A method for screening modified enzyme labels and detecting intracellular RNA molecular events.

Abstract

This invention belongs to the field of RNA molecular event detection technology, specifically disclosing a method for screening and detecting intracellular RNA molecular events using modified enzyme labeling. This invention screens RNA-modifying enzymes that may be used for RNA molecular event labeling, and uses the screened modified enzymes to label the RBP binding, translation, and subcellular localization processes of RNA molecules within cells. Finally, high-throughput sequencing is used to detect the labels. In RBP binding event detection, this method is antibody-independent, simpler to use, and compatible with long-read sequencing and single-cell sequencing, thus broadening its application range. In ribosome translation event detection, it is compatible with long-read sequencing, improving the resolution of translation event resolution. In RNA subcellular localization detection, it can label RNA components in membrane-free organelles that are difficult to separate; compared with other current proximity labeling methods, it has higher labeling efficiency, and the labeling does not affect cell survival, allowing for continuous monitoring of RNA localization.

Description

Technical Field

[0001] This invention belongs to the field of RNA molecular event detection technology, specifically relating to a method for screening modified enzyme labels and detecting intracellular RNA molecular events. Background Technology

[0002] RNA undergoes various processes in cells, including transcription, processing, localization, translation, and degradation. Dysfunction in any of these processes can lead to various diseases. Therefore, recording the events that RNA undergoes is crucial for understanding the physiological functions of RNA.

[0003] Interaction with RNA-binding proteins (RBPs) is a crucial event in the RNA lifecycle. Currently, methods such as RIP and CLIP based on immunoprecipitation, and TRIBE, STAMP, and REMORA based on RNA editing enzymes, have been developed to record RBP binding events. However, the dependence on immunoprecipitation means that CLIP-like methods typically require large amounts of material and are highly dependent on high-performance antibodies. The requirement for RNA fragmentation also makes these methods incompatible with long-read sequencing. While RNA editing enzyme-based methods avoid some of the drawbacks of immunoprecipitation, the widely used deaminases still suffer from high background noise, sequence bias, and the fact that deaminases directly alter the RNA sequence, potentially affecting RNA function and leading to human bias. Translation is a core step in mRNA function. Methods like ribo-seq can measure ribosome occupancy at the transcriptome level, but fragmentation and high-volume processing requirements lead to the loss of isoform information and compatibility issues with long-read platforms. Ribo-STAMP utilizes the fusion of the cytidine deaminase APOBEC1 with the small ribosomal subunit protein RPS2 to record translation events and has successfully combined with long-read sequencing. However, deaminases alter the RNA molecule sequence and directly affect protein coding, potentially impacting cellular translation behavior. Subcellular RNA localization is also a crucial characteristic of RNA molecules, connecting the fundamental processes of gene expression with cellular structure and function. Component-based methods can analyze a limited number of organelles and may suffer from inherent contamination from biochemical methods. FISH and in situ sequencing are also rapidly advancing, but they typically require well-designed oligonucleotide sequences and high-precision imaging instruments, limiting their application. Proximity labeling methods such as APEX-seq offer novel intracellular labeling of subcellular RNA localization; however, current proximity labeling enzymes generally suffer from low activity and can only label snapshots at specific time points, as labeling can lead to cell death. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a method for screening RNA-modifying enzymes for labeling and detecting intracellular RNA molecular events. This method can screen RNA-modifying enzymes that may be used for labeling RNA molecular events, and use the screened enzymes to label events such as RBP binding and translation of RNA molecules within the cell, as well as subcellular localization information. Finally, high-throughput sequencing is used to detect the labels.

[0005] The method of the present invention for screening modified enzyme labels and detecting intracellular RNA molecular events includes the following steps:

[0006] S1. Synthesize gene fragments containing candidate modifying enzyme sequences and construct plasmids for the fusion expression of RNA-binding proteins and candidate modifying enzymes;

[0007] S2. Transfect the plasmid into the cells to enable the fusion protein to be expressed in the cells;

[0008] S3. Harvest cells, extract RNA, and perform LC-MS analysis to identify the activity of modified enzymes.

[0009] S4. After obtaining highly active candidate modifying enzymes, the modifying enzymes are fused with RNA-binding proteins of interest for expression, thereby marking the RNA-binding protein binding sites in the cell; fused with ribosomal proteins for expression, thereby marking ribosomal translation events in the cell; and fused with specific subcellular location signals for expression, thereby marking the subcellular location of RNA molecules in the cell.

[0010] S5. Harvest the labeled cells, extract RNA, and use high-throughput sequencing to detect the modifications of the labeled RNA and perform data analysis to resolve the corresponding RNA molecular events.

[0011] Preferably, the RNA-binding protein in S1 includes the ribosomal small subunit protein component RPS2, which can enhance the RNA-binding ability of the modifying enzyme.

[0012] Preferably, after extracting total RNA from cells in step S3, the total RNA is subjected to rRNA removal or mRNA extraction, and then the modifications on the RNA are detected by LC-MS.

[0013] Preferably, the LC-MS detection in S3 uses the MRM (parallel reaction monitoring) method.

[0014] Preferably, the highly active candidate modifying enzymes in S4 include m6A modifying enzymes M.EcoGII and SUPREM, m5C modifying enzyme rlmI, ac4C modifying enzyme TK0754, and D modifying enzyme Dus2.

[0015] Preferably, the high-throughput sequencing method in S5 includes detecting modifications using next-generation sequencing and nanopore direct RNA sequencing technologies.

[0016] Preferably, the data analysis in S5 includes the alignment of sequencing data, the identification and quantification of modification sites, and the identification of corresponding molecular events through differential modification analysis between the experimental group and the control group.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] The method of this invention first screens RNA-modifying enzymes that may be used for RNA molecular event labeling, then uses the screened RNA-modifying enzymes for labeling, and finally detects the modifications using high-throughput sequencing to resolve RNA molecular events. Regarding RBP binding event detection, compared to traditional immunoprecipitation-based methods, this invention does not rely on antibodies, is simpler to use, and is compatible with long-read sequencing and single-cell sequencing, thus having a wider range of applications; furthermore, this invention can monitor dynamic changes in intracellular RBP binding. Regarding ribosome translation event detection, compared to traditional ribosome analysis methods, this invention is compatible with long-read sequencing, improving the resolution of translation event resolution; compared to deaminase-based methods, the RNA-modifying enzymes used in this invention cause less interference with RNA translation, improving the reliability of translation event detection. Regarding RNA subcellular localization detection, compared to biochemical separation methods, this invention can label RNA components in membrane-free organelles that are difficult to separate; compared to other current proximity labeling methods, this invention has higher labeling efficiency, and labeling does not affect cell survival, allowing for continuous monitoring of RNA localization. In addition, the enzyme screening method proposed in this invention can effectively expand the tool enzyme library that can be used for RNA labeling. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0020] Figure 1 This is a flowchart for screening RNA-modifying enzymes.

[0021] Figure 2 The images show the mass spectrometry results of nucleic acid modification in RPS2-modifying enzyme-transfected and wild-type 293T cells. In the images, a represents the detection result of m6A, b represents the detection result of m5C, c represents the detection result of ac4C, and d represents the detection result of D.

[0022] Figure 3 A flowchart for RNA molecular event labeling and detection.

[0023] Figure 4 A differential analysis plot for COX-M.EcoGII and M.EcoGII-NES markers.

[0024] Figure 5A schematic diagram of the distribution pattern of binding sites for RBFOX2-M.EcoGII markers.

[0025] Figure 6 Motif analysis of the binding site of RBFOX2-M.EcoGII labeled.

[0026] Figure 7 The correlation plot shows the mTotal value of RPS2-M.EcoGII samples and the correlation between Ribo-seq. Detailed Implementation

[0027] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions of embodiments are for the purpose of helping to understand the present invention, but do not constitute a limitation thereof.

[0028] Example 1

[0029] See Figure 1 The method for screening modified enzyme labels and detecting intracellular RNA molecular events includes the following steps:

[0030] (1) Synthesis of candidate modifying enzymes

[0031] The sequences of the candidate modifying enzymes were obtained from the Uniprot and NCBI databases, and the gene synthesis was commissioned to Beijing Qingke Biotechnology Co., Ltd.

[0032] (2) Construction of RPS2- candidate modified enzyme plasmid

[0033] The "pcDNA3.1-RPS2-modifying enzyme" vector was constructed using the Gibson recombination seamless cloning method. The recombinant product was transformed into E. coli competent cells top10, single colonies were picked and cultured, and plasmids were extracted for sequencing verification to ensure that the inserted nucleic acid sequence was consistent with the designed sequence.

[0034] (3) Transfection of recombinant plasmids

[0035] A. Cell Culture

[0036] The frozen 293T cells (derived from human embryonic kidney cells, purchased from the Cell Bank of the Chinese Academy of Sciences) were removed from the liquid nitrogen tank, thawed, and cultured in a 37°C, 5% CO2 incubator. They were passaged when the cell confluence reached 70-80%.

[0037] B. Cell transfection

[0038] a) For each dish of cells, take 100 μL of Opti-MEM into an EP tube, add 5 μg of RPS2-candidate modifying enzyme plasmid to each tube, and mix thoroughly. This is the "plasmid dilution buffer".

[0039] b) For each dish of cells, add 100 μL of Opti-MEM to an EP tube, then add 10 μL of Lipo2000 transfection reagent and mix thoroughly. This is the "transfection dilution".

[0040] c) Add the "transfection dilution buffer" to the "plasmid dilution buffer", mix quickly, and let stand at room temperature for 20 minutes to form a transfection complex; gently and evenly add the transfection complex to the cell culture dish.

[0041] d) After culturing the culture dishes in an incubator at 37°C and 5% CO2 for 48 hours, the cells were collected for subsequent experiments.

[0042] (4) Extraction of Total RNA

[0043] a) Take cells 48 hours after transfection, discard the original culture medium, and wash once with PBS;

[0044] b) Add 1 mL of TRIZOL reagent to collect cells and mix by pipetting;

[0045] c) Add 200 μl of chloroform, mix vigorously, and let stand for 2-5 minutes;

[0046] d) Centrifuge at 4 ℃, 12000×g for 15 min;

[0047] e) Take the upper aqueous phase and add an equal volume of anhydrous ethanol;

[0048] f) After mixing, extract RNA using the RNA Clean & Concentrator-5 kit.

[0049] (5) Remove rRNA

[0050] a) Sample and probe annealing

[0051] The system configuration is shown in Table 1;

[0052] Table 1

[0053]

[0054] Place the mixture in a PCR instrument and perform a standard annealing procedure: heat at 95°C for 2 minutes, then slowly cool to 22°C at a rate of 0.1°C / second, and hold at 22°C for 5 minutes. This process ensures that the sample is fully hybridized with the probe.

[0055] b) Rnase H digestion

[0056] The mixtures are shown in Table 2:

[0057] Table 2

[0058]

[0059] The mixture was placed in a 37°C water bath for 30 minutes to remove the hybridized RNA portion.

[0060] c) Digestion of Dnase 1

[0061] The mixtures are shown in Table 3:

[0062] Table 3

[0063]

[0064] The mixture was placed in a 37°C water bath for 30 minutes to remove DNA.

[0065] d) 2.2× RNA clean beads purification

[0066] i.beads joining and incubating

[0067] Add 110 μl of RNA clean beads to each sample, gently blow to mix, and incubate for 20 minutes to ensure that the beads are fully bound to the nucleic acid.

[0068] ii. Ethanol preparation

[0069] Prepare 80% ethanol, and prepare 400 μl for each sample.

[0070] iii. Washing

[0071] After incubation, place the sample on a magnetic rack and let it stand until the beads are completely adsorbed to one side of the magnetic rack. Carefully remove the supernatant. Wash the beads twice with 200 μl of 80% ethanol for 30 seconds each time to remove impurities.

[0072] iv. Drying and elution

[0073] After removing the ethanol, place the sample on ice and air dry until the surface of the beads is completely cracked. Add 22 μl of ultrapure water for elution and let stand for 2 minutes to fully elute the nucleic acids.

[0074] Place the sample on the magnetic rack, carefully aspirate the supernatant into a new EP tube, and take 1 μl for concentration determination.

[0075] (6) Mass spectrometry detection

[0076] Take 200 ng of sample and first subject it to heat shock: 95 ℃ for 5 min, then quickly place it on ice for more than 2 min; prepare the digestion system as shown in Table 4 below;

[0077] Table 4

[0078]

[0079] Incubate overnight at 37 ℃ (more than 8 h);

[0080] Add 25 μL of water to the digested sample to bring the total volume to 50 μL, mix well, and centrifuge at 14800 × g for 5 min at RT.

[0081] Take the supernatant and transfer it into the injection vial sleeve, then run it on the LC-MS instrument.

[0082] The LC-MS method used a liquid chromatography-triple quadrupole mass spectrometer (AB SCIEX 5500 / WATERSACQUITY UPLC) with a mobile phase of water containing 0.1% formic acid (solvent A) and methanol containing 0.1% formic acid (solvent B). The MRM acquisition mode was used to detect the corresponding modification signals.

[0083] (7) Data Analysis

[0084] The intensity of each modification signal was analyzed using software and compared with that of wild-type control cells to confirm the modification efficiency of the enzymes. Modifying enzymes with higher modification efficiency were selected for subsequent experiments. Figure 2 As shown, the level of the corresponding modification was significantly increased in cells transfected with RPS2-modifying enzyme, indicating that this method can effectively evaluate the ability of the modifying enzyme to modify RNA.

[0085] Example 2: Labeling and Detection of RNA in Mitochondrial Matrix

[0086] like Figure 3 As shown, the specific steps include:

[0087] (1) Construction of cox-M.EcoGII and M.EcoGII-NES plasmids

[0088] The plasmids “pcDNA3.1-COX-M.EcoGII” and “pcDNA3.1-M.EcoGII-NES” were constructed using Gibson recombination seamless cloning. The recombination products were transformed into E. coli competent cells top10, single colonies were selected for culture, and the plasmids were extracted for sequencing verification to ensure that the inserted nucleic acid sequence was consistent with the designed sequence.

[0089] (2) Transfection of recombinant plasmids

[0090] Recombinant plasmid transfection is the same as in Example 1.

[0091] (3) Extraction of Total RNA

[0092] Total RNA extraction was the same as in Example 1.

[0093] (4) Remove rRNA

[0094] rRNA removal is the same as in Example 1.

[0095] (5) MeRIP library construction using mixed samples

[0096] a) RNA fragmentation

[0097] Configure the fragmentation system as shown in Table 5 below;

[0098] Table 5

[0099]

[0100] After incubating the mixture at 94°C for 4 min, quickly place it on ice. Once the liquid has cooled, add 2 μl of 10×frag stop buffer to terminate the reaction, and then purify it using the Zymo RNA-clean kit.

[0101] b) End-of-pipe repair

[0102] Take the fragmented RNA and prepare the end repair system shown in Table 6 below;

[0103] Table 6

[0104]

[0105] The above system was placed at 37°C and reacted for 1 hour.

[0106] c)3' - Connector Connection

[0107] Take the fragmented RNA and prepare the end repair system shown in Table 7 below;

[0108] Table 7

[0109]

[0110] The above system was incubated at 25°C for 2 hours, and then at 4°C overnight.

[0111] After the connection is completed, the excess connectors are digested, and the products are shown in Table 8 below:

[0112] Table 8

[0113]

[0114] Incubate at 30℃ for 30 minutes; at 37℃ for 30 minutes; at 70℃ for 5 minutes.

[0115] After the reaction is complete, all reaction products are combined into one sample tube.

[0116] The merged products were purified using the Zymo RNA-clean kit, and the purified products were eluted with 22 μL of ultrapure water. The concentration of the purified products was determined using a Qubit fluorometer.

[0117] d) First round of IP

[0118] i. Add 2 μL of spike-in to the purified ligation product, mix well, and retain 2 μL of the sample as input. Add 7 μL of ultrapure water.

[0119] ii. Prepare the magnetic bead mixture as shown in Table 9;

[0120] Table 9

[0121]

[0122] The above magnetic bead mixture was placed at 4°C and incubated by rotation for 50 minutes.

[0123] iii. Place the sample in a 70°C heat shock for 2 minutes, then quickly transfer it to ice to cool for more than 2 minutes.

[0124] iv. Wash the magnetic beads twice with 200 μL of reaction buffer, rotating for 5 minutes each time at 4°C. Finally, resuspend the magnetic beads with 250 μL of reaction buffer.

[0125] v. As shown in Table 10, the resuspended magnetic beads were mixed with 20 μL of sample and 5 μL of RRI. The mixture was then incubated at 4°C for 2 hours by rotation.

[0126] Table 10

[0127] vi.

[0128] vii. Wash twice each with reaction buffer, low-salt buffer, and high-salt buffer, 200 μL each time, and rotate at 4°C for 5 minutes.

[0129] viii. Elute the sample using Buffer RLT (QIAGEN, #79216), 50 μL each time, twice.

[0130] ix. Combine the elution products and purify them using the Zymo RNA clean kit, eluting with 21 μL of ultrapure water.

[0131] e) Second round of IP

[0132] i. Prepare the magnetic bead mixture shown in Table 11 below:

[0133] Table 11

[0134]

[0135] The above magnetic bead mixture was placed at 4°C and incubated by rotation for 45 minutes.

[0136] ii. Place the sample in a 70°C heat shock for 2 minutes, then quickly transfer it to ice to cool for more than 2 minutes.

[0137] iii. Wash the magnetic beads twice with 200 μL of reaction buffer, rotating for 5 minutes each time at 4°C. Finally, resuspend the magnetic beads with 250 μL of reaction buffer.

[0138] iv. As shown in Table 12, the resuspended magnetic beads were mixed with 20 μL of sample and 5 μL of RRI. Incubated at 4°C for 2 hours by rotation.

[0139] Table 12

[0140] v.

[0141] vi. Wash twice each with reaction buffer, low-salt buffer, and high-salt buffer, 200 μL each time, and rotate at 4°C for 5 minutes.

[0142] vii. Elute the sample with Buffer RLT (QIAGEN, #79216), 50 μL each time, twice.

[0143] viii. Combine the elution products and purify them using the Zymo RNA clean kit, eluting with 9 μL of ultrapure water.

[0144] ix. Take 1 μL of purified IP sample and 1 μL of input sample for qPCR verification, and use the remaining sample for subsequent 5′ adapter ligation reaction.

[0145] f)5'-Connector Connection

[0146] i. Joint pretreatment

[0147] Place the connector at 70°C for 3 minutes and immediately transfer it to ice to cool.

[0148] ii. Connection system configuration, as shown in Table 13:

[0149] Table 13

[0150]

[0151] The mixture was placed at 25°C and reacted for 2 hours.

[0152] g) Reverse transcription

[0153] The reverse transcription system is shown in Table 14 below:

[0154] Table 14

[0155]

[0156] React at 50℃ for 1 hour; react at 85℃ for 1 minute; after the reaction is complete, store the sample at 4℃ for later use.

[0157] h) Expanding the warehouse

[0158] The reaction system for PCR amplification is shown in Table 15 below:

[0159] Table 15

[0160]

[0161] The reaction conditions for PCR amplification are shown in Table 16 below:

[0162] Table 16

[0163]

[0164] After the reaction was completed, the DNA was purified using 0.9×VAHTS DNA clean beads, yielding 30 μL of purified IP and Input libraries. After the library concentration (greater than 4 ng / μL) and fragment size (200-500 bp) were found to be within acceptable limits, the libraries were sent to Mingma Biotechnology Co., Ltd. for sequencing. High-throughput sequencing was performed on the NovaSeq X Plus sequencing platform.

[0165] (6) Data Analysis

[0166] Download the sequencing results and perform data analysis. First, use FastQ-Multx to split the data according to the correspondence between barcodes and samples. Then, use FastQC to perform quality control on the sequencing data, use Cutadapt to remove adapters, and use FastP to remove index and barcode sequences that may affect alignment, obtaining clean data. Use Hisat2 to align the processed data to the reference genome, and use samtools to process it to obtain BAM files. Use featureCounts to count samples, obtain the count matrix of each gene in each sample, and calculate the FPKM value. Perform differential methylation analysis on the COX-M.EcoGII and M.EcoGII-NES groups to identify methylated enriched genes in the COX-M.EcoGII group, which are RNAs that may be present in the mitochondrial matrix.

[0167] Current research indicates that mitochondrial genome-encoded RNA is primarily located in the mitochondrial matrix, and most studies on subcellular spatial transcriptomics technologies use this distribution pattern as the basis for evaluating the feasibility of their methods. In this example, our analysis of the sequencing results of COX-M. EcoGII samples and the control M. EcoGII-NES samples revealed that, as Figure 4 As shown, the m6A methylation signal on 13 mitochondrial-encoded mRNAs in the COX-M.EcoGII sample was significantly higher than that in the control group, while most other nuclear-encoded genes did not show this enrichment, which is consistent with the current research results.

[0168] Example 3: Labeling and detecting RFBOX2 binding sites

[0169] like Figure 3 As shown, the specific steps include:

[0170] (1) Construction of RBFOX2-M.EcoGII and M.EcoGII lentiviral plasmids

[0171] The plasmids “Tetone-RBFOX2-M.EcoGII” and “Tetone-M.EcoGII” were constructed using Gibson recombination seamless cloning. The recombination products were transformed into E. coli competent cells top10, single colonies were picked and cultured, and the plasmids were extracted for sequencing verification to ensure that the inserted nucleic acid sequence was consistent with the designed sequence.

[0172] (2) Construction of stable cell lines

[0173] a) Cell culture

[0174] The frozen 293T cells (derived from human embryonic kidney cells, purchased from the Cell Bank of the Chinese Academy of Sciences) were removed from the liquid nitrogen tank, thawed, and cultured in a 37°C, 5% CO2 incubator. They were passaged when the cell confluence reached 70-80%.

[0175] b) Cell transfection

[0176] i. For each dish of cells, take 100 μL of Opti-MEM into an EP tube. Then, add 0.58 μg of PMD2.G plasmid, 1.16 μg of psPAX2 plasmid, and 1.7 μg of the corresponding M.EcoGII and RBFOX2-M.EcoGII lentiviral plasmids to each EP tube in sequence, and mix thoroughly to obtain the "plasmid dilution buffer".

[0177] ii. For each dish of cells, take another 100 μL of Opti-MEM into an EP tube, add 7 μL of transfection reagent Lipo2000 and mix thoroughly to make the "transfection dilution".

[0178] iii. Add the "transfection dilution buffer" to the "plasmid dilution buffer", mix quickly, and let stand at room temperature for 20 minutes to form a transfection complex; gently and evenly add the transfection complex to the cell culture dish;

[0179] iv. Place the culture dish in an incubator at 37°C and 5% CO2, and change the medium to complete culture medium 12 hours after transfection;

[0180] v. 48 h after transfection, collect the virus solution from each dish, centrifuge at 500×g for 5 min, take the supernatant and pass it through 0.22 μm to collect the filtered virus solution;

[0181] vi. Pre-coat 293T cells for infection;

[0182] vii. Prepare a complete culture medium containing 16ug / ml polybrene. Take 2mL of filtered virus solution and mix it with 2mL of complete culture medium containing 16ug / ml polybrene. Add 4mL of virus solution to the culture dish to infect the cells.

[0183] viii. Change the medium to complete culture 24 hours after infection. When the confluence reaches 70-80%, passage the cells and add the corresponding antibiotic for selection. After one week of selection, a stable cell line is obtained.

[0184] ix. Add 1 μg / ml doxycycline to induce exogenous protein expression for 48 h.

[0185] (3) Extraction of Total RNA

[0186] Total RNA extraction was the same as in Example 1.

[0187] (4) Remove rRNA

[0188] rRNA removal is the same as in Example 1.

[0189] (5) MeRIP library construction using mixed samples

[0190] Mixed-sample MeRIP library preparation and sequencing are the same as in Example 2.

[0191] (6) Data Analysis

[0192] The process of preprocessing the data to obtain clean data and aligning it to the reference genome is the same as in Example 2. After obtaining the bam file, peak calling was performed on the data using MACS2 to identify the m6A site, and bedtools was used to identify peaks in the RFBOX2-M.EcoGII group that did not overlap with the control M.EcoGII group. These peaks were identified as method-labeled RFBOX2 binding sites, and then homer2 was used to perform de novo motif discovery on sequences near the RFBOX2 binding sites.

[0193] Current research indicates that RBFOX2 binding sites on mature RNA are mainly located at the 3utr region, and these binding sites exhibit a distinct UGCAUG motif. In this embodiment, data analysis of RBFOX2-M.EcoGII and the control M.EcoGII shows that, as... Figure 5 As shown, the RBFOX2 binding sites identified in this invention are enriched at 3utr, and as... Figure 6 As shown, the sequences near the binding site are enriched with the UGCAUG motif, consistent with current research.

[0194] Example 4: Labeling and Detection of Intracellular Translational Events

[0195] like Figure 3 As shown, the specific steps include:

[0196] (1) Construction of RPS2-M.EcoGII and M.EcoGII-NES lentiviral plasmids

[0197] The plasmids “Tetone-RPS2-RBFOX2” and “Tetone-M.EcoGII-NES” were constructed using Gibson recombination seamless cloning. The recombination products were transformed into the top 10 competent cells of E. coli, and single colonies were selected for culture. The plasmids were then extracted and sequenced to verify that the inserted nucleic acid sequence was consistent with the designed sequence.

[0198] (2) Construction of stable cell lines

[0199] The construction of stable cell lines is the same as in Example 3.

[0200] (3) Extraction of Total RNA

[0201] Total RNA extraction was the same as in Example 1.

[0202] (4) Remove rRNA

[0203] rRNA removal is the same as in Example 1.

[0204] (5) MeRIP library construction using mixed samples

[0205] Mixed-sample MeRIP library preparation and sequencing are the same as in Example 2.

[0206] (6) Data Analysis

[0207] The process of preprocessing the data to obtain clean data and aligning it to the reference genome is the same as in Example 2. After obtaining the BAM file, featureCounts is used to count the IP samples and input samples. The count of each gene in each IP sample divided by the total count and gene length of the corresponding input sample is called the mTotal value. The mTotal value of RPS2-M.EcoGII sample minus the mTotal value of M.EcoGII-NES can be used as a measure of gene translation activity. The count ratio of each gene in the IP sample to the corresponding input sample is called the mScore value. The mScore value of RPS2-M.EcoGII sample minus the mScore value of M.EcoGII-NES can be used as a measure of gene translation efficiency.

[0208] Currently, among methods for analyzing translation events using sequencing, Riboseq is the gold standard method, such as... Figure 7 As shown, the mTotal value obtained by this method has a good correlation with riboseq, indicating that this method can effectively capture translation activity.

[0209] The embodiments of the present invention are merely examples to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent structural transformations made under the guidance of the technical concept of the present invention and based on the content disclosed in the present invention specification, or any direct or indirect application of the present invention to other related technical fields, should be considered to fall within the scope of protection of the present invention.

Claims

1. A method for screening RNA-modifying enzyme markers and detecting intracellular RNA molecular events, characterized in that, The RNA-modifying enzyme does not alter the RNA molecule sequence and includes the following steps: S1. Synthesize a gene fragment containing a candidate modifying enzyme sequence and construct a plasmid for fusion expression of an RNA-binding protein and the candidate modifying enzyme; the RNA-binding protein includes the ribosomal small subunit protein component RPS2, which can enhance the RNA-binding ability of the modifying enzyme. S2. Transfect the plasmid into 293T cells to express the fusion protein in the cells; S3. Harvest cells. After extracting total RNA from the cells, remove rRNA or extract mRNA from the total RNA. Then, detect the modifications on the RNA using LC-MS. The LC-MS detection uses a parallel reaction monitoring method. S4. After obtaining highly active candidate modifying enzymes, the modifying enzymes are fused with RNA-binding proteins of interest for expression, and RNA-binding protein binding sites are marked in cells; fused with ribosomal proteins for expression, and ribosomal translation events are marked in cells; fused with specific subcellular location signals for expression, and RNA molecule subcellular localization events are marked in cells; the RNA molecule events are RNA-binding protein binding events, ribosomal translation events, and RNA molecule subcellular localization events. The highly active candidate modifying enzymes include m6A modifying enzymes M.EcoGII and SUPREM, m5C modifying enzyme rlmI, ac4C modifying enzyme TK0754, and D modifying enzyme Dus2. S5. Harvest the labeled cells, extract RNA, and use high-throughput sequencing to detect the modifications of the labeled RNA and perform data analysis to resolve the corresponding RNA molecular events.

2. The method for screening RNA-modifying enzyme markers and detecting intracellular RNA molecular events according to claim 1, characterized in that, The high-throughput sequencing method in S5 includes the use of next-generation sequencing and nanopore direct RNA sequencing technology to detect modifications.

3. The method for screening RNA-modifying enzyme markers and detecting intracellular RNA molecular events according to claim 2, characterized in that, The data analysis in S5 involves the alignment of sequencing data, the identification and quantification of modification sites, and the identification of corresponding molecular events through differential modification analysis between the experimental and control groups.

Images