RNA co-immunoprecipitation-based RNA binding protein aggregate molecular function detection method

Abstract

The invention relates to an RNA (Ribonucleic Acid) co-immunoprecipitation-based RNA binding protein aggregate molecular function detection method, which comprises the following steps: S1) RNA co-immunoprecipitation: firstly, splitting tissues or cells, then cleaning by using magnetic beads to remove a splitting solution, and then preparing a detection sample; s2) protein and RNA detection: firstly carrying out semi-denaturation gel electrophoresis on the sample, confirming the IP efficiency of the sample, and then recovering RNA on the membrane; s3) releasing RNA from the membrane: incubating by adopting a prepared PK buffer solution to perform protein digestion so as to extract RNA, measuring the concentration of RNA, and performing reverse transcription to obtain RNA formed by combining a DAZL polymer and a DAZL monomer; s4) performing qPCR (quantitative polymerase chain reaction) detection on a target target: performing function detection, and verifying whether RNA (ribonucleic acid) exists in the aggregate formed by combining the DAZL polymer and the monomer. The method is simple, efficient and accurate, and provides a more convenient platform for functional research of the polyamyloid protein.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to a method for detecting the function of RNA-binding protein aggregates based on RNA immunoprecipitation. Background Technology

[0002] Protein phase separation is currently a subject of widespread interest. Amyloid aggregates have long been considered pathological protein aggregates leading to neurodegeneration. However, increasing evidence suggests that non-pathological amyloid proteins are present during normal animal development. The formation of amyloid aggregates was initially thought to be a conserved feature of animal gametogenesis. This hypothesis was based on research indicating that regulated amyloid formation controls yeast meiosis through meiosis-specific RNA-binding proteins. Current research shows that many RNA-binding proteins exist and function as aggregates in the testes and other tissues.

[0003] However, how these RNA-binding proteins, existing in aggregate form, participate in life activities remains to be studied. Numerous studies have shown that RNA-binding proteins function by binding to a large number of downstream target RNAs and regulating RNA fate. RNA immunoprecipitation is a commonly used technique for detecting RNA-protein binding. However, this experiment cannot effectively distinguish the targets bound to aggregate proteins from those bound to monomeric proteins, thus hindering the study of the life activities involved in aggregate-form proteins.

[0004] RNA-binding proteins, as a wide range of transcriptional and post-transcriptional regulators, play crucial roles in various life activities, especially gametogenesis. The formation of amyloid aggregates was initially considered a conserved feature of animal gametogenesis. To investigate the impact of this conserved feature on protein function, researchers have used the eCLIP assay to sequence and analyze RNA bound to the multimers and monomers of the germ cell-specific protein BOLL. However, the eCLIP assay requires approximately one week of experimental time and is technically complex, limiting its widespread use in laboratories with varying conditions.

[0005] Considering the need for such detection, it is necessary to propose a simple, efficient, and accurate method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation, which will provide a more convenient platform for functional studies of amyloid proteins. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation. This method requires no special instruments or reagents and can help researchers verify functional hypotheses through qPCR. It is highly efficient and accurate, and will provide a more convenient platform for functional studies of amyloid proteins.

[0007] To address the aforementioned technical issues, the present invention provides a method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation, specifically comprising the following steps: S1) RNA immunoprecipitation: First, the tissue or cells are lysed, then the lysate is removed by washing with magnetic beads, and then the test sample is prepared. S2) Protein and RNA detection: First, the sample is subjected to semi-denaturing gel electrophoresis to confirm the IP efficiency of the sample, and then the RNA on the membrane is recovered. S3) Release RNA from the membrane: Protein digestion was performed by incubation with a prepared proteinase K buffer to extract RNA, and the RNA concentration was measured. Reverse transcription was then performed to obtain RNA with DAZL multimers and monomers bound. S4) qPCR detection target: Functional detection of the obtained DAZL multimer and monomer-bound RNA to verify whether RNA exists in the aggregates of DAZL multimer and monomer-bound RNA.

[0008] Preferably, the specific steps of step S1 are as follows: S11: Select the target protein and prepare lysis buffer and high-salt buffer; S12: Dyslyce the selected tissue or cells. S13: Use magnetic beads to clean and remove the pyrolysis solution; S14: Regroup and prepare samples.

[0009] Preferably, the target protein in step S11 is selected as an RNA-binding protein capable of forming physiological state multimers; The lysis buffer formulation includes: 50 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, 150 mM sodium chloride solution, 10% glycerol, 0.5% Triton-X100, 1× protease inhibitor, and 400 units of RNase inhibitor. The high-salt buffer solution is formulated as follows: 50 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, 500 mM NaCl, 10% glycerol, and 0.5% Triton-X100.

[0010] Preferably, the specific steps of step S12 are as follows: S121: Add lysis buffer to tissues or cells at a ratio of 20:1, mix by pipetting, and sonicate at a low energy level to prevent affecting subsequent RNA detection, until the lysis buffer is free of obvious clumps. S122: Lyse on ice for 15 min, then centrifuge at 8000g for 15 min at 4℃, remove the precipitate and keep the supernatant, and keep 10% of the supernatant as the input group, i.e. denatured sample. S123: Add antibody or IgG (negative control) to the remaining supernatant from step S122, and then incubate at 4°C for 6-8 hours; S124: Take 50 μL of protein G beads (Thermo Fisher Scientific, catalog number: 10003D), i.e. magnetic beads, and wash the beads twice with 1 mL of lysis buffer; then incubate the lysis buffer supernatant with the beads overnight.

[0011] Preferably, the specific steps of step S13 are as follows: using a magnetic base to separate the beads from the lysis buffer and remove the lysis buffer; then washing the beads twice with 1 mL of high-salt buffer and twice with 1 mL of lysis buffer.

[0012] Preferably, the specific steps of step S14 are as follows: S141 Sample separation: Add 100 μL of lysis buffer to the beads and divide the beads into a Hot group (RNA recovery group) and a Cold group (electrophoresis verification group), with a ratio of 9:1 between the Hot group and the Cold group. S142 Sample Preparation Hot Group (Hot Membrane): Add 20 μL of acidic elution buffer (pH < 4), elute for 5 min, add 7 μL of neutralization buffer (pH 9.8), add 9 μL of 4x loading buffer, mix well, and remove beads. S143 Sample Preparation (Cold Group, i.e., Cold Membrane): Add 20 μL of acidic elution buffer (pH < 4), elute for 5 min, add 7 μL of neutralization buffer, add 6.75 μL of 5x loading buffer, mix well, and remove beads; take 10 μL of sample and boil at 100℃ for 5 min to obtain denatured sample. S144 Sample Preparation Input Group: Add 5x protein loading buffer to dilute to 1x, take 10μL of sample and boil for 5min to obtain denatured sample.

[0013] Preferably, the specific steps of step S2 are as follows: S21 Gel Preparation: The gel formulation includes: 2.25g of low-osmotic agarose (Beyotime, catalog number: ST004M), 3mL of 50x Tris acetate-EDTA buffer, and 147mL of double-distilled water. Mix the low-osmotic agarose and 50x Tris acetate-EDTA buffer, place in a container, seal, weigh, and microwave to boiling three times. Add double-distilled water at approximately 70℃ until the weight reaches the initial microwave weight. Microwave to boiling three times, then gently shake. Clean and dry the rotor with double-distilled water, place it in a flask, and add 0.1% sodium dodecyl sulfate (Sangon Biotech, catalog number: 151-21-3). Pour the gel into a plate and allow it to set at room temperature for 1 hour. S22 Sample loading: Add the sample to the gel well, or use sample loading electrophoresis buffer to keep it stable. The electrophoresis buffer formula is: 12 mL of 50x Tris acetate EDTA buffer, 6 mL of 0.1% sodium dodecyl sulfate, and 588 mL of double-distilled water. S23 gel electrophoresis: Place the gel in the electrophoresis tank and inject the electrophoresis buffer into the electrophoresis tank. Turn on the power and apply an electric field. The voltage should not exceed 1V / cm. Keep it at 4℃ for 10h to allow the protein to be loaded into the gel and then separated. S24 Transfer: After electrophoresis, transfer was performed. First, rinse with tap water, cut the gel in 1xTBS, and cut the membrane and filter paper according to the size of the gel. Then, place the filter paper, gel, NC membrane, filter paper, and absorbent paper in the order of bottom to top. Remove excess air bubbles. Block the membrane of the COLD group with 5% milk, incubate with primary antibody at 4°C overnight, incubate with secondary antibody at room temperature for 1 hour, expose, and determine the IP effect. Freeze the membrane of the HOT group at -80°C. After exposure, according to the position of polymer and monomer bands on the Cold membrane, cut the corresponding membrane on the HOT membrane into 1 mm2 small paper pieces for recovery. The RNA on the membrane was recovered.

[0014] Preferably, the specific steps of step S3 are as follows: S31 Preparation of Proteinase K Buffer: The proteinase K buffer formulation is as follows: 100mM tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 7.5 (Solepro, catalog number: T1140), 50mM sodium chloride solution, and 10mM ethylenediaminetetraacetic acid (Thermo Fisher Scientific, catalog number: R1021). S32 protein digestion: Add 160 μL of proteinase K buffer and 40 μL of proteinase K (Solepro, catalog number: P9460) to each tube, mix well, and incubate at 37°C for 20 min; then add 420 mg of urea (Beyotime, catalog number: ST1731) to proteinase K buffer to a final volume of 1 mL, add 200 μL to each tube, and incubate at 37°C for 20 min; S33 RNA extraction: First, incubate, then centrifuge, collect the aqueous phase above the gel, then add isopropanol and glycogen for precipitation to obtain RNA precipitate, then centrifuge, discard the supernatant, wash and centrifuge again to discard the supernatant, and then air-dry the obtained RNA. S34 Transcription: Add 20 μL of DEPC water, place on ice, dissolve RNA on ice for 5 min to prevent RNA degradation at room temperature, measure RNA concentration, and then perform reverse transcription.

[0015] Preferably, the specific steps of step S33 are as follows: S331: Add 400 μL of acidic phenol / chloroform / isoamyl alcohol to the PK buffer after protein digestion, maintain the pH at 6.5, shake well, and incubate at 37°C for 5 min; S332: Transfer the liquid in the tube to a pHaselock gel HEAVY tube and incubate at 37°C for 5 minutes; S333: Centrifuge at 13000g for 15 minutes at room temperature and collect the aqueous phase above the gel. S334: Add 500 μL isopropanol and 0.5 μL glycogen, mix well, and precipitate RNA at -20℃ for 3 hours to improve RNA precipitation efficiency. S335: Centrifuge at 10000g for 10 minutes at 4℃, then discard the supernatant. The RNA will settle at the bottom of the tube. S336: Add 1 mL of 75% ethanol to the centrifuge tube from step S335, gently shake the centrifuge tube, and suspend the RNA precipitate. S337: Centrifuge at 8000g for 5 minutes at 4℃, discard the supernatant, and air dry at room temperature for 10 minutes to obtain RNA.

[0016] Preferably, the placement order in step S23 is as follows: First, immerse two long filter papers, symmetrically arranged on both sides, in triethanolamine buffered saline (Solebo, product number: T1080); soak four square filter papers and set aside for use; then place the adhesive upside down; take one NC membrane, soak it, and attach one corner to the adhesive surface first, then place the rest upright; take two more square filter papers, soak them and set aside for use; prepare several absorbent papers of the same size as the square or long filter papers, and weigh them down with weights; then place them in the following order from bottom to top: adhesive plate, two long filter papers, multiple layers of ordinary filter paper (Solebo, product number: YA0166), adhesive, NC membrane, multiple layers of ordinary filter paper, several absorbent papers, plate, and weights; with both sides in triethanolamine buffered saline, soak for 10 hours.

[0017] Compared with the prior art, the beneficial effects of this invention are: the detection method of RNA-binding protein aggregate molecular function based on RNA immunoprecipitation can complete the experiment and obtain results in a shorter time (2-3 days), which is faster than the prior art, requires fewer related reagents and instruments, and can help experimenters to preliminarily detect the molecular function of polymers.

[0018] Meanwhile, this method is simple to operate, requires no special instruments or reagents, and can help experimenters verify functional hypotheses through qPCR. It is highly efficient and accurate, and will provide a more convenient platform for functional studies of polyamyloid proteins. Attached Figure Description

[0019] Figure 1 The SDD-AGE results are from the RNA-binding protein aggregate molecular function detection method based on RNA immunoprecipitation of the present invention. Figure 2 This is a schematic diagram of sample preparation during electrophoresis testing in the RNA-binding protein aggregate molecular function detection method based on RNA immunoprecipitation of the present invention. Figure 3 The images show the exposure and processing of the COLD and HOT membranes after exposure in the RNA-binding protein aggregate molecular function detection method based on RNA immunoprecipitation of the present invention. Figure 4 This figure shows the relative expression levels of mRNA in the RNA-binding protein aggregate molecular function detection method based on RNA immunoprecipitation of the present invention. Detailed Implementation

[0020] To enhance understanding of the present invention, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. These embodiments are only used to explain the invention and do not limit the scope of protection of the invention.

[0021] Example: This method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation specifically includes the following steps: S1, RNA immunoprecipitation: First, the tissue or cells are lysed, then the lysate is removed by washing with magnetic beads, and then the test sample is prepared. The specific steps of step S1 are as follows: S11: Select the target protein and prepare lysis buffer and high-salt buffer; In step S11, the target protein is selected as an RNA-binding protein capable of forming physiologically multimers. The lysis buffer formulation includes: 50 mM Tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5 (Solepro, catalog number: T1140), 150 mM sodium chloride solution, 10% glycerol, 0.5% Triton-X100 (immunostaining permeabilization solution, Beyotime, catalog number: P0096), 1× protease inhibitor (Roche, catalog number: 05892970001), 400 units RNase inhibitor (Thermo Fisher Scientific, catalog number: AM2694); the high-salt buffer formulation includes: 50 mM Tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5 (Solepro, catalog number: T1140), 500 mM NaCl, 10% glycerol, 0.5% Triton-X100 (immunostaining permeabilization solution, Beyotime, catalog number: P0096). S12: Dyslyce the selected tissue or cells. The specific steps of step S12 are as follows: S121: Add lysis buffer to tissues or cells at a ratio of 20:1, mix by pipetting, and sonicate at a low energy level to prevent affecting subsequent RNA detection, until the lysis buffer is free of obvious clumps. S122: Lyse on ice for 15 min, then centrifuge at 8000g for 15 min at 4℃, remove the precipitate and keep the supernatant, and keep 10% of the supernatant as the input group, i.e. denatured sample. S123: Add antibody or IgG (negative control) to the remaining supernatant from step S122, and then incubate at 4°C for 6-8 hours; S124: Take 50 μL of protein G beads (Thermo Fisher Scientific, catalog number: 10003D), i.e. magnetic beads, and wash the beads twice with 1 mL of lysis buffer; then incubate the lysis buffer supernatant with the beads overnight. S13: Use magnetic beads to clean and remove the lysis buffer; the specific steps of step S13 are as follows: use a magnetic base to separate the beads from the lysis buffer and remove the lysis buffer; then wash the beads twice with 1 mL of high-salt buffer and twice with 1 mL of lysis buffer. S14: Regroup and prepare samples; The specific steps of step S14 are as follows: S141 Sample separation: Add 100 μL of lysis buffer to the beads and divide the beads into a Hot group (RNA recovery group) and a Cold group (electrophoresis verification group), with a ratio of 9:1 between the Hot group and the Cold group. S142 Sample Preparation Hot Group (Hot Membrane): Add 20 μL of acidic elution buffer (pH < 4), elute for 5 min, add 7 μL of neutralization buffer (pH 9.8), add 9 μL of 4x loading buffer, mix well, and remove beads. S143 Sample Preparation (Cold Group, i.e., Cold Membrane): Add 20 μL of acidic elution buffer (pH < 4), elute for 5 min, add 7 μL of neutralization buffer, add 6.75 μL of 5x loading buffer, mix well, and remove beads; take 10 μL of sample and boil at 100℃ for 5 min to obtain denatured sample. S144 Sample Preparation Input Group: Add 5x protein loading buffer to dilute to 1x, take 10μL of sample and boil for 5min to obtain denatured sample; S2, Protein and RNA detection: First, the sample is subjected to semi-denaturing gel electrophoresis to confirm the IP efficiency of the sample, and then the RNA on the membrane is recovered. The specific steps of step S2 are as follows: S21 Gel Preparation: The gel formulation includes: 2.25g of low-osmotic agarose (Beyotime, catalog number: ST004M), 3mL of 50x Tris acetate-EDTA buffer, and 147mL of double-distilled water. Mix the low-osmotic agarose and 50x Tris acetate-EDTA buffer, place in a container, seal, weigh, and microwave to boiling three times. Add double-distilled water at approximately 70℃ until the weight reaches the initial microwave weight. Microwave to boiling three times, then gently shake. Clean and dry the rotor with double-distilled water, place it in a flask, and add 0.1% sodium dodecyl sulfate (Sangon Biotech, catalog number: 151-21-3). Pour the gel into a plate and allow it to set at room temperature for 1 hour. S22 Sample Loading (Loading Samples): Add the sample to the gel well, or use the sample loading electrophoresis buffer to keep it stable. The electrophoresis buffer formula is: 12 mL of 50x Tris acetate EDTA buffer, 6 mL of 0.1% sodium dodecyl sulfate, and 588 mL of double-distilled water. S23 gel electrophoresis: Place the gel in the electrophoresis tank, add electrophoresis buffer, turn on the power, apply an electric field (voltage not exceeding 1V / cm), and maintain at 4℃ for 10 hours to allow proteins to be loaded into the gel and separated. The specific order of placement in step S23 is as follows: First, immerse two long strips of filter paper, symmetrically arranged on both sides, in triethanolamine-buffered saline (Solepro, catalog number: T1080); wet four square filter papers and set aside for use; then invert the gel. Take one NC membrane, wet it, and attach one corner to the adhesive surface first, then place the rest upright; take two square filter papers, wet them, and set them aside for use; prepare several absorbent papers of the same size as the square or rectangular filter papers, and weigh them down; then place them in the following order from bottom to top: adhesive plate, two strips of rectangular filter paper, multiple layers of ordinary filter paper (Solebo, item number: YA0166), adhesive, NC membrane, multiple layers of ordinary filter paper, several absorbent papers, plate, and weight; with triethanolamine buffered saline on both sides, soak for 10 hours; Figure 2 As shown; S24 Transfer: After electrophoresis, transfer was performed. First, rinse with tap water, cut the gel in 1xTBS, and cut the membrane and filter paper according to the size of the gel. Then, place the membrane in the following order from bottom to top: filter paper, gel, NC membrane, filter paper, and absorbent paper. Remove excess air bubbles. Block the membrane of the COLD group with 5% milk, incubate with primary antibody at 4°C overnight, incubate with secondary antibody at room temperature for 1 hour, and expose to confirm the IP effect. Freeze the membrane of the HOT group at -80°C. After exposure, according to the position of polymer and monomer bands on the Cold membrane, cut the corresponding membrane on the HOT membrane into 1mm² pieces for recovery. Recover the RNA from the membrane. Figure 3 As shown, Figure 3 In this context, SDD-AGE is a semi-denaturing detergent agarose gel electrophoresis; SDS-PAGE is sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DAZL IP polymer is immunoprecipitation of the DAZL protein polymer fraction; DAZL IP monomer is immunoprecipitation of the DAZL protein monomer fraction; igG is immunoglobulin G, serving as a negative control for the immunoprecipitation group; COLD represents the membrane used for exposure; and HOT represents the membrane used for RNA recovery. S3, RNA release from the membrane: Protein digestion is performed by incubation with a prepared proteinase K buffer to extract RNA, and the RNA concentration is measured. Reverse transcription is then performed to obtain RNA with DAZL multimers and monomers bound. The specific steps of step S3 are as follows: S31 Preparation of Proteinase K Buffer: The proteinase K buffer formulation is as follows: 100mM tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 7.5 (Solepro, catalog number: T1140), 50mM sodium chloride solution, and 10mM ethylenediaminetetraacetic acid (Thermo Fisher Scientific, catalog number: R1021). S32 protein digestion: Add 160 μL of proteinase K buffer and 40 μL of proteinase K (Solepro, catalog number: P9460) to each tube, mix well, and incubate at 37°C for 20 min; then add 420 mg of urea (Beyotime, catalog number: ST1731) to proteinase K buffer to a final volume of 1 mL, add 200 μL to each tube, and incubate at 37°C for 20 min; S33 RNA extraction: First, incubate, then centrifuge, collect the aqueous phase above the gel, then add isopropanol and glycogen for precipitation to obtain RNA precipitate, then centrifuge, discard the supernatant, wash and centrifuge again to discard the supernatant, and then air-dry the obtained RNA. The specific steps of step S33 are as follows: S331: Add 400 μL of acidic phenol / chloroform / isoamyl alcohol to the PK buffer after protein digestion, maintain the pH at 6.5, shake well, and incubate at 37°C for 5 min; S332: Transfer the liquid in the tube to a pHaselock gel HEAVY tube and incubate at 37°C for 5 minutes; S333: Centrifuge at 13000g for 15 minutes at room temperature and collect the aqueous phase above the gel. S334: Add 500 μL isopropanol and 0.5 μL glycogen, mix well, and precipitate RNA at -20℃ for 3 hours to improve RNA precipitation efficiency. S335: Centrifuge at 10000g for 10 minutes at 4℃, then discard the supernatant. The RNA will settle at the bottom of the tube. S336: Add 1 mL of 75% ethanol to the centrifuge tube from step S335, gently shake the centrifuge tube, and suspend the RNA precipitate. S337: Centrifuge at 8000g for 5 minutes at 4℃, discard the supernatant, and air dry at room temperature for 10 minutes to obtain RNA; S34 Transcription: Add 20 μL of DEPC water, place on ice, dissolve RNA on ice for 5 min to prevent RNA degradation at room temperature, measure RNA concentration, and then perform reverse transcription.

[0022] S4, qPCR detection target: to perform functional detection on the RNA obtained by binding DAZL multimers and monomers, and to verify whether there is RNA in the aggregates of DAZL multimers and monomers. Detecting the presence of these RNAs allows for the selection of reported or related molecularly functional targets to verify the functional differences between polymers and monomers. The results of the relative expression level test for mRNA are as follows: Figure 4 As shown, Figure 4 In this context, DAZL IPpolymer represents the immunoprecipitation of the DAZL protein polymeric portion; DAZL IP monomer represents the immunoprecipitation of the DAZL protein monomeric portion; and igG represents immunoglobulin G, serving as a negative control for the immunoprecipitation group. Figure 4 In the testes, it was observed that DAZL polymers bind to Pum1 and Lin28a, while DAZL monomers bind to Suz12, and Gapdh acts as a yin agonist. This result demonstrates that different forms of the same protein perform different functions, providing important results for subsequent biological research.

[0023] This method is an improvement on the experiment, with an experimental cycle of 3 days and no special instruments or reagents required. It can help experimenters verify functional hypotheses through qPCR and has the characteristics of high efficiency and accuracy. It will provide a more convenient platform for functional research of polyamyloid proteins.

[0024] For those skilled in the art, the specific embodiments are merely illustrative descriptions of the present invention. Obviously, the specific implementation of the present invention is not limited to the above-described manner. Any non-substantial improvements made using the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution to other situations without modification, are all within the protection scope of the present invention.

Claims

1. A method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation, characterized in that, The specific steps are as follows: S1) RNA immunoprecipitation: First, the tissue or cells are lysed, then the lysate is removed by washing with magnetic beads, and then the test sample is prepared. S2) Protein and RNA detection: First, the sample is subjected to semi-denaturing gel electrophoresis to confirm the IP efficiency of the sample, and then the RNA on the membrane is recovered. S3) Release RNA from the membrane: Protein digestion was performed using a prepared PK buffer to extract RNA, and the RNA concentration was measured. Reverse transcription was then performed to obtain RNA with DAZL multimers and monomers bound. S4) qPCR detection target: Functional detection of the obtained DAZL multimer and monomer-bound RNA to verify whether RNA exists in the aggregates of DAZL multimer and monomer-bound RNA.

2. The method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation according to claim 1, characterized in that, The specific steps of step S1 are as follows: S11: Select the target protein and prepare lysis buffer, high-salt buffer and 4x protein loading buffer; S12: Dysly the selected tissue or cells; S13: Use magnetic beads to clean and remove the pyrolysis solution; S14: Regroup and prepare samples.

3. The method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation according to claim 2, characterized in that, In step S11, the target protein is selected as an RNA-binding protein that can form physiological state multimers. The lysis buffer formulation includes: 50 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, 150 mM sodium chloride solution, 10% glycerol, 0.5% Triton-X100, 1× protease inhibitor, and 400 units of RNase inhibitor. The high-salt buffer solution is formulated as follows: 50 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, 500 mM NaCl, 10% glycerol, and 0.5% Triton-X100.

4. The method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation according to claim 2, characterized in that, The specific steps of step S12 are as follows: S121: Add lysis buffer to tissues or cells, mix thoroughly by pipetting, and sonicate to break up the lysis buffer to prevent it from affecting subsequent RNA detection, until there are no obvious clumps in the lysis buffer. S122: Lyse on ice for 15 min, then centrifuge at 8000g for 15 min at 4℃, remove the precipitate and keep the supernatant, and keep 10% of the supernatant as the input group, i.e. denatured sample. S123: Add antibody or IgG to the remaining supernatant from step S122, and then incubate at 4°C for 6-8 hours; S124: Take 50 μL of protein G beads (magnetic beads), wash the beads twice with 1 mL of lysis buffer; then incubate the lysis buffer supernatant with the beads overnight.

5. The method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation according to claim 4, characterized in that, The specific steps of step S13 are as follows: use a magnetic base to separate the beads from the lysis buffer and remove the lysis buffer; then wash the beads twice with 1 mL of high-salt buffer and twice with 1 mL of lysis buffer.

6. The method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation according to claim 4, characterized in that, The specific steps of step S14 are as follows: S141 Sample division: Add 100 μL of lysis buffer to the beads and divide the beads into a Hot group (RNA recovery group) and a Cold group (electrophoresis verification group), with a ratio of 9:1 between the Hot group and the Cold group. S142 Sample Preparation Hot Group (Hot Membrane): Add 20 μL of acidic elution buffer (pH < 4), elute for 5 min, add 7 μL of neutralization buffer (pH 9.8), add 9 μL of 4x loading buffer, mix well, and remove beads. S143 Sample Preparation (Cold Group, i.e., Cold Membrane): Add 20 μL of acidic elution buffer (pH < 4), elute for 5 min, add 7 μL of neutralization buffer, add 9 μL of 4x loading buffer, mix well, and remove beads; take 10 μL of sample and boil at 100℃ for 5 min to obtain denatured sample. S144 Sample Preparation Input Group, also known as Input Membrane: Add 5x protein loading buffer to dilute to 1x, take 10μL of sample and boil for 5min to obtain denatured sample.

7. The method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation according to claim 4, characterized in that, The specific steps of step S2 are as follows: S21 Gel Preparation: The gel formulation includes: 2.25 g of low-osmotic agarose, 3 mL of 50x Tris acetate-EDTA buffer, and 147 mL of double-distilled water. Mix the low-osmotic agarose and 50x Tris acetate-EDTA buffer, place them in a container, seal, weigh, and microwave to boiling three times. Add double-distilled water at approximately 70°C until the weight reaches the original weight before microwave microwaving. Microwave to boiling three times again, then gently shake. Clean and dry the rotor with double-distilled water, place it in a flask, and add 0.1% sodium dodecyl sulfate. Pour the gel into a plate and allow it to set at room temperature for 1 hour. S22 Loading Samples: Add the sample to the gel well, or use sample loading electrophoresis buffer to stabilize it. The electrophoresis buffer formula is: 12 mL of 50x Tris acetate EDTA buffer, 6 mL of 0.1% sodium dodecyl sulfate, and 588 mL of double-distilled water. S23 gel electrophoresis: Place the gel in the electrophoresis tank and inject the electrophoresis buffer into the electrophoresis tank. Turn on the power and apply an electric field. The voltage should not exceed 1V / cm. Keep it at 4℃ for 10h to allow the proteins to be loaded into the gel and separated. S24 Transfer: After electrophoresis, the membrane was transferred. First, rinsed with tap water, and the gel was cut in 1xTBS. The membrane and filter paper were then cut according to the size of the gel and placed in the following order from bottom to top: filter paper, gel, NC membrane, filter paper, and absorbent paper. Excess air bubbles were removed. The membranes of the COLD group were blocked with 5% milk, incubated with primary antibody at 4°C overnight, and incubated with secondary antibody at room temperature for 1 hour. Exposure was then performed to determine the IP effect. The membranes of the HOT group were frozen at -80°C. After exposure, the corresponding membranes on the HOT membrane were cut into 1mm pieces according to the positions of the polymer and monomer bands on the Cold membrane. 2 Small pieces of paper are used for recycling to recover RNA from the membrane.

8. The method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation according to claim 7, characterized in that, The specific steps of step S3 are as follows: S31 Preparation of proteinase K buffer: The proteinase K buffer is formulated as follows: 100mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, 50mM sodium chloride solution, and 10mM ethylenediaminetetraacetic acid; S32 protein digestion: Add 160 μL of proteinase K buffer and 40 μL of proteinase K to each tube, mix well, and incubate at 37°C for 20 min; then add 420 mg of urea to proteinase K buffer to bring the volume to 1 mL, add 200 μL to each tube, and incubate at 37°C for 20 min. S33 RNA extraction: First, incubate, then centrifuge, collect the aqueous phase above the gel, then add isopropanol and glycogen for precipitation to obtain RNA precipitate, then centrifuge, discard the supernatant, wash and centrifuge again to discard the supernatant, and then air-dry the obtained RNA. S34 Transcription: Add 20 μL of DEPC water, place on ice, and dissolve RNA on ice for 5 minutes to prevent RNA degradation at room temperature. Measure the RNA concentration and perform reverse transcription.

9. The method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation according to claim 8, characterized in that, The specific steps of step S33 are as follows: S331: Add 400 μL of acidic phenol / chloroform / isoamyl alcohol to the PK buffer after protein digestion, maintain the pH at 6.5, shake well, and incubate at 37°C for 5 min; S332: Transfer the liquid in the tube to the root tube and incubate at 37°C for 5 min; S333: Centrifuge at 13000g for 15 minutes at room temperature and collect the aqueous phase above the gel. S334: Add 500 μL isopropanol and 0.5 μL glycogen, mix well, and precipitate RNA at -20℃ for 3 hours to improve RNA precipitation. S335: Centrifuge at 10000g for 10 minutes at 4℃, then discard the supernatant. The RNA will settle at the bottom of the tube. S336: Add 1 mL of 75% ethanol to the centrifuge tube from step S335, gently shake the centrifuge tube, and suspend the RNA precipitate. S337: Centrifuge at 8000g for 5 minutes at 4℃, discard the supernatant, and air dry at room temperature for 10 minutes to obtain RNA.

10. The method for detecting the molecular function of RNA-binding protein aggregates based on RNA immunoprecipitation according to claim 8, characterized in that, The specific placement order in step S23 is as follows: First, immerse two long filter papers, symmetrically arranged on both sides, in triethanolamine buffered saline; soak four square filter papers and set them aside for use; then place the adhesive upside down; take one NC membrane, soak it, and attach one corner to the adhesive surface first, then place the rest upright; take two more square filter papers, soak them and set them aside for use; prepare several absorbent papers of the same size as the square or long filter papers, and weigh them down with weights; then place them in the following order from bottom to top: adhesive plate, two long filter papers, multiple layers of filter paper, adhesive, NC membrane, multiple layers of filter paper, several absorbent papers, plate, weights; with TBS on both sides, soak for 10 hours.

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