Method for detecting RNA binding protein and RNA binding strength in cell, tissue or organism

Abstract

The invention relates to a method for detecting RNA binding protein and RNA binding strength in cells, tissues or organisms. Specifically, the present invention provides a method of detecting RNA binding protein in a cell, tissue or organism, the method comprising: a) covalently cross-linking the content of the cell, tissue or organism comprising RNA binding protein using radiation to produce a biological material comprising a cross-linked RNA protein complex; b) cleaving the biological material comprising the cross-linked RNA protein complex; c) performing phase separation on the cross-linked RNA protein compound, separating, washing and dissolving a middle layer solid phase, cleaning a precipitation product by using ethanol, and removing DNA (Deoxyribose Nucleic Acid); d) repeating step c) at least once; e) carrying out enzymolysis by using FASP to generate released RNA binding protein; and f) detecting the released RNA binding protein.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, this invention relates to a method for detecting RNA-binding proteins and RNA binding strength in cells, tissues or organisms. Background Technology

[0002] RNA-binding proteins (RBPs) are a class of proteins with broad biological functions. They can regulate the processing, localization, translation, and stability of various RNA molecules within the cell by binding to specific motifs or structural elements on RNA molecules. The dynamic interactions (changes in affinity) between RBPs and RNA molecules are crucial in plant growth, development, disease resistance, and stress tolerance. For example, the RNA-binding protein RBGD2 / 4 in Arabidopsis thaliana can significantly enhance heat tolerance, while HRLP regulates flowering through phase separation. Therefore, identifying and studying RBPs not only helps us to better understand the regulatory mechanisms of RNA metabolism and gene expression but also provides new strategies for developing high-yielding, high-quality, and multi-resistant germplasm resources.

[0003] To detect RNA-binding proteins, researchers have developed various methods. One such method is the Oligo(T)-dependent RNA-binding protein capture technique, represented by RNA Interactome Capture (RIC). RIC technology forms a covalently bonded complex between RNA and RBPs through ultraviolet irradiation and then uses Oligo(dT) to separate the RNA-protein complex from the lysate. However, because many non-coding RNAs (ncRNAs) and pre-mRNAs do not contain Poly(A) tail modifications, RIC technology may lose proteins that interact with these RNAs, thus limiting the comprehensive analysis of RBP function. To overcome this limitation of RIC technology, researchers have recently developed a Plant Phase Extraction (PPE) RNA-binding protein extraction technique that is independent of Oligo(dT) capture. The main steps of this method include: cross-linking cells with ultraviolet light followed by cryogenic grinding with liquid nitrogen; phase separation using acidic guanidine thiocyanate-phenol (commercially available Trizol reagent)-chloroform; separation, washing, and dissolution of the intermediate solid phase RNA-protein complex; repeating the phase separation and dissolution process 4-5 times on the washed RNA-protein complex; protein electrophoresis and in-gel enzymatic digestion; and mass spectrometry detection. The principle of this technique is based on the difference in affinity between free RNA and protein for water and organic solutions, allowing free RNA to enter the aqueous phase and free protein to enter the organic phase, thus separating the RNA-protein complex in the intermediate phase. However, PPE RNA-binding protein extraction technology also has some problems. For example, the in-gel enzymatic digestion method may result in the loss of high molecular weight proteins. Furthermore, the multiple phase separations are not only redundant and significantly increase costs, but may also lead to the loss of low-abundance and low-RNA-affinity RBPs. Additionally, this method can only perform qualitative analysis of RNA-binding proteins, not quantitative analysis.

[0004] Therefore, there is an urgent need in this field to improve the identification methods for RNA-binding proteins in order to solve the above problems and to establish a system capable of quantitatively analyzing RNA-binding proteins. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting RNA-binding proteins and RNA binding strength in cells, tissues or organisms.

[0006] In a first aspect of the present invention, a method for detecting RNA-binding proteins in cells, tissues, or organisms is provided, the method comprising:

[0007] a) Using radiation to covalently crosslink the contents of cells, tissues, or organisms containing RNA-binding proteins to produce biological material containing crosslinked RNA-protein complexes;

[0008] b) Lysing of biological material containing the cross-linked RNA-protein complex;

[0009] c) The cross-linked RNA-protein complex is subjected to phase separation, the intermediate solid phase is taken for separation, washing and dissolution, and the precipitate is washed with ethanol to remove DNA.

[0010] d) Repeat step c) at least once;

[0011] e) Using FASP enzymatic hydrolysis, RNA-binding proteins are released; and

[0012] f) Detect the released RNA-binding proteins.

[0013] In one or more embodiments, step c) includes: phase separation of the cross-linked RNA-protein complex using Trizol and chloroform.

[0014] In one or more embodiments, step c) includes: using Trizol and chloroform to perform phase separation on the cross-linked RNA-protein complex, the cross-linked RNA-protein complex is phase separated into three layers, the middle solid phase is taken, the RNA-protein complex in the middle solid phase is separated sequentially using low-salt SDS buffer and high-salt SDS buffer, the supernatant is taken, the supernatants from the two separations are mixed in a ratio of 2:1 to 1:2, and precipitated using a precipitant, and the precipitated product is washed with ethanol to remove DNA.

[0015] In one or more embodiments, the weight percentage of SDS in the low-salt SDS buffer is less than 0.2%, more preferably 0.15% to 0.1%.

[0016] In one or more embodiments, the low-salt SDS buffer also contains Tris and / or EDTA.

[0017] In one or more embodiments, the concentration of Tris is 10–100 mM, 10–90 mM, 20–80 mM, 30–70 mM, or 40–60 mM.

[0018] In one or more embodiments, the concentration of EDTA is 0.1–10 mM, 0.5–5 mM, 0.6–3 mM, or 0.8–2 mM.

[0019] In one or more embodiments, the low-salt SDS buffer also contains 50 mM Tris and 1 mM EDTA.

[0020] In one or more embodiments, the weight percentage of SDS in the high-salt SDS buffer is greater than 0.3%, more preferably 0.5% to 1%.

[0021] In one or more embodiments, the high-salt SDS buffer also contains Tris and / or EDTA.

[0022] In one or more embodiments, the concentration of Tris is 10–100 mM, 10–90 mM, 20–80 mM, 30–70 mM, or 40–60 mM.

[0023] In one or more embodiments, the concentration of EDTA is 0.1–10 mM, 0.5–5 mM, 0.6–3 mM, or 0.8–2 mM.

[0024] In one or more embodiments, the low-salt SDS buffer also contains 50 mM Tris and 1 mM EDTA;

[0025] In one or more embodiments, the supernatant obtained by separating the RNA-protein complex from the intermediate solid phase using low-salt SDS buffer is mixed in a 1:1 ratio with the supernatant obtained by separating the RNA-protein complex from the intermediate solid phase using high-salt SDS buffer.

[0026] In one or more embodiments, the precipitant is NH4AC and / or Invitrogen. TM GlycoBlue TM .

[0027] In one or more embodiments, step c) further includes one or more of the following reaction conditions:

[0028] (1) The volume ratio of Trizol to chloroform is 100:1 to 30:1, preferably 80:1 to 50:1;

[0029] (2) Trizol and chloroform are mixed by vortexing before the reaction is carried out;

[0030] (3) Add Trizol first, react for a period of time, and then add chloroform; preferably, the reaction time of Trizol is 5 to 30 min, more preferably 10 to 20 min;

[0031] (5) The reaction temperature of Trizol is 25-35℃, preferably carried out in a metal bath;

[0032] (6) Trizol was reacted under shaking conditions at 500 rpm to 1500 rpm;

[0033] (7) The precipitate is washed with ethanol at least once, preferably twice, three times or more;

[0034] (8) DNA is removed by DNase, preferably by adding DNase and then removing DNA by PCR reaction; more preferably, the temperature of PCR reaction is 30-40℃ and the time of PCR reaction is 10-30 min.

[0035] In one or more embodiments, in step a), the radiation covalent crosslinking is ultraviolet crosslinking.

[0036] In one or more embodiments, the intensity of the UV crosslinking is 500–2000 mJ / cm. 2 More preferably 600–1500 mJ / cm 2 800~1300mJ / cm 2 Or 900~1200mJ / cm 2 .

[0037] In one or more embodiments, step a) further includes a control group, wherein the control group is not subjected to radiation covalent crosslinking.

[0038] In one or more embodiments, the control group is not subjected to UV crosslinking.

[0039] In one or more embodiments, the UV crosslinking intensity of the control group is 0 mJ / cm. 2 ;

[0040] In one or more embodiments, the number of radiation covalent crosslinks is at least once.

[0041] In one or more embodiments, the number of radiation covalent crosslinks is at least two.

[0042] In one or more embodiments, step d) is repeated at least once, preferably only once.

[0043] In one or more embodiments, in step e), FASP enzymatic hydrolysis is performed using trypsin; preferably, the hydrolysis temperature is 30–40°C; more preferably, urea is used to dissolve the product in step d), and then FASP enzymatic hydrolysis is performed; more preferably, the concentration of urea is 5–10 M.

[0044] In one or more embodiments, in step b), the lysis is liquid nitrogen cryogenic grinding, preferably grinding the biomaterial containing the cross-linked RNA-protein complex under RNase-free conditions and after pre-cooling with liquid nitrogen.

[0045] In one or more embodiments, in step f), the detection includes: detection using mass spectrometry; preferably, the mass spectrometry is GC-MS or LC-MS.

[0046] A second aspect of the present invention provides a method for quantitatively analyzing the binding strength of RNA-binding proteins, the method comprising:

[0047] 1) Use radiation covalently crosslinked contents of cells, tissues or organisms containing RNA-binding proteins to produce biological materials containing crosslinked RNA-protein complexes; use contents of cells, tissues or organisms that have not undergone radiation covalent crosslinking as control biological materials.

[0048] 2) Lysis of biological material containing the cross-linked RNA-protein complex, and control biological material;

[0049] 3) Phase separation was performed on the pyrolyzed biomaterial from step 2);

[0050] 4) Extract RNA-binding proteins from the middle solid phase and free proteins from the lower organic phase, respectively;

[0051] 5) Calculate the RNA binding activity index of the radiation-covalently cross-linked biomaterial and the RNA binding activity index of the control biomaterial, respectively:

[0052] RNA binding activity index of radiation covalently cross-linked biomaterials = amount of intermediate phase protein ÷ amount of free protein in precipitated phase;

[0053] RNA binding activity index of control biological material = amount of intermediate phase protein ÷ amount of free protein in precipitate phase;

[0054] RNA binding activity index = RNA binding activity index of radiation covalently cross-linked biological material - RNA binding activity index of control biological material.

[0055] In one or more embodiments, a higher RNA binding activity index indicates a stronger binding strength of the RNA-binding protein.

[0056] In one or more embodiments, the intermediate layer RNA-binding protein is an RNA-binding protein detected using the method described in any embodiment of the present invention.

[0057] A third aspect of the present invention provides a kit comprising reagents for performing the method for detecting RNA-binding proteins as described in any embodiment of the present invention, or reagents for the method for quantitatively analyzing the binding strength of RNA-binding proteins as described in any embodiment of the present invention, wherein the reagents comprise one or more of the following: Trizol reagent, chloroform, low-salt SDS buffer, high-salt SDS buffer, ethanol, DNase, and trypsin.

[0058] In one or more embodiments, the kit may further include reagents, buffer solutions, and washing solutions for mass spectrometry detection.

[0059] In one or more embodiments, the buffer and washing solution do not contain RNase.

[0060] In one or more embodiments, the kit may further include: instructions for use of the kit to guide technicians in operation.

[0061] A fourth aspect of the invention provides the use of the kit described in any embodiment of the invention for detecting RNA-binding proteins in cells, tissues or organisms, or for detecting the binding strength of RNA-binding proteins.

[0062] In one or more embodiments, the cells, tissues, or organisms are derived from plants, animals, or microorganisms.

[0063] In one or more embodiments, the plant includes: dicotyledonous plants, monocotyledonous plants, or gymnosperms.

[0064] In one or more embodiments, the plants include: Arabidopsis thaliana, wheat, barley, rye, rice, corn, sorghum, sugar beet, apple, pear, plum, peach, apricot, cherry, strawberry, raspberry, blackberry, bean, lentil, pea, soybean, rapeseed, mustard, poppy, artemisia annua, oleander, sunflower, coconut, castor oil plant, cocoa bean, peanut, gourd, tobacco, oil palm, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, grapefruit, spinach, velvetleaf, asparagus, cabbage, Chinese cabbage, bok choy, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugarcane, tea, pepper, grapevine, oyster grass, banana, poplar, willow, pine, fir, eucalyptus, sago palm, simonium, natural rubber tree, and ornamental plants.

[0065] In one or more embodiments, the plant is Arabidopsis thaliana.

[0066] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0067] Figure 1 A schematic diagram of the method for extracting RNA-binding proteins (RBPs) and calculating their binding activity according to the present invention.

[0068] Figure 2 The results of SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining of the RNA-RBP complex extracted in Example 1 of this invention.

[0069] Figure 3 Repeatability test of LC / MS mass spectrometry results of the RBP group and free protein group extracted in Example 1 of this invention.

[0070] Figure 4 The LC / MS mass spectrometry results of the RBP group and free protein group extracted in Examples 1 and 2 of this invention, and the difference analysis between the groups.

[0071] Figure 5 The RBP groups extracted in Example 1 of this invention are compared with the results obtained by the previous RIC and PPE methods. The numbers at the intersection of the two circles represent the RBPs common to different experimental methods. The numbers on both sides represent the number of RBPs captured by each method. "Random" indicates that an equal number of RBPs identified by the method of this invention were randomly selected from the Arabidopsis genome and compared with the RIC or PPE methods.

[0072] Figure 6 The analysis of the RBP group and RBI results extracted in Example 3 of this invention, as well as the RBI display of proteins previously confirmed as RBPs. The RNA binding activity index (RBI) was calculated as (amount of mesophase protein) / (amount of free protein in the precipitate phase) under radiation covalent crosslinking conditions minus (amount of mesophase protein) / (amount of free protein in the precipitate phase) under non-radiation covalent crosslinking conditions.

[0073] Figure 7 The number of RBP peptides that directly bind to RNA extracted in Example 4 of this invention is shown, as well as the protein domains of representative RBPs are shown. Detailed Implementation

[0074] To address the problems of low specificity, low capture efficiency, and inability to quantitatively assess the RNA-binding activity of proteins in current methods for detecting RNA-binding proteins, this invention makes several improvements to the Plant Phase Extraction (PPE) method. These improvements include two consecutive phase separations using Trizol and chloroform, separation, washing, and dissolution of the intermediate solid phase, and a refined phase separation process using ethanol to wash away the precipitate and remove DNA. The efficiency of the phase separation is enhanced to 1000 J / cm³. 2 The cross-linking strength was improved, and the cross-linking was repeated once. The FASP enzymatic digestion method was used instead of the in-gel digestion method. These improvements combined to simplify the experimental steps and effectively overcome the limitations of the Oligo(dT) capture technology in the RIC method. It can capture RBPs with weak binding ability and identify RNA-protein complexes with large molecular weights. Compared with the existing RIC and PPE methods, it increases the number of RBPs identified. It can detect RNA-binding proteins and quantify their RNA binding activity (RNA Binding Index, RBI) at the whole transcriptome level.

[0075] Methods for detecting RNA-binding proteins

[0076] This invention provides a method for detecting RNA-binding proteins in cells, tissues, or organisms, the method comprising:

[0077] a) Using radiation to covalently crosslink the contents of cells, tissues, or organisms containing RNA-binding proteins to produce biological material containing crosslinked RNA-protein complexes;

[0078] b) Lysing of biological material containing the cross-linked RNA-protein complex;

[0079] c) The cross-linked RNA-protein complex is subjected to phase separation, the intermediate solid phase is taken for separation, washing and dissolution, and the precipitate is washed with ethanol to remove DNA.

[0080] d) Repeat step c) at least once;

[0081] e) Using FASP enzymatic hydrolysis, RNA-binding proteins are released; and

[0082] f) Detect the released RNA-binding proteins.

[0083] In step a), the radiation covalent crosslinking can be, for example, ultraviolet crosslinking. The intensity of the ultraviolet crosslinking can be 500–2000 mJ / cm. 2 The preferred strength is 600–1500 mJ / cm³. 2 800~1300mJ / cm 2 Or 900~1200mJ / cm 2 The number of radiation-induced covalent crosslinking processes is at least one, preferably two, three, or more. Preferably, the number of radiation-induced covalent crosslinking processes is at least two. The wavelength of the ultraviolet light is typically 254 nm to 365 nm. This invention improves the plant ultraviolet crosslinking method by using an enhancement of 1000 J / cm². 2 The cross-linking strength was improved, and repeated cross-linking once was able to capture RBPs with weak binding ability. A broad-spectrum and universal cross-linking system was established and successfully applied to a variety of plants with good results.

[0084] In this article, “radiative covalent crosslinking”, “covalent crosslinking”, “CrossLink” and “CL” can be used interchangeably, as can “non-radiative covalent crosslinking”, “non-covalent crosslinking”, “NonCrossLink” and “NonCL”.

[0085] It should be understood that, typically, in some embodiments, the radiation covalent crosslinking refers to ultraviolet crosslinking. The terms "ultraviolet crosslinking" and "UV" are used interchangeably, as are the terms "non-ultraviolet crosslinking" and "nonUV".

[0086] The contents of a cell, tissue, or organism containing RNA-binding proteins can be derived from plants, animals, or microorganisms. The term "plant" can be, for example, (but is not limited to): dicotyledons, monocotyledons, or gymnosperms. More specifically, the plants include (but are not limited to): Arabidopsis thaliana, wheat, barley, rye, rice, corn, sorghum, sugar beet, apple, pear, plum, peach, apricot, cherry, strawberry, raspberry, blackberry, bean, lentil, pea, soybean, rapeseed, mustard, poppy, artemisia annua, oleanum, sunflower, coconut, castor oil plant, cocoa bean, peanut, gourd, tobacco, oil palm, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, grapefruit, spinach, velvetleaf, asparagus, cabbage, Chinese cabbage, bok choy, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugarcane, tea, pepper, grapevine, oystergrass, banana, poplar, willow, pine, fir, eucalyptus, sago palm, simonium, natural rubber tree, and ornamental plants, preferably, the plants are Arabidopsis thaliana.

[0087] In step b), the lysis can be performed by liquid nitrogen cryogenic grinding, preferably grinding the biomaterial containing the cross-linked RNA-protein complex under RNase-free conditions and after pre-cooling with liquid nitrogen.

[0088] In step c), the volume ratio of Trizol to chloroform can be 100:1 to 30:1, preferably 80:1 to 50:1, for example, 60:1. Trizol and chloroform can be vortexed before the reaction. Trizol and chloroform are usually added separately and sequentially, for example, Trizol is added first, and chloroform is added after a certain reaction time. The reaction time for Trizol can be 5 to 30 minutes, for example, 10 to 20 minutes. The reaction temperature for Trizol can be 25 to 35°C, preferably carried out in a metal bath. The reaction of Trizol can be carried out with shaking at 500 rpm to 1500 rpm.

[0089] In step c), after treatment with Trizol reagent and chloroform, the cross-linked RNA-protein complex is phase-separated into three layers: an upper aqueous phase, a middle white solid phase, and a lower dark red organic phase. The RNA-protein complex in the middle solid phase is then separated, washed, and dissolved sequentially using low-salt SDS buffer and high-salt SDS buffer. Low-salt SDS buffer refers to an SDS buffer with a weight percentage of SDS less than 0.2%, for example less than 0.15%, preferably 0.1%. High-salt SDS buffer refers to an SDS buffer with a weight percentage of SDS greater than 0.3%, for example greater than 0.4%, preferably 0.5–1%. Typically, low-salt and high-salt SDS buffers also contain Tris and EDTA, such as 10–100 mM Tris, 10–90 mM Tris, 20–80 mM Tris, 30–70 mM Tris, or 40–60 mM Tris, and 0.1–10 mM EDTA, 0.5–5 mM EDTA, 0.6–3 mM EDTA, or 0.8–2 mM EDTA, preferably 50 mM Tris (pH = 7.5) and 1 mM EDTA. In some embodiments, after treating the low-salt and high-salt SDS buffers separately, the supernatants are mixed in a 1:1 ratio and precipitated using a precipitant. Exemplary precipitants may be NH4AC and Invitrogen. TM GlycoBlue TM .

[0090] In this document, the weight percentage refers to the percentage of the mass of solute contained in the solution. For example, a weight percentage of 0.1% means that 100 mL of solution contains 0.1 g of solute.

[0091] In the method of this invention, Trizol is used as the phase separation extraction buffer. Compared with traditional protein extraction cell lysis buffers, commercially available Trizol is readily available and does not require fresh preparation, simplifying the preparation process. Furthermore, phase separation with Trizol yields RNA-protein complexes with higher specificity, reducing contamination from free proteins.

[0092] In step c), the precipitate is washed with ethanol at least once, for example, two, three, or more times. DNA can be removed using DNase, for example, by adding DNase followed by PCR. The PCR reaction temperature can be 30–40°C, for example, 37°C; the PCR reaction time can be 10–30 min, for example, 15 min.

[0093] In step d), step c) is repeated at least once, for example, two, three, or more times. Preferably, step c) is repeated only once in step d).

[0094] Compared to the RIC method, the method for detecting RNA-binding proteins in this invention effectively overcomes the limitations of the Oligo(dT) capture technique in the RIC method. The RIC method may lose proteins that interact with RNAs that do not contain poly(A) tail modifications, while the method of this invention can significantly improve proteome quality and detect RBPs that bind to RNAs without poly(A) modifications, thus providing new possibilities for the research and application of RNA-binding proteins.

[0095] Compared to the Plant Phase Extraction (PPE) method, which requires 4-5 repeated phase separation processes, step c) in step d) is repeated only once. This significantly reduces the amount of Trizol reagent used and greatly simplifies the extraction process, shortening the working time. The main reason for using 4-5 repeated phase separation processes is to avoid contamination from free proteins in the RBP fraction and improve data specificity. In this invention, the intermediate layer RNA-protein complex and free proteins in the organic phase were simultaneously extracted and identified. Comparison of the two sets of mass spectrometry identification results showed a significant difference in composition between the RBP and free protein fractions after two phase separation extractions. This indicates that two precise phase separation extractions are sufficient to obtain a highly specific RBP fraction.

[0096] In step e), FASP hydrolysis can be performed using trypsin. The hydrolysis temperature can be 30–40°C, for example, 37°C. The product from step d) can be dissolved in urea before FASP hydrolysis. The urea concentration can be 5–10 M, for example, 8 M.

[0097] Because some large molecular weight RNA-protein complexes cannot migrate electrophoretically within an SDS-PAGE gel, and previous PPE methods using in-gel enzymatic digestion resulted in the loss of large molecular weight complexes such as THO, AtCSP2, GRRBP3, eIF3C, NTF2, PDR7, RDM16, SR34, and SR34A, this invention employs the FASP enzymatic digestion method for RBP components. Compared to multiple phase separation-in-gel digestion methods, this significantly increases the number of RBPs identified, achieving large-scale global RBP identification for the first time in plants.

[0098] In step f), mass spectrometry is typically used to detect the released RBPs. The mass spectrometry methods include, but are not limited to, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). Detection of the released RBPs using mass spectrometry allows for the identification of peptides.

[0099] Methods for quantifying the binding strength of RNA-binding proteins

[0100] The present invention also provides a method for quantifying RNA binding activity, the method comprising: using (amount of intermediate phase protein) / (amount of precipitated phase free protein) as the RNA binding activity index (RBI), and using the RNA binding activity index to quantitatively analyze the binding strength of RNA-binding proteins.

[0101] The intermediate phase refers to the intermediate solid phase obtained after the separation treatment with Trizol reagent and chloroform phase in step c). The precipitated phase refers to the lower dark red organic phase obtained after the separation treatment with Trizol reagent and chloroform phase in step c).

[0102] Specifically, the method for quantitatively analyzing the binding strength of RNA-binding proteins includes:

[0103] 1) Use radiation covalently crosslinked contents of cells, tissues or organisms containing RNA-binding proteins to produce biological materials containing crosslinked RNA-protein complexes; use contents of cells, tissues or organisms that have not undergone radiation covalent crosslinking as control biological materials.

[0104] 2) Lysis of biological material containing the cross-linked RNA-protein complex, and control biological material;

[0105] 3) Phase separation was performed on the pyrolyzed biomaterial from step 2);

[0106] 4) Extract RNA-binding proteins from the middle solid phase and free proteins from the lower organic phase, respectively;

[0107] 5) Calculate the RNA binding activity index of the radiation-covalently cross-linked biomaterial and the RNA binding activity index of the control biomaterial, respectively:

[0108] RNA binding activity index of radiation covalently cross-linked biomaterials = amount of intermediate phase protein ÷ amount of free protein in precipitated phase;

[0109] RNA binding activity index of control biological material = amount of intermediate phase protein ÷ amount of free protein in precipitate phase;

[0110] RNA binding activity index = RNA binding activity index of radiation covalently cross-linked biological material - RNA binding activity index of control biological material.

[0111] The RNA binding activity index can be used to quantitatively analyze the binding strength of RNA-binding proteins. For example, for a specific RNA-binding protein, a high RNA binding activity index indicates that the RNA binds strongly to the protein, and that it exists more as an RNA-binding protein in the intermediate phase, while its free protein form in the precipitate phase is less common.

[0112] The method according to the invention further includes detecting changes in the RNA binding strength of the detected RBP when compared with a control sample or a sample obtained under different experimental conditions. Therefore, in some embodiments, the above method can be used to obtain and compare profiles of RNA interactions in two or more different samples. In these embodiments, results obtained from, for example, the above method can be normalized to, for example, control RNA and then compared.

[0113] The method according to the invention further includes detecting changes in the RNA binding strength of the detected RBP when compared under different experimental conditions. Therefore, in some embodiments, two or more experimental conditions can be used to detect and compare RNA interactions in the same sample.

[0114] In certain implementations, the binding strength of RNA-binding proteins in two or more different samples can be compared to identify interactions associated with a specific disease or condition (e.g., interactions induced by the disease or condition). The different samples may consist of an "experimental" sample, i.e., the sample of interest, and a "control" sample, which can be compared to the experimental sample.

[0115] The method of this invention can be applied to plants, including but not limited to Arabidopsis thaliana, rice, corn, soybean, and tomato. This method can be used to quantitatively analyze the binding strength of RNA-binding proteins in plants, and to perform batch analysis of the binding properties of different RNA-binding proteins, thereby providing a reference for elucidating the binding domains of RNA-binding proteins.

[0116] Kits and Uses

[0117] The present invention also provides a kit comprising materials for performing the method for detecting RNA-binding proteins described herein, such as Trizol reagent, chloroform, low-salt SDS buffer, high-salt SDS buffer, ethanol, DNase, trypsin, etc.

[0118] The kit may also contain other reagents, such as reagents for mass spectrometry detection, buffer solutions (e.g., PBS), and washing solutions. Preferably, the buffer solutions (e.g., PBS) and washing solutions do not contain RNase.

[0119] The kit may also include: instructions for use of the kit to guide technicians in operation.

[0120] The present invention also provides the use of the kit for detecting RNA-binding proteins (RBPs) in the contents of cells, tissues or organisms, or for detecting the binding strength of RBPs.

[0121] Compared with existing plant RBP extraction and identification methods, the advantages of this invention include:

[0122] This invention improves upon the Plant Phase Extraction (PPE) method in several ways, including performing phase separation twice using Trizol reagent-chloroform, separating, washing, and dissolving the intermediate solid phase RNA-protein complex, using ethanol to wash the precipitate and remove DNA, and using a refined phase separation enhanced to 1000 J / cm³. 2 The cross-linking strength was improved, and the cross-linking was repeated once. The FASP enzymatic digestion method was used instead of the in-gel enzymatic digestion method. These improvements combined to simplify the experimental steps and effectively overcome the limitations of the Oligo(dT) capture technology in the RIC method. It can capture RBPs with weak binding ability and identify RNA-protein complexes with large molecular weights. Compared with the existing RIC method and PPE method, it increases the number of RBPs identified.

[0123] This invention proposes for the first time the concept of RNA binding affinity (RBI) for RBP. By performing bioinformatics analysis on two sets of peptide datasets, namely RBP and free protein, the protein information corresponding to the peptides is obtained. Based on the ratio of the mass spectrometry intensity peak of a certain protein in the two sets of data, the RBI quantification of RBP is realized on a large scale for the first time.

[0124] This invention is the first to identify a peptide in plants that directly binds to RBP and RNA. This has significant reference value for better understanding the RBP domain. Combined with pre-treatment steps, it helps to identify RBPs accurately on a large scale and enables batch analysis of different RBP properties.

[0125] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods and reagents in the following embodiments that do not specify specific conditions are generally performed under conventional conditions and with conventional reagents, or under conditions and with reagents recommended by the manufacturer.

[0126] Example 1: Extraction of RNA-RBP complex

[0127] In this embodiment, taking Arabidopsis thaliana as an example, a method for extracting RNA-binding proteins (RBPs) is provided, as illustrated in the schematic diagram below. Figure 1 As shown, the RNA-RBP complex obtained by the method of this invention is also known as the RBP complex, RBP group, or binding group. The steps of this method are as follows:

[0128] 1. Sampling: Arabidopsis thaliana was sown on 1 / 2 MS medium and cultured at 22℃ for 15 days before harvesting. Note: Immediately after harvesting, the seedlings were spread evenly on filter paper, ensuring that the roots were completely covered to prevent drought stress.

[0129] 2. Radiation-covalently cross-linked cells: Seedlings evenly distributed on filter paper were placed in an ultraviolet cross-linking instrument and subjected to radiation at 1000 mJ / cm². 2 The parameters are used for ultraviolet crosslinking, and the crosslinking is repeated once.

[0130] 3. Liquid nitrogen cryogenic grinding: Rinse the cross-linked seedlings once with ddH2O, drain the water with kitchen paper, put them into an RNase-free mortar pre-cooled with liquid nitrogen, add liquid nitrogen for quick freezing and grind, repeat grinding 3 times until the plant fragment powder is close to white.

[0131] 4. Collect the ground sample: Use a small spoon made from a 1ml RNase-free pipette tip to collect the thoroughly ground tissue powder into a 50mL RNase-free centrifuge tube pre-cooled with liquid nitrogen.

[0132] Phase separation is performed using Trizol reagent-chloroform in steps 5-6:

[0133] 5. Add 12 mL of Takara RNAiso PLUS (Trizol) vortex mixer and mix thoroughly. Then aliquot the mixture into 1.5 mL RNase-free centrifuge tubes and place them in a 30°C metal bath. Shake at 1000 rpm for 10 min.

[0134] 6. Add 200 μL of chloroform, vortex for one minute, let stand, and then centrifuge at 12,000 rpm for 10 minutes at 4°C in a low-temperature centrifuge.

[0135] Steps 7-12 involve separating, washing, and dissolving the intermediate solid-phase RNA-protein complex:

[0136] 7. After centrifugation, the liquid in the EP tube separates into three layers: an upper aqueous phase, a middle white solid phase, and a lower dark red organic phase. Use a pipette tip to aspirate and discard the upper aqueous phase. Insert a 1mL syringe into the lower layer to aspirate all the organic phase and collect it into a new 15mL RNase-free centrifuge tube for the extraction of free proteins. Retain the middle solid phase.

[0137] 8. Add 100 μL ddH2O, mix well by pipetting, vortex for 2 seconds, and centrifuge at 25°C and 5000g for 2 minutes.

[0138] 9. Remove and discard the upper layer of water. Use a new 1mL syringe to remove and discard the remaining lower organic phase.

[0139] 10. Add 100 μL of low-salt SDS buffer (50 mM Tris (pH 7.5), 1 mM EDTA, 0.1% SDS), mix well by pipetting, vortex for 2 seconds, and centrifuge at 5000 g for 2 minutes at 25°C. Collect the supernatant aqueous phase into a new 1.5 mL centrifuge tube. Name it IE1.

[0140] 11. Add 100 μL of high-salt SDS buffer (50 mM Tris (pH 7.5), 1 mM EDTA, 1% SDS), mix well by pipetting, vortex for 2 seconds, and centrifuge at 5000 g for 2 minutes at 25°C. Collect the supernatant aqueous phase into a new 1.5 mL centrifuge tube. Name it IE2.

[0141] 12. Centrifuge the collected products from steps 10 and 11 at 25°C and 5000g for 2 minutes. Add 200 μL of IE1 + 200 μL of IE2 to each tube, along with 40 μL of 5M NH4AC and 2 μL of Invitrogen. TM GlycoBlue TM Add a co-precipitant, and finally add 1200 μL of pre-cooled anhydrous ethanol and let it precipitate overnight at -80°C.

[0142] Steps 13-17: Wash the precipitate with ethanol to remove DNA.

[0143] 13. Centrifuge the overnight precipitate at 14,000 rpm for 30 minutes at 4°C using a low-temperature centrifuge. Discard the upper layer of ethanol, filter the tube opening with kitchen paper, and add 500 μL of 75% ethanol to wash the tube once by inverting it.

[0144] 14. After washing the product with 75% ethanol, centrifuge at 4°C and 14,000 rpm for 10 minutes using a low-temperature centrifuge. Discard the upper layer of ethanol, dry the tube opening with kitchen paper, and remove the residual ethanol with a pipette tip.

[0145] 15. Place in a vacuum apparatus and vacuum at 1000 rpm and 25°C for 10 minutes to dry.

[0146] 16. After complete evacuation, add 180 μL of Invitrogen to each tube. TM RNase-free ddH2O (1 μL of promega's RNasin was added to each 1 mL). After vortexing, the mixture was placed in a 4°C refrigerator to allow the product to dissolve completely.

[0147] 17. Add 20 μL of Invitrogen to each tube. TM Add 4 μL of Invitrogen after TURBO DNase buffer. TMAliquot 100 μL of TURBO DNase into 200 μL RNase-free PCR tubes. Incubate in a PCR instrument at 37°C for 15 min to remove DNA.

[0148] Steps 18-19 repeat phase separation:

[0149] 18. Aliquot the reaction product into new 1.5 mL RNase-free centrifuge tubes in 300 μL each, add 1 mL of Ltakara RNAiso PLUS (Trizol), vortex thoroughly, and place in a 30 °C metal bath and shake at 1000 rpm for 5 min.

[0150] 19. Add 200 μL of chloroform, vortex for one minute, let stand for 5 minutes, and then centrifuge at 12,000 rpm for 10 minutes at 4°C in a low-temperature centrifuge. Repeat steps 7-12 once.

[0151] Steps 20-21: Wash the precipitate with ethanol.

[0152] 20. Centrifuge the overnight precipitate at 14,000 rpm for 30 minutes at 4°C using a low-temperature centrifuge. Discard the upper layer of ethanol, filter the tube opening with kitchen paper, and add 500 μL of 75% ethanol to wash the tube once by inverting it.

[0153] 21. After cleaning, the product was placed on the mass spectrometry platform and centrifuged at 4°C and 14,000 rpm for 10 minutes using a low-temperature centrifuge. All ethanol was discarded and the sample was dissolved in 8M urea.

[0154] 22. FASP digestion and LC-MS separation and detection: Promega trypsin was used for FASP digestion at 37°C. The samples were then completely digested using a Thermo Scientific Zeba offline column for desalting and purification before being processed by BRUKER. TM timsTOF Pro 2 was used for LC-MS separation and detection.

[0155] The RNA-RBP complex obtained from Arabidopsis thaliana using the method described in this embodiment was detected by SDS-PAGE gel electrophoresis combined with Coomassie brilliant blue staining. The results are as follows: Figure 2 As shown in the figure, the method in this embodiment yields a large number of RNA-RBP complexes with a wide molecular weight distribution.

[0156] Example 2: Extraction of free proteins after phase separation

[0157] This embodiment provides a method for extracting free proteins after phase separation. The extracted free proteins are also referred to as the free phase. This embodiment also uses Arabidopsis thaliana as an example, and steps 1-7 are the same as in Example 1. After obtaining the dark red organic phase, the following steps are followed:

[0158] 1. Centrifugation: Aliquot the dark red organic phase into new 1.5 mL RNase-free centrifuge tubes at 500 μL each. Centrifuge at 12,000 rpm for 10 minutes at 4°C in a low-temperature centrifuge.

[0159] 2. After washing the precipitate with ethanol, repeat the centrifugation: Use a pipette tip to transfer the bottom 400 μL of dark red organic phase to a new 2 mL RNase-free centrifuge tube. Add 300 μL of anhydrous ethanol to precipitate the DNA. After adding, invert the tube to mix well, let it stand at room temperature for 3 minutes, and then centrifuge at 2000 g for 10 minutes at 4°C in a low-temperature centrifuge.

[0160] 3. After washing the precipitate with isopropanol, repeat the centrifugation: Carefully aspirate the supernatant along the tube wall and transfer it to a new 2mL RNase-free centrifuge tube. Add 1.125mL of isopropanol to precipitate the protein. After adding, invert the tube to mix well, let it stand at room temperature for 10 minutes, and then centrifuge at 2000g for 10 minutes at 4℃.

[0161] 4. Wash the precipitate with washing buffer and then repeat centrifugation: After centrifugation, a white cake-like precipitate will appear at the bottom of the tube. Discard the supernatant completely and add 2 mL of washing buffer (95% ethanol containing 3M guanidine sulfate) for washing. After adding the washing buffer, invert the tube and wash. Let it stand at room temperature for 20 minutes, then centrifuge at 7500g for 5 minutes at 4°C and discard the supernatant. Repeat the washing process three times. Finally, add 2 mL of anhydrous ethanol and vortex thoroughly. Let it stand at room temperature for 20 minutes, then centrifuge at 7500g for 5 minutes at 4°C and discard the supernatant.

[0162] 5. Dissolve the sample after vacuum drying: Dry the sample using a vacuum concentrator, and then dissolve it by adding 100 μL of 0.5% SDS + 8M urea solution.

[0163] 6. FASP digestion and LC-MS separation and detection: Promega trypsin was used to completely digest the sample using the FASP method. After purification using a Thermo Scientific Zeba offline column for desalting, the sample was then analyzed using a BRUKER column. TM LC-MS separation and detection were performed using timsTOFPro 2.

[0164] Example 3: Comparison of the intermediate layer RNA-RBP complex and free protein after phase separation

[0165] In this embodiment, the RNA-RBP complex is compared with free protein.

[0166] (1) Visual observation

[0167] After the first phase separation, the liquid was visible to the naked eye to be divided into three layers: an upper aqueous phase, a middle white solid phase, and a lower dark red organic phase. The RNA-RBP complex mainly aggregated in the middle white solid phase, while the free protein mainly aggregated in the lower dark red organic phase.

[0168] (2) Comparison of LC-MS spectra

[0169] The RNA-RBP complex was extracted from the intermediate layer after the first phase separation using the method in Example 1, and the free protein was extracted using the method in Example 2. The LC-MS patterns of the free protein and the intermediate RNA-RBP complex were compared, revealing significant component differences between the intermediate RNA-protein complex and the free protein in the organic phase. Figure 3 The correlation analysis results show that the correlation between the intermediate phase and the free phase is weaker than the correlation between the intermediate phases themselves. Repeated extraction of free protein and RNA-RBP complex from the intermediate layer were performed three times, followed by repeatability testing and inter-group difference analysis. The results are as follows: Figure 3 and Figure 4 As shown. Among them, Figure 3 The different biological replicates obtained showed a high correlation. Figure 4 It compares the binding activity of proteins under UV and non-UV conditions to screen out proteins that are significantly enriched under UV conditions, namely RBPs.

[0170] (3) Analyze the activity of RNA-binding proteins

[0171] The RNA-RBP complex (i.e., the intermediate phase protein under radiation covalent cross-linking conditions) was obtained according to the method of Example 1, with the RNA-RBP complex (intermediate phase protein under non-radiation covalent cross-linking conditions) obtained without radiation covalent cross-linking (non-radiation covalent cross-linking conditions) and with all other steps performed according to the method of Example 1 serving as a control. Free proteins in the precipitate phase were extracted from both groups according to the method of Example 2, namely, free proteins in the precipitate phase under radiation covalent cross-linking conditions and free proteins in the precipitate phase under non-radiation covalent cross-linking conditions.

[0172] The RNA binding activity index (RBI) was used to analyze the binding strength of RNA-binding proteins. The ratio of (amount of intermediate phase protein) / (total amount of free protein in precipitate phase) under radiation covalent crosslinking conditions to (amount of intermediate phase protein) / (total amount of free protein in precipitate phase) under non-radiation covalent crosslinking conditions was used.

[0173] Plotting the binding strength of RNA-binding proteins on the x-axis and RNA density on the y-axis, we can obtain results such as... Figure 6 The curves shown are further labeled with the RNA-binding proteins THO, AtCSP2, GRRBP3, eIF3C, NTF2, PDR7, RDM16, SR34, and SR34A identified in this invention. Figure 6 As shown.

[0174] Example 4: Identification of RNA-binding proteins obtained by the method of the present invention

[0175] In this embodiment, the RNA-binding protein (RBP) obtained from Arabidopsis thaliana using the method of Example 1 was identified, and the peptides in which RBP directly binds to RNA were analyzed. Steps 1-22 were the same as in Example 1. After obtaining the RBP trypsin product by FASP digestion of the sample, the following steps were performed:

[0176] 1. Aliquot 300 μL of the enzymatically digested sample into 1.5 mL RNase-free centrifuge tubes, add 30 μL of 5 M NH4AC and 2 μL of Invitrogen. TM GlycoBlue TM Add a co-precipitant, and finally add 1200 μL of pre-cooled anhydrous ethanol and let it precipitate overnight at -80°C.

[0177] 2. Centrifuge the overnight precipitate at 14,000 rpm for 40 minutes at 4°C using a low-temperature centrifuge. After discarding the upper layer of ethanol, filter the tube opening with kitchen paper and add 500 μL of 75% ethanol to wash the tube once by inverting it.

[0178] 3. After washing the product with 75% ethanol, centrifuge at 4°C and 14,000 rpm for 10 minutes using a low-temperature centrifuge. Discard the upper layer of ethanol, dry the tube opening with kitchen paper, and remove the residual ethanol with a pipette tip.

[0179] 4. Place it in a vacuum chamber and dry it at 1000 rpm and 25°C for 10 minutes.

[0180] 5. After complete evacuation, add 100 μL of Invitrogen to each tube. TM RNase-free ddH2O (1 μL of promega's RNasin was added to each 1 mL). After vortexing, the mixture was placed in a 4°C refrigerator to allow the product to dissolve completely.

[0181] 6.2.4 ml of the dissolved product was added to 8.4 ml of QIAGEN RNeasy Plant Mini Kit RLT buffer (provided in the QIAGEN RNeasy kit) (3.5 times), 200 μL of 1M DTT, and mixed thoroughly. The mixture was then aliquoted into 1.5 ml RNase-free centrifuge tubes at 700 μL each and incubated in a 60°C metal bath for 15 minutes.

[0182] 7. After adding 700 μL of anhydrous ethanol to each tube, centrifuge at 3000 g for 5 minutes at room temperature in 18 QIAGEN RNeasy mini spin columns (provided in the QIAGEN RNeasy kit).

[0183] 8. Add 500 μL of RPE buffer (provided in the QIAGEN RNeasy kit) to each tube and centrifuge at 3000g for 2 minutes at room temperature. Repeat step 8 once.

[0184] 9. After discarding the RPE buffer, centrifuge at 3000g for 2 minutes at room temperature.

[0185] 10. Elute the product with RNase-free water (provided in the QIAGEN RNeasy kit). Repeat once.

[0186] 11. After treating the eluent in an 85°C metal bath for 5 minutes, place it on ice for 2 minutes.

[0187] 12. Add the following to every 400 μL of reaction system:

[0188]

[0189] 13. After RNase digestion, the sample was completely digested using Promega's trypsin via FASP digestion. After desalting and offline purification using a Thermo Scientific Zeba column, the sample was then processed using a BRUKER... TM timsTOF Pro 2 was used for LC-MS separation and detection.

[0190] 14. Using Bruker ProteoScape software with the Arabidopsis thaliana proteome in the uniprot library as a library, protein information was retrieved to identify the RNA-binding protein peptides obtained by the method of this invention.

[0191] Upon identification, a total of 1260 RNA-binding protein peptides were obtained using the method of this invention. A bar chart showing the peptide distribution with peptide length on the x-axis and peptide number on the y-axis is shown below. Figure 7 The left figure shows a schematic diagram of a representative RNA-RBP complex domain. Figure 7 As shown in the figure on the right.

[0192] Example 5: Comparison of the method of the present invention with existing RIC and PPE methods

[0193] In this embodiment, RNA-binding proteins obtained by the RIC method described in "The increasing diversity and complexity of the RNA-binding protein repertoire in plants" (royalsocietypublishing.org) and the PPE method described in "Plant phase extraction: A method for enhanced discovery of the RNA-binding proteome and its dynamics in plants" were used as controls. The RBPs obtained in Arabidopsis thaliana in Example 4 were compared with RBPs obtained by existing RIC and PPE methods.

[0194] Correlation analysis was performed on the RBPs captured by the PPE method, the RBPs captured by the RIC method, and the RBPs captured in Example 4. The results are as follows: Figure 5 As shown, the number of RBPs obtained by the method of this invention that overlap with known RBPs is significantly higher than that of the randomized control group, indicating that the RBPs captured by the method of this invention are authentic and reliable. Simultaneously, the method of this invention also captured some RBP proteins not previously identified in PPE, such as double-stranded RNA binding proteins HYL1, RH36, and SR45a.

[0195] The various technical features of the above embodiments can be freely arranged and combined according to actual needs to achieve different experimental purposes. As long as these technical features are not combined in a contradictory manner, they should all be considered to be within the scope of this invention.

[0196] The above embodiments only illustrate several implementation routes of the present invention. In order to better demonstrate the technical features of the present invention, the process has been described in detail. These limited embodiments do not represent a limitation on the scope of protection of the present invention. Modifications and adjustments made by relevant researchers without departing from the conceptual framework of the method described in the present invention are all within the scope of protection of the present invention.

Claims

1. A method for detecting RNA-binding proteins in cells, tissues, or organisms, the method comprising: a) Using radiation to covalently crosslink the contents of cells, tissues, or organisms containing RNA-binding proteins to produce biological material containing crosslinked RNA-protein complexes; b) Lysing of biological material containing the cross-linked RNA-protein complex; c) The cross-linked RNA-protein complex is subjected to phase separation, the intermediate solid phase is taken for separation, washing and dissolution, and the precipitate is washed with ethanol to remove DNA. d) Repeat step c) at least once; e) Using FASP enzymatic hydrolysis, RNA-binding proteins are released; and f) Detect the released RNA-binding proteins.

2. The method as described in claim 1, characterized in that, Step c) includes: performing phase separation on the cross-linked RNA-protein complex using Trizol and chloroform; preferably, step c) includes: performing phase separation on the cross-linked RNA-protein complex using Trizol and chloroform, the cross-linked RNA-protein complex being phase-separated into three layers, taking the middle solid phase, and sequentially separating the RNA-protein complex in the middle solid phase using low-salt SDS buffer and high-salt SDS buffer, taking the supernatant, mixing the supernatants from the two separations in a ratio of 2:1 to 1:2, precipitating with a precipitant, and washing the precipitate with ethanol to remove DNA; Preferably, the weight percentage of SDS in the low-salt SDS buffer is less than 0.2%, more preferably 0.15% to 0.1%; preferably, the low-salt SDS buffer also contains Tris and / or EDTA; more preferably, the concentration of Tris is 10-100 mM, 10-90 mM, 20-80 mM, 30-70 mM or 40-60 mM, and the concentration of EDTA is 0.1-10 mM, 0.5-5 mM, 0.6-3 mM or 0.8-2 mM; more preferably, the low-salt SDS buffer also contains 50 mM Tris and 1 mM EDTA. Preferably, the weight percentage of SDS in the high-salt SDS buffer is higher than 0.3%, more preferably 0.5% to 1%; preferably, the high-salt SDS buffer also contains Tris and / or EDTA; more preferably, the concentration of Tris is 10-100 mM, 10-90 mM, 20-80 mM, 30-70 mM or 40-60 mM, and the concentration of EDTA is 0.1-10 mM, 0.5-5 mM, 0.6-3 mM or 0.8-2 mM; more preferably, the low-salt SDS buffer also contains 50 mM Tris and 1 mM EDTA. Preferably, the supernatant obtained by separating the RNA-protein complex from the intermediate solid phase using low-salt SDS buffer and the supernatant obtained by separating the RNA-protein complex from the intermediate solid phase using high-salt SDS buffer are mixed in a 1:1 ratio. Preferably, the precipitant is NH4AC and / or Invitrogen. TM GlycoBlue TM .

3. The method as described in claim 2, characterized in that, Step c) further includes one or more of the following reaction conditions: (1) The volume ratio of Trizol to chloroform is 100:1 to 30:1, preferably 80:1 to 50:1; (2) Trizol and chloroform are mixed by vortexing before the reaction is carried out; (3) Add Trizol first, react for a period of time, and then add chloroform; preferably, the reaction time of Trizol is 5 to 30 min, more preferably 10 to 20 min; (5) The reaction temperature of Trizol is 25-35℃, preferably carried out in a metal bath; (6) Trizol was reacted under shaking conditions at 500 rpm to 1500 rpm; (7) The precipitate is washed with ethanol at least once, preferably twice, three times or more; (8) DNA is removed by DNase, preferably by adding DNase and then removing DNA by PCR reaction; more preferably, the temperature of PCR reaction is 30-40℃ and the time of PCR reaction is 10-30 min.

4. The method according to any one of claims 1-3, characterized in that, In step a), the radiation covalent crosslinking is ultraviolet crosslinking; Preferably, the strength of the ultraviolet crosslinking is 500–2000 mJ / cm. 2 More preferably 600–1500 mJ / cm 2 800~1300mJ / cm 2 Or 900~1200mJ / cm 2 ; Preferably, step a) further includes a control group, wherein the control group does not undergo radiation covalent crosslinking; more preferably, the control group does not undergo ultraviolet crosslinking; even more preferably, the ultraviolet crosslinking intensity of the control group is 0 mJ / cm. 2 ; Preferably, the number of radiation covalent crosslinkings is at least one; more preferably, the number of radiation covalent crosslinkings is at least two.

5. The method according to any one of claims 1-4, characterized in that, In step d), step c) is repeated at least once, preferably only once.

6. The method according to any one of claims 1-5, characterized in that, In step e), FASP enzymatic hydrolysis is performed using trypsin; preferably, the hydrolysis temperature is 30–40°C; more preferably, urea is used to dissolve the product from step d), followed by FASP enzymatic hydrolysis; even more preferably, the urea concentration is 5–10 M; and / or, In step b), the lysis is liquid nitrogen cryogenic grinding, preferably grinding the biomaterial containing the cross-linked RNA-protein complex under RNase-free conditions and after pre-cooling with liquid nitrogen. And / or, In step f), the detection includes: detection using mass spectrometry; preferably, the mass spectrometry is GC-MS or LC-MS.

7. A method for quantitatively analyzing the binding strength of RNA-binding proteins, the method comprising: 1) Use radiation to covalently crosslink the contents of cells, tissues, or organisms containing RNA-binding proteins to produce biological materials containing crosslinked RNA-protein complexes; Cell, tissue, or biological contents that have not undergone radiation covalent cross-linking were used as control biological materials; 2) Lysis of biological material containing the cross-linked RNA-protein complex, and control biological material; 3) Phase separation was performed on the pyrolyzed biomaterial from step 2); 4) Extract RNA-binding proteins from the middle solid phase and free proteins from the lower organic phase, respectively; 5) Calculate the RNA binding activity index of the radiation-covalently cross-linked biomaterial and the RNA binding activity index of the control biomaterial, respectively: RNA binding activity index of radiation covalently cross-linked biomaterials = amount of intermediate phase protein ÷ amount of free protein in precipitated phase; RNA binding activity index of control biological material = amount of intermediate phase protein ÷ amount of free protein in precipitate phase; RNA binding activity index = RNA binding activity index of radiation covalently cross-linked biological material - RNA binding activity index of control biological material; Preferably, a higher RNA binding activity index indicates a stronger binding strength of the RNA-binding protein; More preferably, the intermediate layer RNA-binding protein is an RNA-binding protein detected using the method described in any one of claims 1-6.

8. A kit comprising reagents for performing the method for detecting RNA-binding proteins according to any one of claims 1-6, or reagents for the method for quantitatively analyzing the binding strength of RNA-binding proteins according to claim 7, wherein the reagents comprise one or more of the following: Trizol reagent, chloroform, low-salt SDS buffer, high-salt SDS buffer, ethanol, DNase, and trypsin. Preferably, the kit may further include reagents, buffer solutions, and washing solutions for mass spectrometry detection; more preferably, the buffer solutions and washing solutions do not contain RNase. Preferably, the kit may further include: Instructions for use of the reagent kit to guide technicians in its operation.

9. The use of the kit as described in claim 8 for detecting RNA-binding proteins in cells, tissues or organisms, or for detecting the binding strength of RNA-binding proteins.

10. As described in any one of claims 1-9, characterized in that, The cells, tissues, or organisms mentioned are derived from plants, animals, or microorganisms; Preferably, the plant includes: dicotyledonous plants, monocotyledonous plants, or gymnosperms; More preferably, the plants include: Arabidopsis thaliana, wheat, barley, rye, rice, corn, sorghum, sugar beet, apple, pear, plum, peach, apricot, cherry, strawberry, raspberry, blackberry, bean, lentil, pea, soybean, rapeseed, mustard, poppy, artemisia annua, oleanum, sunflower, coconut, castor oil plant, cocoa bean, peanut, gourd, tobacco, oil palm, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, grapefruit, spinach, velvetleaf, asparagus, cabbage, Chinese cabbage, bok choy, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugarcane, tea, pepper, grapevine, oyster grass, banana, poplar, willow, pine, fir, eucalyptus, purslane, jade tree, sago palm, simonium, natural rubber tree, and ornamental plants; Most preferably, the plant is Arabidopsis thaliana.

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