Method for analyzing single ribosome and double ribosome maps of drosophila melanogaster

Abstract

The invention belongs to the technical field of high-throughput sequencing, and particularly relates to a method for analyzing single ribosome and double ribosome maps of drosophila melanogaster. According to the method, P1 nuclease is used for carrying out ribosome atlas analysis on a drosophila sample, in consideration of particularity of ribosome rRNA of the drosophila, a translation inhibitor actinomycete ketone is added into a drosophila sample lysate and a sucrose density gradient solution, and the actinomycete ketone can inhibit extension of ribosome on mRNA and play a role in fixing the ribosome, so that the ribosome can be fixed in the lysate; finally, the ribosome footprint is successfully reserved. In addition, RNA enzyme or protease inhibitors are not added into the lysate and the sucrose density gradient solution, so that the experiment cost can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of high-throughput sequencing technology, specifically relating to a method for analyzing the monoribosomes and diribosomes of Drosophila. Background Technology

[0002] Protein translation is a key step in gene expression regulation, and its efficiency and accuracy directly determine the level of protein synthesis and cellular functional state. Therefore, in-depth analysis of the translation process is crucial for understanding biological processes such as gene function, cell fate determination, and stress responses. Ribosome profiling, invented in 2009, has been continuously optimized and improved, enabling the capture of ribosome distribution characteristics on mRNA at nucleotide resolution, revealing multi-layered mechanisms of translation regulation (DOI: 10.1126 / science.1168978). The traditional ribosome profiling process is as follows: Figure 1 As shown, this technique typically involves cell lysis, RNase digestion of unprotected ribosome regions, and centrifugation using a sucrose density gradient to separate the ribosome-mRNA complex. Subsequent purification of ribosome-protected mRNA fragments allows for library construction and sequencing. Compared to traditional transcriptome sequencing, ribosome mapping provides dynamic information at the translational level, revealing key aspects such as post-transcriptional regulation, translation initiation site usage, new open reading frames (ORFs), and translation efficiency. It has become an important tool for studying gene expression regulation mechanisms (DOI: 10.1016 / j.ymeth.2017.05.028).

[0003] Yeast, fruit fly, and human cell lines are common models for analyzing translation dynamics and regulatory mechanisms. In ribosome mapping analysis in yeast and humans, RNase I is commonly used to cleave mRNA, preserving the ribosome footprint; however, as... Figure 2As shown, Drosophila ribosomal RNA (rRNA) is abnormally sensitive to RNase I treatment and cannot retain a complete ribosomal footprint, which may be related to the abnormal sequence and structure of Drosophila rRNA. A "cryptic cleft" exists on Drosophila 28S ribosomal RNA, which leads to structural instability of Drosophila ribosomes during enzyme digestion (DOI: 10.1673 / 031.010.14119). This characteristic also poses a significant challenge to ribosomal mapping analysis in Drosophila. In 2013, a study on ribosome mapping analysis in Drosophila was published (DOI: 10.7554 / eLife.01179). This study successfully enriched single ribosome footprints by cleaving Drosophila mRNA with the nuclease MNase. However, sequencing results of the single ribosome library showed that MNase cleavage exhibited a strong 3' A / T base bias, leading to uncertainty in the location of some MNase-cleaved ribosome P sites. The sequencing data also showed poor tribasicity, failing to achieve the subcodon-level resolution of ribosome location information generated by RNase I cleavage. Furthermore, MNase cleavage requires the addition of RNase inhibitors and protease inhibitors to the lysis buffer and sucrose density gradient solution, resulting in high costs. Figure 2 (As shown).

[0004] In 2023, research on ribosome profiling using the P1 endonuclease was developed. Compared with commonly used RNase I and MNase, P1 cleavage exhibits no base bias, produces less rRNA contamination than RNase I, and preserves intact monoribosome and diribosome footprints. This suggests that P1 can also be used to study ribosome arrest and collision during protein translation in yeast and humans (DOI: 10.1038 / s41592-023-02028-1). However, a stable and cost-effective method for ribosome profiling in Drosophila is still lacking.

[0005] Ribosome profiling can detect translational activity at high resolution across the entire genome, revealing the precise location of ribosomes and directly providing information on the dynamics of protein translation. Drosophila, a common model for protein translation research, is unsuitable for RNase I digestion in traditional ribosomal RNA studies due to the unique sequence and structure of its ribosomal RNA. Although recent literature has addressed this digestion issue to some extent, certain limitations and shortcomings remain.

[0006] (1) The 2013 literature (DOI: 10.7554 / eLife.01179) successfully preserved the single ribosome footprint for the first time using MNase. However, MNase cleavage has a strong A / T base bias and poor tri-base periodicity, so the experimental data cannot accurately reflect the actual intracellular translation situation.

[0007] (2) When using MNase to perform ribosome profiling experiments on fruit flies, although it can preserve the monoribosome footprint well, there is a lot of rRNA contamination generated during the enzyme digestion process.

[0008] (3) Traditional Ribosome profiling experiments require the addition of reagents such as RNase inhibitors, protease inhibitors, and translation inhibitors to the lysis buffer and ribose density gradient solution, which is costly. Summary of the Invention

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0010] This invention discloses a method for analyzing the monoribosomes and diribosomes of Drosophila, the method comprising the following steps:

[0011] (1) Sample preparation

[0012] Preparation of stock solution for Drosophila tissue samples:

[0013] After mixing the fruit fly tissue with the tissue lysis buffer, centrifugation was performed to obtain the original fruit fly tissue sample solution. The components of the tissue lysis buffer were: 20 mM Tris-Cl, 150 mM KCl, 5 mM MgCl2, 1% Triton-X100, 1 mM DTT, 25 U / mL TurboDNase I, and 100 ug / mL CHX.

[0014] Preparation of Drosophila cell sample stock solution:

[0015] Drosophila suspension cells and adherent cells were lysed using a cell lysis buffer, the composition of which is as follows:

[0016] 20mM Tris-Cl, 150mM KCl, 5mM MgCl2, 1% Triton-X100, 1mM DTT, 25U / mL TurboDNase I, 100ug / mL CHX;

[0017] (2) P1 nuclease digestion of Drosophila tissue sample original solution

[0018] The original solutions of Drosophila tissue samples and Drosophila cell samples were obtained by enzymatic digestion step (1) using P1 nuclease at 27℃-30℃.

[0019] (3) Preparation of linear sucrose density gradient solution

[0020] Prepare a sucrose density gradient solution, wherein the components of the sucrose density gradient solution are: 20 mM HEPES-KOH, 150 mM KCl, 5 mM MgCl2, Sucrose, 1 mM DTT, and 100 ug / mL CHX;

[0021] (4) Separation of monoribosomes or diribosomes

[0022] After adding the enzyme-digested Drosophila tissue sample stock solution and Drosophila cell sample stock solution to the sucrose density gradient solution of step (3), centrifuge to separate monoribosomes or diribosomes;

[0023] (5) Release of ribosomal mRNA protective fragments

[0024] The ribosomes or diribosomes were treated with SDS solution and proteinase K, then extracted with acid-phenol-form and precipitated with anhydrous ethanol to obtain the protected mRNA fragment.

[0025] (6) Sequencing analysis after isolating ribosome-protected mRNA fragments.

[0026] Ribosome-protected mRNA fragments were separated using TBU polyacrylamide gel electrophoresis.

[0027] Preferably, in step (1), the mass-to-volume ratio of fruit fly tissue to tissue lysis fluid is 1:3; the centrifugation conditions are 14000 rcf, 4℃ for 10 min.

[0028] Preferably, in step (2), the original solutions of Drosophila tissue samples and Drosophila cell samples are adjusted to pH 6.5 with 300mM Bis-Tris solution of pH=6.0, and P1 nuclease is added at 1.5U / ug RNA. The digestion is carried out at 27℃-30℃ for 1h. After digestion, the digestion is stopped by rapidly cooling down.

[0029] Preferably, in step (3), the linear sucrose density gradient solution is a 10%-50% linear sucrose density gradient solution.

[0030] Preferably, the centrifugation conditions in step (4) are 35,000 rpm, 4°C, and 3 h.

[0031] Preferably, the mass fraction of the SDS solution in step (5) is 10%, and the reaction is carried out at 42°C for 30 min.

[0032] Preferably, in step (6), electrophoresis is performed at 200V for 90 min using a 15% TBU polyacrylamide gel. After staining, a gel block containing a 30-40 nt fragment of monoribosomes and a gel block containing a 45-80 nt fragment of diribosomes are selected. RNA Elution Buffer and SUPERase In are added to the gel block, and the gel block is rotated at room temperature for at least 7-8 h to precipitate the mRNA from the gel block. The mRNA is then precipitated with ethanol to obtain a purified ribosomal mRNA protected fragment for sequencing analysis.

[0033] The beneficial effects of this invention are:

[0034] (1) Adding 100ug / ml CHX to the lysis buffer and sucrose density gradient solution prepared for the sample improved the stability of the ribosome-mRNA complex and solved the problem of abnormal sensitivity of Drosophila rRNA to nuclease digestion.

[0035] (2) Using P1 nuclease to perform ribosome profiling experiments in Drosophila eliminated the 3' A / T base bias of MNase digestion and improved the quality of sequencing data;

[0036] (3) No protease or RNase inhibitors are added to the experimental reagents, which is lower in cost compared with traditional Ribosome profiling. Attached Figure Description

[0037] Figure 1 Traditional ribosome profiling process (DOI:10.1101 / cshperspect.a032698);

[0038] Figure 2 Separation map of sucrose density gradient in Drosophila cells treated with RNase I (DOI: 10.7554 / eLife.01179);

[0039] Figure 3 Original CMt sample and ribosome curves after P1 digestion;

[0040] Figure 4 Original sample S125 and ribosome curves after P1 digestion;

[0041] Figure 5 CMt document library segment comparison chart;

[0042] Figure 6 Length distribution of WA30Mt single ribosomal library;

[0043] Figure 7 CHXMt single ribosomal library length distribution;

[0044] Figure 8 CHXDt dual-ribosomal library length distribution

[0045] Figure 9 Base configuration of the MHribo1 restriction site;

[0046] Figure 10 Base information of the IH0_3M restriction site. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0048] This invention uses P1 nuclease to perform ribosome profiling on Drosophila samples. Considering the unique characteristics of Drosophila ribosomal rRNA, the translation inhibitor cycloheximide (CHX) is added to the Drosophila sample lysis buffer and sucrose density gradient solution. CHX can inhibit the elongation of ribosomes on mRNA, thus immobilizing the ribosomes and successfully preserving the ribosomal footprint. Furthermore, no RNase or protease inhibitors are added to the lysis buffer or sucrose density gradient solution, reducing experimental costs. During the separation and purification of the ribosomal footprint, high-resolution denaturing gels are used to separate the corresponding ribosomal footprints. For monoribosomes, bands at 30-40 nt are selected, and for diribosomes, bands at 45-80 nt are selected. During gel excision, the removal of rRNA bands is minimized, reducing contamination in the library.

[0049] The experimental steps are as follows:

[0050] I. Sample Preparation

[0051] (1) Preparation of original solution of Drosophila tissue sample

[0052] Prepare fresh tissue lysis buffer with the following components: 20 mM Tris-Cl, 150 mM KCl, 5 mM MgCl2, 1% Triton-X100, 1 mM DTT, 25 U / mL Turbo DNase I, and 100 ug / mL CHX.

[0053] Grind the Drosophila tissue into powder in a mortar; transfer the powder to a glass homogenizer, add 200 μL to 1 ml of fresh tissue lysis buffer (powder to fresh lysis buffer volume ratio of 1:3), and homogenize thoroughly. Centrifuge the homogenized lysis buffer at 14000 rcf, 4°C for 10 min, and collect the middle layer liquid to obtain the original Drosophila tissue sample solution for enzyme digestion, which can be stored at -80°C.

[0054] (2) Preparation of original Drosophila cell sample solution (S2 cells, semi-adherent and semi-suspended cells)

[0055] Prepare fresh cell lysis buffer with the same composition as tissue lysis buffer;

[0056] 1. For suspension cells: collect suspension cells by centrifugation, resuspend cells in 1xPBS, centrifuge at 2000g, 4℃ for 3min, and discard the PBS; for adherent cells: rinse with 1xPBS and discard the PBS.

[0057] 2. Resuspend the suspended cells in the centrifuged pellet with cell lysis buffer, inject the resuspended cells into the adherent cells, collect all cells with a cell scraper, and continuously process 400 μL of cells in 4 10 cm culture dishes. Lyse on ice for 30 min.

[0058] 3. Centrifuge at 14000 rcf at 4℃ for 10 min, and take the middle layer liquid to obtain the cell stock solution for enzyme digestion, which can be stored at -80℃;

[0059] II. Concentration Measurement and P1 Nuclease Digestion Treatment

[0060] 1. Use Qubit to measure the concentration of the original solution of Drosophila tissue sample or Drosophila cell sample. Take a portion of the original solution of Drosophila tissue sample or Drosophila cell sample as the original sample. The original sample does not need to be digested with nuclease.

[0061] 2. Take 150-300 μL of the stock solution and adjust the pH to 6.5 with Bis-Tris (300 mM, pH = 6.0). Add P1 nuclease at 1.5 U / μg RNA and digest at 27℃-30℃ for 1 hour. After digestion, quickly cool down to stop the enzyme digestion.

[0062] III. Preparation of 10%–50% linear sucrose density gradient solution

[0063] 1. Prepare 10% and 50% (mass-volume ratio) sucrose density gradient solutions in advance. The composition of 50ml sucrose density gradient solution is: 20mM HEPES-KOH, 150mM KCl, 5mM MgCl2, 5g / 25g Sucrose, 1mM DTT, and 100ug / mL CHX.

[0064] 2. Take a clean 15ml centrifuge tube, use a syringe to draw 8ml of 10% sucrose density gradient solution and add it to the centrifuge tube, then take 8ml of 50% sucrose density gradient solution, insert it into the bottom of the centrifuge tube and add it to the centrifuge tube, so that the concentration boundary line coincides with the streak line, and then put on the cap;

[0065] 3. Prepare 10%-50% linear density gradients using the BioComp fully automated density gradient preparation and separation system (model: 108-T & 152-T).

[0066] IV. Ultracentrifugation for the separation of different ribosome components

[0067] 1. Pre-cool the ultra-high-speed refrigerated centrifuge to 4°C;

[0068] 2. Load the enzyme-digested sample or the original sample onto the top of a 10%–50% linear sucrose density gradient solution and balance it; hang the balanced centrifuge tube on the rotor and incubate at 35,000 rpm, 4°C, for 3 hours.

[0069] V. Gradient Profiler Detection of Sample Separation

[0070] 1. After ultracentrifugation, the separation of ribosomes in the sample was detected using the BioComp fully automated density gradient preparation and separation system;

[0071] 2. After clicking SCAN on the red detection instrument, wait 4 seconds, open the Flowcell software on your computer, set the parameters, and calibrate:

[0072] (1)Number of fractions: 80;

[0073] (3) Perform calibration and zeroing;

[0074] (4) Adjust the OFF value to 60-70 so that the LED1 (260nm) value is in the range of 800-900k.

[0075] 3. Begin detection. Collect samples based on the absorption peaks and times of monoribosomes or diribosomes. Store samples at -80℃ and save the data.

[0076] VI. Release of ribosome protective fragments

[0077] 1. Aliquot the recovered monoribosomes or diribosomes into 534ul tubes, add 60ul of 10% SDS and 6ul of proteinase K, and react at 42℃ for 30min;

[0078] 2. Extract with acid-phenol-formaldehyde, precipitate with anhydrous ethanol, and let stand at -80℃ for more than 3 hours.

[0079] VII. 15% TBU Gel Separates RPFs

[0080] Dissolve RNA in 1.10 μL of ultrapure water for 7 min.

[0081] 2. Prepare 15% TBU polyacrylamide gel and pre-run at 180V for 15 minutes.

[0082] 3. Sample preparation: After the precipitate dissolves, add an equal volume of 2×gel loading buffer; take 0.5ul of Lowrange ssRNA marker, add 9.5ul of UP water, and then add 10ul of 2×gel loading buffer; take 1ul of 30nt+40nt mixed marker, add 5ul of water, and then add 10ul of 2×gel loading buffer.

[0083] 4. Incubate RNA and marker in a 95°C metal bath for 2–3 min, then immediately place on ice for 2–3 min; add samples at 200°C for 90 min.

[0084] 5. After staining, cut off the appropriate length with a disposable blade. For monoribosomes, select a fragment at 30-40 nt; for diribosomes, select a fragment at 45-80 nt.

[0085] 6. Break up the recovered gel block and add 400ul RNA Elution Buffer and 1ul SUPERase In. Place it on a gyroscope and rotate at room temperature for at least 7-8 hours to allow the RNA to precipitate from the gel.

[0086] 7. Ethanol precipitation to obtain purified ribosome-protected mRNA fragments: precipitate with anhydrous ethanol and let stand at -80℃ for more than 3 hours.

[0087] 8. The purified ribosome-protected mRNA fragments can be used for library construction and then subjected to next-generation high-throughput sequencing.

[0088] The results showed that, taking MHribo1 (sample digested with MNase), WA30Mt (sample digested with P1 nuclease without CHX), and four Drosophila tissue samples (CMt, CHXMt, CHXDt, IH0_3M) and cell sample S125 digested using this method as examples, the sucrose density gradient separation curves of the original solutions and P1 digested solutions of CMt and S125 samples are shown in the figures below. Figure 3 , 4 As shown, the document segment comparison results are as follows: Figure 5 As shown; the length distribution of the WA30Mt footprint is as follows Figure 6 As shown; the length distributions of the CHXMt single-ribosomal library and the CHXDt double-ribosomal library are as follows. Figure 7 ,8 As shown; the base composition of the restriction enzyme sites in the MHribo1 and IH0_3M libraries is as follows. Figure 9 , 10 As shown in the figure, compared with samples digested with MNase and those digested with P1 without CHX, the samples obtained by this method for Ribosome profiling showed better results. The length distribution and read alignment of the library were consistent with those of high-quality ribo-seq libraries, and there was no base bias in the digestion. This indicates that this method can better preserve the single and double ribosome footprints, more accurately reflect the position of ribosomes on mRNA, reveal translation dynamics, and reduce experimental costs.

Claims

1. A method for analyzing the monoribosomes and diribosomes of Drosophila, characterized in that, The method includes the following steps: (1) Sample preparation Preparation of stock solution for Drosophila tissue samples: After mixing the Drosophila tissue with the tissue lysis buffer, centrifugation was performed to obtain the original Drosophila tissue sample solution. The components of the tissue lysis buffer were: 20 mM Tris-Cl, 150 mM KCl, 5 mM MgCl2, 1% Triton-X100, 1 mM DTT, 25 U / mL Turbo DNase I, and 100 ug / mL CHX. Preparation of Drosophila cell sample stock solution: Drosophila suspension cells and adherent cells were lysed using a cell lysis buffer, the composition of which is as follows: 20mM Tris-Cl, 150mM KCl, 5mM MgCl2, 1% Triton-X100, 1mM DTT, 25U / mL Turbo DNaseI, 100ug / mL CHX; (2) P1 nuclease digestion of Drosophila tissue sample original solution and Drosophila cell sample original solution The original solutions of Drosophila tissue samples and Drosophila cell samples obtained by enzymatic digestion step (1) using P1 nuclease at a temperature of 27℃-30℃; (3) Preparation of linear sucrose density gradient solution Prepare a sucrose density gradient solution, wherein the components of the sucrose density gradient solution are: 20 mM HEPES-KOH, 150 mM KCl, 5 mM MgCl2, Sucrose, 1 mM DTT, and 100 ug / mL CHX; (4) Separation of monoribosomes or diribosomes After adding the enzyme-digested Drosophila tissue sample stock solution and Drosophila cell sample stock solution to the sucrose density gradient solution of step (3), centrifuge to separate monoribosomes or diribosomes; (5) Release of ribosomal mRNA protective fragments The ribosomes or diribosomes were treated with SDS solution and proteinase K, then extracted with acid phenolform, and finally precipitated with anhydrous ethanol to obtain the protected mRNA fragment. (6) Sequencing analysis after isolating ribosome-protected mRNA fragments. Ribosome-protected mRNA fragments were separated using TBU polyacrylamide gel electrophoresis.

2. The method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of the fruit fly tissue to the tissue lysis solution is 1:3; the centrifugation conditions are 14000 rcf, 4℃ for 10 min.

3. The method according to claim 1, characterized in that, In step (2), the original solutions of the Drosophila tissue sample and the original solutions of the Drosophila cell sample were adjusted to pH 6.5 with 300mM Bis-Tris solution of pH=6.

0. P1 nuclease was added at 1.5U / ug RNA and digested at 27℃-30℃ for 1h. After digestion, the enzyme digestion was terminated by rapidly cooling down.

4. The method according to claim 1, characterized in that, The linear sucrose density gradient solution mentioned in step (3) is a 10%-50% linear sucrose density gradient solution.

5. The method according to claim 1, characterized in that, The centrifugation conditions in step (4) are 35,000 rpm, 4°C, and 3 h.

6. The method according to claim 1, characterized in that, In step (5), the mass fraction of the SDS solution is 10%, and the treatment conditions are: reaction at 42°C for 30 min.

7. The method according to claim 1, characterized in that, In step (6), electrophoresis was performed at 200V for 90 min using a 15% TBU polyacrylamide gel. After staining, a gel block containing a fragment of 30-40 nt for monoribosomes and a gel block containing a fragment of 45-80 nt for diribosomes was selected. RNA Elution Buffer and SUPERase In were added to the gel blocks, and the gel blocks were rotated at room temperature for at least 7-8 h to precipitate the mRNA from the gel blocks. The mRNA was then precipitated with ethanol to obtain purified ribosomal mRNA protected fragments for sequencing analysis.

Images