 pl 100 mM Tris-HCl buffer (pH 9) at 37 °C for 30 min. Equal amount (10 pl) of 2* denaturing RNAloading buffer (7M urea, 0.03% (w / v) Bromophenol blue, 0.03% (w / v) Xylene cyanol, 12% (w / v) Ficoll, 2* TBE) was added to each sample. All samples were loaded onto a pre-run 10% denaturing PAGE gel with 0.5% (w / v) APB (Frontier Scientific). The gel was run at constant power in the cold room (4 °C) for ~2 hours until the Bromophenol blue bands came out. The gel was then stained with 1 x SYBR gold, and the subsequent Northern blot steps were performed as above.

[0158] PreQl Injection and mouse tissue harvest: Mice were inj ected intraperitoneally with 100 pL of either sterile saline or 10 mg / mL preQi in sterile saline every 24 hours for three consecutive days. After 24 hours following the final injection, mouse tissues (liver, lung, kidney, heart) were harvested, frozen and stored as previously described150’151. Mice were anesthetized with 2,2,2-tribromoethanol (250-500 mg / kg) (ThermoFisher) and perfused transcardially with PBS containing 10 mM EDTA. Immediately after perfusion and dissection, tissues were placed in RNA-preserving solution (5.3 M ammonium sulfate, 25 mM sodium citrate, 20 mM EDTA) and kept at 4°C overnight prior to transfer at -80°C for storage.

[0159] Mouse whole-tissue RNA extraction: Whole-tissue RNA extraction was performed as previously described15°. Briefly, tissues stored in RNA-preserving solution were thawed 300397066.2 - 57 -and transferred to 2 mL tubes containing 700-1500 pL (depending on tissue) of PureZOL (BioRad). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (Qiagen). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane, ThermoFisher), buffer RLT (Qiagen), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher) treatment at 37°C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent)150. Sample concentrations were measured using a Nanodrop One (Thermo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent).

[0160] Mouse Tumor Xenograft Model: The abdomen of mice used for experiments were shaved using a pet trimmer (Wahl Bravmini) on the day before tumor cell injections. Ovalbumin-expressing B16.F10 (B16-OVA) cell lines were cultured with complete DMEM medium with 10% dialyzed FBS at 37°C. For consistency across experiments, tumor cells were thawed from liquid nitrogen stocks frozen in 90% FBS and 10% DMSO two days prior to injections and passaged twice in total. For preQi treated B16-OVA cells, additional preQl-spiked complete DMEM media was added such that the final preQi concentration was 10 pM for 12 hours. Mice were injected with 100,000 B16-OVA cells resuspended in 100 pL of sterile saline in the flank. Mice were injected intraperitoneally every other day beginning on day 2 with 100 pL of sterile saline or 100 pL of 2.5 mg / mL preQi in sterile saline. Tumor volumes were calculated using the formula 1 / 2 * D * d2, where D is the major axis and d the minor axis (in mm). Mice were sacrificed when tumors reached 1000 mm3or upon ulceration.

[0161] MSR-seq of preQl and / or queuine treated total RNA samples: 0Q HEK293T cells were cultured to 80% confluency and treated with preQi and / or queuine at concentrations as indicated in Fig. 4A for 24 hours. Total RNA was extracted using TRIzol reagent. 1 pg total RNA from each sample was used to build MSR-seq sequencing libraries as previously reported 78

[0162] tRNA abundance and charging level measurements by Northern blot: The measurement of tRNA abundance by Northern blot was similar to the Q modification measurement described above. To measure charging, 3 pg of total RNA in 5 pl from different 300397066.2 - 58 -preQl / queuine treatment condition was mixed with 5 pl 2* acidic denaturing RNA loading buffer. ±Deacylated total RNA samples were prepared with ± 100 mM Tris-HCl buffer (pH 9) treatment as above and used as controls. All samples were loaded onto a pre-run 10% acidic denaturing sequencing PAGE gel containing 0.1 MNaOAc / HOAc, pH 4.8. The gel was run in cold room (4 °C) for ~24 hours using 1 x TAE buffer containing 0.1 M NaOAc / HOAc, pH 4.8. The gel was then transferred and blotted as described above.

[0163] Sucrose gradient polysome profiles of HEK293T cells in mock and 1 uM treated preQl: Polysome profiling was derived from previously reported protocol with modifications152. Briefly, 4 15-cm plates (5x 106cells / plate) of 0Q HEK293T cells per sample were seeded 3 days before collection. PreQl was added to haff of the plates to 1 pM final at the time of seeding cells and incubated for 3 days, lx PBS was added to the other haff of the plates. On the day of collection, 5%-50% sucrose gradient was prepared in ultra-centrifuge tolerant tube (Seton) by combining 5% sucrose buffer (20 mM HEPES (pH 7.6), 100 mM KC1, 5 mM MgCh, 5% sucrose, 100 pg / ml CHX, 1% protease inhibitor (Roche), 1% RNase inhibitor (ThermoFisher)) with 50% sucrose buffer (20 mM HEPES (pH 7.6), 100 mM KC1, 5 mM MgCh, 50% sucrose, 100 pg / ml CHX, 1% protease inhibitor, 1% RNase inhibitor (ThermoFisher)) using Gradient Station (Biocomp). The sucrose gradient for all samples was stored in cold room (4 °C) before use. Cells were then treated with 100 pg / ml cycloheximide (CHX, Fisher Scientific) in complete medium for 7 min at 37 °C followed by immediate wash with ice-cold PBS buffer containing 100 pg / ml CHX twice. The cells were collected in 10 ml ice-cold PBS buffer containing 100 pg / ml CHX and pelleted by centrifugation at 500x g for 5 min. Cells from 4x 15-cm plates were combined as one sample. 4x volumes of lysis buffer (20 mM HEPES (pH 7.6), 100 mM KC1, 5 mM MgCh, 1% Triton X-100, 100 pg / ml CHX, 1% protease inhibitor, 1% RNase inhibitor) were added to the combined cell pellet and the cells were lysed on ice for 20 min with periodic perturbation (or rotating at 4 °C). Supernatant lysate of each sample was collected after centrifugation at 16,000x g for 15 min. 4 pl Turbo DNase (ThermoFisher) was added to the lysate and incubated at room temperature for 15 min. The lysate samples were centrifuged at 16,000x g for 15 min after DNase treatment to remove any debris. OD260 of each lysate sample was measured using Nanodrop (ThermoFisher). The OD260 values of all samples were adjusted to the same using lysis buffer. 20 pl of the lysate was saved for Western blot validation. One-fifth of the lysate was saved as the input and the total RNA was extracted using TRIzol reagent (ThermoFisher). All the sucrose gradient tubes were weighed and balanced and 500 pl gradient solution was slowly removed from the top before loading. Equal amount (-500 pl) of the leftover 4 / 5 lysate was slowly and horizontally added 300397066.2 - 59 -to the top of the gradient while gently rotating the tube. The gradient tubes were then centrifuged at 28,000 rpm and 4 °C under vacuum for 3 hours using ultracentrifuge (Beckman Coulter Optima L-100XP, SW28.1 rotor). The sucrose gradient fractions (30 fractions, 590 pl each) were collected automatically using Sucrose Gradient Station and fraction collector (Gilson). All fractions were flash frozen using liquid nitrogen before RNA extraction and Western blot validation. 400 pl aliquot from disome and above (polysome) fractions were used to extract total RNA using TRIzol reagent. Total RNA from these fractions were combined for polysome mRNA extraction using polyA+ RNA extraction kit (Promega) and sequencing. 1 pg total RNA from the input and polysome fractions was also used for mass spectrometry for the detection of preQi sine in polysome.

[0164] Polysome mRNA sequencing by MSR-seq: Polysome mRNA sequencing libraries were constructed using MSR-seq protocol as described78. Briefly, 400 ng input and polysome polyA+ RNA was fragmented at 94 °C for 3 min using Magnesium fragmentation buffer (NEB) to get -300 nt fragment RNA. Fragment RNA larger than 200 nt was then purified using RNA clean and concentrator kit (Zymo) and eluted in 8 pl sterile H2O. 1 pl T4 PNK buffer and 1 pl 10 U / pl T4 PNK (NEB) were added to the fragment RNA and incubated at 37° C for 30 min. T4 PNK was inactivated by incubation at 75 °C for 10 minutes followed by immediately incubation on ice. The entire reaction mixture (-10 pl) was used as the RNA input for MSR-seq. The MSR-seq libraries after PCR amplification were purified using AMPure XP beads or gel.

[0165] Generation of OQ HEK293T cells that stably express Cas9 (Cas9 stable cells):LentiCas9-Blast lentiviral prep (Addgene viral prep # 52962 -LV) was used to generate Cas9 stable cells as previously reported153. Briefly, several LentiCas9-Blast lentiviral prep dilutions were prepared using complete DMEM containing 8 pg / ml polybrene and added to 12-well plate together with no virus control medium. 10000Q HEK293T cells in 500 pl were added to each well to perform reverse transduction (Final MOIs were 0, 1, 5, 10) followed by incubation at 37 °C for 48-72 hours. The transduced cells were then treated with 5 pg / ml Blasticidin (ThermoFisher) in complete DMEM for several days until the cells without virus transduction were all dead. The resulting Cas9 stable cells (polyclonal) were then expanded for cell stock preparation, Cas9 expression validation and Cas9 stable single clone generation. Cas9 monoclonal cells were selected by limiting dilution. Briefly, the stable polyclonal Cas9 cells were treated with trypsin and separated into individual cells by passing through a serological pipet several times or by passing through a 40 pm cell strainer mesh (Corning). The cell concentration was then measured using cell counter. A serial dilution of the cells was done to 300397066.2 - 60 -get a final concentration of 5 cells / ml. 100 pl of the diluted cell solution was transferred to each well of the 96-well plate (average density was 0.5 cells / well). The cells were then incubated in 37 °C cell incubator without disturbance for 7-14 days. After 7 days, cell growth in each well was checked and wells with single colony were identified and recorded. The single clonal cells were transferred to larger tissue culture plates after they have sufficiently expanded. Cell stocks were prepared and Cas9 expression were checked by Western blot after enough cells were obtained. Single clonal Cas9 stable cell line with the highest Cas9 expression was selected for CRISPR screen.

[0166] Genome-wide CRISPR screen for proteins that mediate preQl effect on cell proliferation: Brunello sgRNA library lentiviral prep (Addgene #73178-LV) and Cas9 stable single clone were used to perform CRISPR screen as previously reported81,154. Briefly, Cas9 stable single clone cells were treated with different concentrations of puromycin to determine the appropriate concentration of puromycin that killed the cells within 2-3 days. Then the Cas9 stable single clone cells were transduced with Brunello sgRNA library lentiviral prep at different MOIs to determine the condition that yields 20-30% infection efficiency. Ten 15-cm plates of Cas9 stable cells were cultured to 80-90% confluency and collected by centrifugation to pellet cells. Cells from all plates were then resuspended in complete DMEM containing 8 pg / ml polybrene and combined in 50-ml conical tube. Cell concentration was determined by cell counter. On average, over 500 copies of each sgRNA (~40 million infected cells) were maintained throughout the screen. 2* 108cells were aliquoted into new 50-ml conical tubes and diluted to 40 ml using complete DMEM containing 8 pg / ml polybrene. Corresponding amount of sgRNA library according to the MOI determined above was added to the cells and mixed well to get ~40 million infected cells. The cells and virus mixture were split equally into 12x 15-cm plates (~1.6><107cells / plate). 48 hours after infection, the culture medium was replaced by compete DMEM without polybrene followed by incubation overnight. Three days after infection, the medium was replaced with complete DMEM containing 4 pg / ml puromycin to select stable cells expressing the sgRNA. Cells were treated with puromycin until cells without virus infection were all dead. Cells at confluency were collected and combined before subculture. When enough stable cells were obtained (~7 days after infection), cells were mixed and split for 500 nM preQi or control PBS treatments. At least 40 million infected cells were used for each replicate to keep the sgRNA coverage. Cells were treated with 500 nM preQi or PBS for 5 days before collection. Cells from each condition (~8* 15-cm plates) were collected and genomic DNA was extracted using Wizard Genomic DNA Purification Kit (Promega).300397066.2 - 61 -

[0167] Next- generation sequencing (NGS) library construction for CRISPR screen:CRISPR screen NGS libraries were constructed in two PCR steps as previously reported155. Briefly, 30* first PCR reactions with 10 pg genomic DNA each were performed using Ex Taq (TaKaRa) for each sample in 100 pl reaction. The sgRNA sequences were amplified for 18 cycles using primers CRISPR-F1: 5’AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG (SEQ ID NO: 1) and CRISPR-R1 : 5’GTAATTCTTTAGTTTGTATGTCTGTTGCTATTATG (SEQ ID NO: 2). All the first PCR reactions for each sample were pooled and mixed. 125 pl of the pooled first PCR reactions was cleaned using DNA clean and concentrator-25 (Zymo) and eluted in 100 pl sterile H2O. Secondary PCR reaction with 5 pl of the purified first PCR reaction mix was amplified for 15-18 more cycles in 100 pl using Ex Taq to add sequencing adapters and indices (See Table S4 for primer sequences). The PCR products were purified using DNA clean and concentrator-25 followed by gel purification with 5% or 6% TBE PAGE gel (Bio-Rad). The resulting libraries (372-379 bp) was quality checked by Bioanalyzer (Agilent) before sequencing on Illumina NextSeq platform with 75 bp flowcell using single end mode. On average, ~40 million reads were obtained for each sample.

[0168] RT-qPCR:Real-time quantitative PCR (qPCR) was performed using Superscript IV (SSIV, ThermoFisher) and LightCycler 480 SYBR Green I Master (Roche Diagnostics). Briefly, for cDNA synthesis, 2 pl 100-200 ng total RNA was mixed with 1 pl 50 pM Poly-d(T) primer (IDT, sequence in Table S5), 1 pl 10 mM dNTP mix (Promega), and 10.5 pl sterile H2O in PCR tubes. The RNA-primer mixture was incubated at 65 °C for 5 min on a thermocycler (Eppendorf) followed by incubation on ice / 4 °C for > 1 min. Superscript IV master mix containing 4 pl 5* SSIV Buffer, 1 pl 100 mM DTT, 0.25 pl RNaseOut RNase inhibitor (ThermoFisher), and 0.25 pl SSIV reverse transcriptase for each reverse transcription reaction was prepared. 5.5 pl of the master mix were then added to the annealed RNA-primer mix. Reverse transcription (RT) was carried out in the thermocycler at 55 °C for 60 min and the SSIV reverse transcriptase was inactivated by incubation at 85 °C for 10 min followed by immediately incubation at 4 °C. The RT mix was diluted with 20 pl sterile H2O and 2 pl RT mix was used for each qPCR reaction. Master mix containing 5 pl LightCycler 480 SYBR Green I Master and 3 pl primers mix (1.5 pM each) for each qPCR reaction was prepared and 8 pl aliquot was added to each well of the qPCR plate. 2 pl diluted RT mix was then added to each well. qPCR reactions were then carried out on Bio-Rad CFX qPCR instrument. Actin mRNA level was used as control. Target gene expression was evaluated by analyzing the qPCR results with AACT method.300397066.2 - 62 -

[0169] CRISPR candidate genes knockdown followed by cell proliferation: Eight candidate genes were chosen for cell proliferation rescue validation. SiRNAs for the 8 genes were obtained from Sigma Mission predesigned siRNA collections (Sigma). MISSION siRNA Universal Negative Control #1 was used as siRNA control (sequence proprietary to Sigma). siRNAs were transfected into the cells by Lipofectamine RNAiMax (ThermoFisher) using the reverse transfection method. Briefly, 120 pmol siRNA was diluted in 2 ml Opti-MEM I reduced serum medium (ThermoFisher) in 10-cm tissue culture plate. 20 pl of Lipofectamine RNAiMax was then added to each plate and mixed followed by incubation at room temperature for 10-15 min. 0Q HEK293T cells were collected and resuspended in complete DMEM without antibiotics at the same time. Cells were then counted, and 1.5x 106cells were added to each plate. Complete DMEM without antibiotics were then added to each plate to 10 ml. Cells were incubated in 37 °C cell incubator for 3-4 days. The initial siRNA knockdown steps depleted the target genes at the beginning of the proliferation assay. Cells for each siRNA knockdown were collected and resuspended in DMEM without antibiotics. Cell concentrations were determined using cell counter. To check the effect of target gene knockdown on preQi inhibition effect, cells from each siRNA knockdown were treated with 0 nM, 100 nM, and 500 nM preQi. Six ml 5* 104cells / ml initial knockdown cells were prepared for each siRNA and each preQi condition. At the same time, transfection mix for each siRNA and each condition was prepared by diluting 12 pmol siRNA and 2 pl Lipofectamine RNAiMax in 200 pl Opti-MEM I reduced serum medium followed by incubation at room temperature for 10-15 min. Reduced amount of siRNA and Lipofectamine RNAiMax were used for each well of 96-well transfection to reduce cell death. The transfection mix was then added to the corresponding 6 ml 5x 104cells / ml cell mixture and mixed. 100 pl of the siRNA and cell mixture was then transferred to each well of the 96-well plate using multi-channel pipet (8 replicates per condition). The subsequent cell proliferation assay steps were then performed as described above. The leftover cells from each initial siRNA knockdown in 10-cm plates were collected and total RNA and lysate was extracted for qPCR and Western blot validation.

[0170] CRISPR candidate genes knockdown Northern blot experiments: Initial candidate gene knockdown in 10-cm plates was performed as above. After 3-4 days incubation of the siRNA, cells from initial knockdown were collected and resuspended in complete DMEM without antibiotics. Cell concentrations for each siRNA knockdown were measured. At the same time, 30 pmol siRNA was diluted in 500 pl Opti-MEM I reduced serum medium in each well of 6-well plate. 5 pl of Lipofectamine RNAiMax was then added to each well followed by incubation at room temperature for 10-15 min. 3.75 x 105cells in 2.5 ml complete DMEM 300397066.2 - 63 -without antibiotics from each initial knockdown experiment were added to corresponding wells of 6-well plates. Cells were then treated with PBS or 100 nM preQi for 4 days before collection (3 or 4 replicates for each condition). Total RNA and lysate were extracted from the collected cells and qPCR, or Western blot was performed to validate target gene knockdown. Northern blot was then performed as above mentioned using the extracted total RNA to check Q-modified tRNAs level changes after candidate gene knockdown.

[0171] Ribosome sucrose cushion: 0Q HEK293T cells were cultured in 15-cm plates to 60-70% confluency before preQi or sterile H2O was added to 1 pM final. Cells were incubated ±preQl for 24 hours before collection. On the day of collection, 37.5% sucrose buffer (20 mM HEPES (pH 7.6), 100 mM KC1, 5 mM MgCh, 37.5% sucrose (1.1M), 100 pg / ml CHX, 1% protease inhibitor (Roche), 1% RNase inhibitor (ThermoFisher)) was prepared and stored on ice. Cells were treated with 100 pg / ml cycloheximide in complete medium for 7 min at 37 °C followed by immediate wash with ice-cold PBS buffer containing 100 pg / ml CHX twice. Cells were then collected in 10 ml ice-cold PBS buffer containing 100 pg / ml CHX and pelleted by centrifugation at 500* g for 5 min. PBS buffer was completely removed and cells were lysed with 1 ml lysis buffer (20 mM HEPES (pH 7.6), 100 mM KC1, 5 mM MgCh, 1% Triton X-100, 100 pg / ml CHX, 1% protease inhibitor, 1% RNase inhibitor) on ice for 20 min with periodic perturbation (or rotating at 4 °C). Cell lysate were then centrifuged at 16,000* g for 15 min and supernatant lysate were saved. 4 pl Turbo DNase Turbo DNase was added to the supernatant lysate followed by incubation at room temperature for 15 min. The DNase treated lysate were centrifuged at 16,000* g for 15 min to clear out debris. OD260 of each cleared lysate sample was measured and adjusted to the same value using lysis buffer. 900 pl of the prepared lysate from each sample was loaded to the bottom of polycarbonate ultracentrifuge tolerant tube (Beckman Coulter). The rest of the lysate was saved as input. 22 ml ice-cold sucrose cushion buffer (20 mM HEPES (pH 7.6), 100 mM KC1, 5 mM MgCh, 100 pg / ml CHX, 1% protease inhibitor (Roche), 1% RNase inhibitor (ThermoFisher)) was then added to the tube. 3 ml 37.5% sucrose buffer was gently loaded to the bottom of the tube using long blunt end needle. The weight of each tube was measured using analytical balance and the tubes were balanced by adding more sucrose cushion buffer to the lighter ones. The sucrose cushion tubes were loaded into fixed angle rotor in ultracentrifuge (Beckman Coulter Optima L-100XP, 70 Ti rotor). Ultracentrifugation was performed at 60,000 rpm and 4 °C under vacuum for 2.5 hours. After centrifugation, supernatant was removed, and the ribosome pellet was rinsed twice with 200 pl of pre-chilled lysis buffer twice. The ribosome pellet was resuspended with 500 pl300397066.2 - 64 -lysis buffer followed by incubation on ice for 5 min with periodic vortex. The ribosome suspension and input samples were stored at -80 °C before Western blot analysis.

[0172] Polysome profiling and validation of SCAP in polysome fractions: Wild-type HEK293T cells, wild-type SCAP overexpression cells, SCAP mutant 1 overexpression cells, and SCAP mutant 2 overexpression cells were seeded in 15-cm plates with ± 1 pM preQi treatment. Cells from 2* 15-cm plates were combined as one sample. Polysome profiling was then performed as above. For Western blot, 14 pl solution from each fraction was mixed with 5 pl 4* LDS loading buffer (ThermoFisher), and 1 pl IM DTT. The mixture was then boiled at 95 °C for 5 min. 10 pl mixture from each sample was loaded on the gel. Western blot was then performed as above. SCAP and RPL8 were checked in the polysome fractions.

[0173] Northern blot quantitation of tRNAs with ^translation inhibitors (emetine and cycloheximide treatment: 0Q HEK293T cells were cultured in 2* 6-well plates to 60-70% confluency. Cells in 1 * 6-well plate were treated with 25 pg / ml Emetine or Cycloheximide for 20 min. 1 * PBS or preQi was then added to half of the wells in each 6-well plate to 1 pM final and incubated for 24 hours. Cells were then collected and total RNA was extracted. 3 pg total RNA from each sample was used for Northern blot as described above.

[0174] Generation of wild-type, mutant 1, and mutant 2 SCAP, and SCAP-N-Tad8e stable overexpression cells: 3* Flag tagged wild-type, mutant 1, and mutant 2 SCAP, and SCAP-N-Tad8e plasmids were obtained from Genscript. Mutant 1 SCAP has loop 6 deletion (aa 452-512) and mutant 2 SCAP has C-terminal WD domain deletion (aa 741-1279). Tad8e-N-SCAP has Tad8e linked to the N-terminal of SCAP through a linker peptide. Lentivirus particles containing these plasmids were prepared. Briefly, HEK293T cells were cultured in T25 flask in 3 ml to 40-50% confluency. Medium were replaced 1 hour before lentiviral plasmids transfection. Transfection plasmid cocktail containing 1.34 pg pCMV-VSV-G (Addgene #8454) plasmid, 2.14 pg pMDLg / pRRE (Addgene #12251) plasmid, 2.14 pg pRSV-Rev (Addgene #12253) plasmid, and 5.63 pg corresponding SCAP lentiviral plasmid was prepared in 11.25 pl (1 pg / pl plasmid). 200 pl Opti-MEM I reduced serum medium (ThermoFisher) was then added to the plasmid mixture and mixed. 19 pl TransIT-LTl Transfection Reagent (Minis Bio MIR 2300) was then added to the plasmid mixture dropwise below the surface of the mixture slowly. The mixture was then mixed and briefly spun followed by incubation at room temperature for 15 min. The transfection mixture was then added to the T-25 flask dropwise below the surface of the medium slowly. Cells were incubated with the transfection mixture in 37 °C cell incubator for 18 hours or overnight. Complete high BSA lentivirus harvesting medium (DMEM, 10% FBS, 1% (w / v) BSA, and lx Pen / Strep) was prepared. Medium in the 300397066.2 - 65 -T25 flask was then replaced with 3 ml high BSA lentivirus harvesting medium followed by incubation for 24 hours. Harvesting medium was collected and fresh harvesting medium was added to the T25 flask for one more round of harvesting. The collected harvesting medium was combined and filtered through 45 pm PES filter (ThermoFisher, 725-2545). The virus containing harvesting medium was then aliquoted and stored at -80°C for future use. Cells that stably overexpress these proteins were generated as above. The stable cells were then used for polysome profiling, Western blot validation, tRNA-seq, CLiP-seq, SCAP-N-Tad8-seq etc.

[0175] SCAP-CLiP-seq using Flag tagged wild-type SCAP overexpression stable cells:CLiP-seq was performed as previously reported with modifications156. Briefly, 3* Flag tagged wild-type SCAP overexpression stable cells were cultured to 60-70% confluency in 8* 15-cm plates. PreQi were then added to 4 * 15-cm plates to 1 pM final followed by incubation for 24 hours. Culture medium was removed and cells were washed with 10 ml ice-cold 1 x PBS once.10 ml ice-cold 1 x PBS were then added to the plate. Cells were irradiated with 400 mJ / cm2UV light at 254nm using the UV crosslinker twice (400 mJ / cm2each time) on ice. Cells were harvested with cell lifter and transferred to 15-ml conical tubes. Cells were pelleted by centrifugation at 500x for 5 min. Cells from 2x 15-cm plates were combined as one sample. Cell pellets were resuspended with 1 ml lysis buffer (50 mM Tris-HCl (pH 7.5), 100 mMNaCl.1% NP-40 (Igepal CA630), 0.1% SDS, 0.5% (w / v) sodium deoxycholate, 2.5% (w / v) Digitonin, 0.5 mM DTT, 1% protease inhibitor). The mixture was rotated for 45 min at 4 °C cold room. RNase I (ThermoFisher, AM2295) were diluted in 1:25 ratio in ice-cold DPBS. 5 pl Turbo DNase (ThermoFisher) and 10 pl diluted RNase I were added to the lysed samples. The samples were immediately mixed and incubated in a Thermomixer preheated to 37 °C for 5 min at 1200 rpm. The samples were transferred to ice and 10 pl RNase Inhibitor (ThermoFisher) were immediately added to each sample and mixed to inhibit remaining RNase activity. Cell lysates were then centrifuged at 12500x for 15 min at 4 °C to get clear supernatant lysate. 50 pl of each clear lysate was saved as input. At the same time, anti-Mouse IgG (ThermoFisher, 11202D) or Anti-Rabbit IgG (ThermoFisher, 11204D) magnetic beads were thoroughly resuspened by gentle inversion. 100 pl beads were then transferred to a new microcentrifuge tube for each sample. The beads were washed three times using 500 pl lysis buffer with protease inhibitor. At the last wash, the beads suspension were divided into 25 pl and 75 pl aliquot. Lysis buffer were completely removed from each tube before use. Supernatant lysate was added to the corresponding 25 pl aliquot and rotated at room temperature for 45 min to preblock the lysate. 10 pg corresponding Flag (Sigma, Fl 804) or IgG (Cell Signaling Technology, 2729S) antibody in 200 pl lysis buffer were added to the 75 300397066.2 - 66 -pl aliquot and rotate at cold room for 45 min. For the tube with antibodies, antibody solution was removed from the beads and saved. The beads were washed three times with 500 pl lysis buffer. For the tube with lysate, the beads were captured with magnetic stand and the supernatant lysate was added into the tube with antibody-beads conjugate. The samples were briefly mixed and rotated at 4 °C overnight. After overnight incubation, 20 pl of each lysate (including beads) was saved into two clean tubes and stored at -80 °C for future use. Beads were captured with magnetic stand and supernatant was saved as flow-through. Beads were washed twice with 500 pl high salt buffer (50 mM Tris-HCl (pH 7.4), 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate). A transition wash was performed by adding 500pl of high-salt wash buffer followed by mixing and the addition of 500pl wash buffer (20 mM Tris-HCl (pH 7.4), 10 mM MgCh, 0.2% Tween-20). Beads were then washed 3 times with 500pl wash buffer. At the last wash, beads were transferred to new tubes. Beads were resuspended with 150 pl RNA elution buffer with proteinase K (100 mM Tris-HCl pH 7.5, 50 mM NaCl, 20 mM EDTA, 0.2% (w / v) SDS, 4 pg / pl proteinase K, 1% RNase Inhibitor (ThermoFisher)). The samples were incubated in a Thermomixer at 37 °C and 1200 rpm for 20 min followed by incubation at 50 °C and 1200 rpm for 20 min. Beads were captured by captured with magnetic stand and the elution product was transferred to clean tubes. 55 pl sterile H2O was added to the tube with beads and mixed by flicking. Beads were then captured again, and the elution solution was transferred to the corresponding previously harvested elution RNA tubes. Crosslinked RNA were extracted from the elution mixture using RNA clean and concentrator-5 kit (Zymo) and eluted in 11 pl sterile H2O. The elute was transferred back to the corresponding columns and the elution was repeated to increase the yield. All eluted RNA samples were stored at -80 °C for future use. Crosslinked RNA sequencing libraries were constructed using MSR-seq method as previously reported78.

[0176] SCAP-N-Tad8 sequencing: HEK293T cells that stably overexpress wild-type SCAP and SCAP-N-Tad8e were cultured to 60-70 confluency in 2* 6-well plates. PreQi or sterile H2O was added to 1 pM final to half of the wells in each 6-well plates. Cells were incubated in 37 °C cell incubator for 24 hours. Cells were collected and total RNA was then extracted using TRIzol reagent. 1 pg total RNA from each sample was used to build sequencing libraries for large RNAs. Briefly, 1 pg total RNA was fragmented to 200 nt using Magnesium RNA Fragmentation module (NEB, E6150S) as follows. 1 pg total RNA was mixed with 2 pl RNA Fragmentation Buffer (10*) and sterile H2O to 20 pl in PCR tube. The tubes were incubated in a preheated thermal cycler for 5 minutes at 94 °C. The tubes were transferred to ice and 2 pl 10x RNA Fragmentation Stop Solution was immediately added to each tube and 300397066.2 - 67 -mixed. 30 pl sterile H2O was added to each tube and mixed. Fragmented RNA was then extracted using RNA clean and concentrator-5 kit (Zymo) and eluted in 7 pl sterile H2O. MSR-seq protocol (Watkins et al., 2022) was then followed to build the sequencing libraries.

[0177] Northern blot quantitation of tRNAs with ± IRE1 ribonuclease inhibitor 4u8C treatment: 0Q HEK293T were cultured to 60-70% confluency in 4* 6-well plates. Sterile H2O or IRE1 ribonuclease inhibitor 4p8C was added to 8 wells of the 6-well plates to 0 pM, 10 pM, or 50 pM 4p8C final. For each IRE1 concentration, cells were treated with 1 pM preQi for Oh, 8h, 16h, or 24h (duplicates). Cells from each treatment were then collected and total RNA were extracted using TRIzol reagent. 3 pg total RNA from each sample was then used to run Northern blot with preQi sine detection as above.

[0178] EIF2a phosphorylation and XBP1 mRNA splicing under ± preQi treatment: 0Q HEK293T were cultured to 60-70% confluency in 2* 6-well plates. Sterile H2O or preQi was added to 1 pM final to half of the wells in each 6-well plates (triplicates) and incubated for 24 hours. Cells from lx 6-well plate were collected, and total RNA was extracted using TRIzol reagent. For XBP1 mRNA splicing detection, RT-PCR followed by gel electrophoresis was performed. Briefly, 5 pg total RNA from each sample was diluted to 52 pl using sterile H2O.6 pl 10x Turbo DNase buffer and 2 pl Turbo DNase (ThermoFisher) were added to each sample and mixed. The samples were incubated at 37 °C for 30 min. RNA was then purified using RNA clean and concentrator-5 kit (Zymo) and eluted in 20 pl sterile H2O. Concentration of the eluted RNA samples were measured by Nanodrop and 500 ng total RNA from each sample was diluted to 9 pl in PCR tubes. 1 pl 50 ng / pl random hexamer and 1 pl 10 mM dNTP mix were added to each sample and mixed. The samples were then incubated at 65 °C for 5 min followed by incubation on ice for > 1 min. 9 pl master cDNA synthesis mix containing 4 pl 5x SSIV RT buffer, 1 pl 0.1 M DTT, 3.75 pl sterile H2O, and 0.25 pl SuperScript IV RT (200 U / pl) was then added to each sample and mixed. The samples were incubated at 25 °C for 10 min followed by incubation at 55 °C for 60 min. Reverse transcription was terminated by incubation at 80 °C for 10 min followed by immediate incubation on ice. PCR reactions were performed with 1 pl cDNA mix using Q5 High-Fidelity DNA Polymerase (NEB, M0491L) in 20 pl for 20 cycles (GAPDH) and 25 cycles (XBP1). The PCR primers used for GAPDH and XBP1 are in the following table. The sizes of PCR products of unspliced XBP1 and spliced XBP1 are 442 bp and 416 bp, respectively. The size of PCR product of GAPDH control is 404 bp. The primer sequences are: XBP1 F, 5’ CCTTGTAGTTGAGAACCAGG (SEQ ID NO: 3), XBP1 R, 5’ GGGGCTTGGTATATATGTGG (SEQ ID NO: 4), GAPDH F, 5’ GGATGATGTTCTGGAGAGCC (SEQ ID NO: 5), GAPDH R, 5’ 300397066.2 - 68 -CATCACCATCTTCCAGGAGC. Cells from the other 6-well plate were collected and whole cell lysate was extracted using CelLytic M lysis buffer (Sigma, C2978). Whole cell lysate from each sample (±preQl treatment) was used to run Western blot as above check the phosphorylation status of EIF2a. Antibodies for EIF2a (Cell Signaling Technology, 9722S), phosphorylated EIF2a (Cell Signaling Technology, 3398S), vinculin (Santa Cruz Biotechnology, sc-73614), and IRE1 (Cell Signaling Technology, 3294S) were used.

[0179] PAQS-seq of microbiome tRNA modification: MSR-seq reads were first mapped to a reference genome composed of bacterial 5S rRNA sequences. Reference sequences for 5S rRNA were downloaded from the 5S rRNA database (http: / / combio.ol / rrna). Sequences were combined from Bacteria (n = 7291), Archaea (n = 319), Eukaryota (n = 2861), mitochondria (n = 110), and plastids (n = 838). Full lineages were assigned to each reference using the ETE3 NCBI Taxa toolkit in python157From here, tRNA reference genomes were retrieved from gtRNAdB158for microbes from the most abundant class taxon: Lachnoclostridium_phytofermentans_ISDg, Ery sipelothrix rhusiopathiae S Y 1027, Camobacterium maltaromaticum, Faecalibacterium_prausnitzii, Bacteroides dorei, Bifidobacterium_longum_subsp_longum_BBMN68, Clostridium beijerinckii, Roseburia_intestinalis_XB6B4, Ruminococcus_bromii_L2-63. These reference tRNA genomes were combined into a single reference fasta and tRNA-seq reads were mapped to this combine reference. Q-tRNA modification was detected by its characteristic deletion signatures75. Further analysis was performed with custom R scripts available on Github (http s : / / github . com / ckatanski / PreQ 1 ) .

[0180] MSR-seq data analysis for tRNA: Data analysis was performed as described previously78. Briefly, starting from index demultiplexed fastq data, paired end reads were split by internal barcode sequence using Je159demultiplex with options BPOS = BOTH BM = READ 1 LEN = 4:6 FORCE = true C = false 6. Barcode sequences are available on Github at https: / / github.com / ckatanski / Q_paper75. Next read 2 files were used to map with bowtie2160with the following parameters: -q -p 10 -local — no-unal. Reads were mapped to curated list of non-redundant tRNA mature genes with tRNAScane score > 40 from hgl9. Bowtie2 output sam files were converted to bam files, then sorted using samtools. Next, IGV161was used to collapse reads into 1 nt window. IGV output.wig files were reformatted using custom python scripts (available on GitHub at https: / / github.com / ckatanski / Q_paper). The bowtie2 output Sam files were also used as input for a custom python script using PySam, a python wrapper for SAMTools162to sum all reads that mapped to each gene. For tRNA fragment analysis, related custom scripts were used to divide reads based on which 10 nt window the 3' end 300397066.2 - 69 -mapped to for each tRNA. Data were visualized with custom R scripts. All custom scripts are available on GitHub (https: / / github.com / ckatanski / CHRIS-seq). R script for the present analysis is available on Github (https: / / github.com / ckatanski / PreQl). Relative tRNA expression levels were calculated as the ratio of reads of a tRNA divided by the total number of reads. The expression level of an iso-acceptor is the sum of expression levels of all isodecoders. The mean expression level of the two replicates were used when visualization.

[0181] MSR-seq data analysis for mRNA: Paired end reads were split by internal barcode sequence using Je demultiplex with options BPOS = BOTH BM = READ 1 LEN = 4:6 FORCE = true C = false 6. Barcode sequences are available on Github at https: / / github.com / ckatanski / Q_paper75. The quality of reads were checked by fastqc vO.11.9163and the reads were aligned to human hg38 genome (GRCh38.pl3. genome. fa) by STAR 2.7.9a164with the human annotation file (gencode.v39. annotation. gtf) from GENCODE database (https: / / www.gencodegenes.org). Then the expression levels were counted and collected by RSEM vl .3.3165. Low expressed genes were filtered by filterByExpr function with default parameters in edgeR v3.36.0166and the CPM (Counts Per Million reads mapped) and TPM (Transcripts Per Million) of each gene were calculated. The TPM in visualization was the mean of the two replicates. Translation efficiency (TE) of a gene was calculated as the expression level in the polysome sample divided by that in the input sample. Codon usage of a gene or a group of genes was defined as the ratio of each amino acid within CDS region of a gene or a group of genes. The gene ontology (GO) analysis was performed using the Gene Ontology Resource (http: / / geneontology.org)167>168.

[0182] CRISPR screen sequencing data analysis: Sequencing libraries containing Illumina sequencing indices and the raw reads were separated by indices right after sequencing. The raw reads were trimmed from 3’ end to 20-nt long by Cutadapt169. The resulting sgRNA sequences were mapped to Brunello sgRNA library reference by Bowtie2160. The mapped reads were sorted and indexed by SAMtools program170. Reads mapped to each sgRNA were counted by SAMtools idxstats function. Low expressed sgRNA were filtered by filterByExpr function with default parameters in edgeR. sgRNA with low replicate consistency (any two replicate expression level ratio > 5 fold) were filtered. The differential sgRNA expression analysis was performed by edgeR. The average difference of sgRNA levels between Ctrl and preQi treated samples were evaluated by STARS vl.381with 100 iterations. Genes with STARS p value < 0.05 and STARS pi score > 2.4503 were selected as significantly affected genes for downstream analysis. The p values in visualization were calculated by two-sided Mann-300397066.2 - 70 -Whitney U test unless specifically indicated. The gene ontology (GO) analysis was performed using the Gene Ontology Resource167>168.

[0183] Clustering of protein-protein interaction enrichment analysis was done using STRING program90for genes with pi value > 2.4503 and STARS p <0.05. The meaning of network edges (interaction lines) was set to be evidence (medium), which the line thickness indicates the strength of data support. The interaction network was further processed by Inkscape (https: / / inkscape.org) to add more information. Proteins in the same pathway were grouped and annotated with colored shadow. Proteins in each pathway that were chosen for further experimental validation were highlighted with colored dots.* * *

[0184] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. 1. Fergus, C., Barnes, D., Alqasem, M.A., and Kelly, V.P. (2015). The queuine micronutrient: charting a course from microbe to man. Nutrients 7, 2897-2929.10.3390 / nu7042897.2. Sarid, L., Sun, J., Chittrakanwong, J., Trebicz-Geffen, M., Ye, J., Dedon, P.C., and Ankri, S. (2022). Queuine Salvaging in the Human Parasite Entamoeba histolytica. Cells 77. 10.3390 / cellsl 1162509.3. Patel, B.I., Heiss, M., Samel-Pommerencke, A., Carell, T., and Ehrenhofer-Murray, A.E. (2022). Queuosine salvage in fission yeast by Qngl-mediated hydrolysis to queuine. 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Claims

WHAT IS CLAIMED IS:

1. A method of treating a disease associated with protein translation deregulation in a patient, the method comprising administering a sterol regulatory element-binding protein cleavageactivating protein (SCAP) gene or gene product to the patient.

2. The method of claim 1, wherein the disease is a neurological disease.

3. The method of claim 1, wherein the disease is a cancer.

4. The method of any one of claims 1 to 3, wherein the SCAP gene or gene product is mutated compared to a wild-type SCAP gene.

5. The method of claim 4, wherein the wild-type SCAP gene is a human SCAP gene.

6. The method of claim 4 or 5, wherein the SCAP gene or gene product comprises a loop 6 deletion and / or a WD domain deletion.

7. The method of any one of claims 4 to 6, wherein the SCAP gene comprises a polynucleotide of SEQ ID NOs: 9-11 or the SCAP gene product comprises a protein of SEQ ID NO: 7 or 8 or a polynucleotide of SEQ ID NOs: 9-11.

8. A method of regulating endoplasmic reticulum ribosome translation in a cell, the method comprising introducing a sterol regulatory element-binding protein cleavage-activating protein (SCAP) gene or gene product to the cell.

9. The method of claim 8, wherein the SCAP gene or gene product is mutated compared to a wild-type SCAP gene.

10. The method of claim 9, wherein the wild-type SCAP gene is a human SCAP gene.

11. The method of claim 9 or 10, wherein the SCAP gene or gene product comprises a loop 6 deletion and / or a WD domain deletion.

12. The method of any one of claims 8 to 11, wherein the cell is a cancer cell.

13. The method of any one of claims 8 to 11, wherein the cell is a neuron.

14. The method of any one of claims 8 to 13, wherein the cell is a human cell.

15. A method of treating a disease associated with protein translation deregulation in a patient, the method comprising administering a sterol regulatory element-binding protein cleavageactivating protein (SCAP) inhibitor.

16. The method of claim 15, wherein the disease is a neurological disease.300397066.2 - 88 -17. The method of claim 15, wherein the disease is a cancer.

18. The method of any one of claims 15 to 17, wherein the SCAP inhibitor is a nucleic acid capable of reducing a SCAP gene product.

19. The method of any one of claims 15 to 18, wherein the SCAP inhibitor comprises a CRISPR-Cas9 system targeting a SCAP gene in a cell in the patient.

20. The method of claim 19, wherein the cell is a tumor cell.

21. The method of claim 19, wherein the cell is a neuron.

22. A method of regulating endoplasmic reticulum ribosome translation in a cell, the method comprising introducing a sterol regulatory element-binding protein cleavage-activating protein (SCAP) inhibitor.

23. The method of claim 22, wherein the cell is a cancer cell.

24. The method of claim 22, wherein the cell is a neuron.

25. The method of any one of claims 22 to 24, wherein the cell is a human cell.

26. The method of any one of claims 22 to 25, wherein the SCAP inhibitor is a nucleic acid capable of reducing a SCAP gene product.

27. The method of any one of claims 22 to 26, wherein the SCAP inhibitor comprises a CRISPR-Cas9 system targeting a SCAP gene in the cell.300397066.2 - 89 -

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