CN114431193B - Application of Prrc2b in preparation of medicine for treating demyelinating diseases - Google Patents
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- CN114431193B CN114431193B CN202111530185.3A CN202111530185A CN114431193B CN 114431193 B CN114431193 B CN 114431193B CN 202111530185 A CN202111530185 A CN 202111530185A CN 114431193 B CN114431193 B CN 114431193B
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Abstract
The invention discloses application of Prrc2b in preparation of a medicament for treating demyelination diseases. Experiments prove that Prrc2b is a novel m 6 A binding protein regulates oligodendrocyte fate determination, prrc2b gene knockout can lead to hypomyelination or demyelination, therefore Prrc2b can be regarded as the pharmaceutical target of the treatment of demyelinating disease. In addition, the invention also discloses application of Prrc2b in construction of an animal model with myelin sheath damage or demyelination.
Description
Technical Field
The invention belongs to the field of biological medicines, and relates to application of Prrc2b in preparation of a medicine for treating demyelination diseases.
Background
Myelin sheaths are a layer of myelin protein which wraps the periphery of nerve fibers and can wrap axons to insulate the axons from each other so as to avoid interference; the LangFeijie formed between myelin sheaths can make action potential carry out 'jump conduction' on axon, and accelerate signal transmission; in addition, myelin sheaths can also play a protective role surrounding axons.
Axon regeneration can be aided in the case of axonal injury. Myelin damage or dysplasia can cause myelin-related diseases such as multiple sclerosis, neuromyelitis optica, and the like, and various nervous system diseases such as Alzheimer's disease, depression, brain trauma, and the like, are found to be accompanied by myelin loss. The cells responsible for myelination in the brain are mainly Oligodendrocytes (OL), which develop from neural progenitor cells into Oligodendrocyte Precursor Cells (OPC) and then proliferate and differentiate into oligodendrocytes, which continue to differentiate into myelinated oligodendrocytes in vivo to wrap around axons, thereby forming myelin sheaths. Thus, normal development of oligodendrocytes is of great significance for normal development of myelin.
By now, studies have found that various factors can influence the developmental differentiation process of oligodendrocytes, including epigenetics, genes, and environment, etc., wherein various influencing factors such as DNA methylation, histone modification, chromosome remodeling, etc. are included in the epigenetics. In recent years, the role of mRNA methylation modification in oligodendrocyte development gradually becomes a research hotspot, but the research results of the mRNA methylation modification are few at present, and the mRNA methylation modification is used as an intervention target to regulate oligodendrocyte differentiation and further regulate myelination, so that the aim of treating demyelination-related diseases is achieved.
N-6 methyladenosine modification in mRNA methylation is a ubiquitous modification mode in eukaryotes, can regulate mRNA variable shearing, degradation and translation, and needs m corresponding to the variable shearing, degradation and translation of mRNA 6 Recognition of binding protein A. In our research, we found a new m 6 It is not clear whether the A binding protein, prrc2b, is involved in the regulation of the developmental differentiation of oligodendrocytesFurther exploration is needed to find whether they can be used as targets for intervention in the treatment of demyelination-related diseases. It has been reported that Prrc2A can regulate myelination, and it is unclear whether Prrc2b, as its homologous protein, plays a role in oligodendrocyte fate determination. The research of the application focuses on whether Prrc2b can regulate and control OPC development and differentiation, and further serves as a target for treating demyelinating diseases.
Disclosure of Invention
This application relies on a liquid chromatography mass spectrometry detection (LC-MS/MS) system and RNA m 6 A methylation sequencing technology (MeRIP-seq) proves that Prrc2b is a novel m 6 A binding protein. Then, animal model research is adopted to find Prrc2b as m 6 The knockout of the A binding protein affects the fate determination of oligodendrocytes and causes pathological damage to the animal, thereby affecting the behavior of the animal. Therefore, prrc2b can be used as an intervention target to regulate the development and differentiation of oligodendrocytes, thereby achieving the purpose of treating demyelination diseases.
According to one aspect of the present invention, there is provided m 6 The application of the A modified binding protein in constructing myelin sheath damage or demyelination animal models.
According to another aspect of the present invention, there is provided m 6 The application of the A modified binding protein in preparing a medicament for repairing myelin sheath damage and promoting myelination or remyelination.
According to yet another aspect of the present invention, there is provided m 6 The application of the A modified binding protein in preparing a medicament for treating demyelinating diseases.
M is 6 The A modified binding protein comprises YTH domain protein, eukaryotic initiation factor (eIF), and heterogeneous nuclear ribonucleoprotein (hnRNP).
In a specific embodiment of the present invention, said m 6 The a modified binding protein is referred to as Prrc2b.
Further, the medicament of the present invention includes an agent that promotes Prrc2b.
According to yet another aspect of the invention, there is provided a method of constructing an animal model of myelin damage or demyelinating disease, the method comprising inhibiting Prrc2b expression.
The agent that inhibits the expression of Prrc2b is not limited as long as it can inhibit the expression level of Prrc2b or inhibit the functional activity of Prrc2b.
The effective method for inhibiting gene expression mainly comprises the following steps: gene knock-out, antisense nucleotide technology and RNAi technology.
The gene knockout method comprises CRISPR/Cas9 technology, zinc Finger Nuclease (ZFN) technology and Transcription Activator Like Effector Nuclease (TALEN) technology.
The antisense oligonucleotide technology is a gene therapy technology which applies antisense oligonucleotide medicine to specifically combine with nucleic acid (DNA or RNA) in cells through Watson-Crick base pairing to form hybrid molecules so as to inhibit the expression of specific genes at the transcription and translation levels.
Introduction of double-stranded RNA consisting of sense and antisense RNA corresponding to mRNA sequences into cells can degrade RNA and silence genes, and this post-transcriptional gene silencing (PTGS) is called RNAi. Commonly used RNAi techniques use reagents including shRNA and siRNA.
According to yet another aspect of the invention, the invention provides a method of producing a polypeptide on RNA 6 A-modified recognition method comprising detecting the interaction of a Prrc2b protein with an RNA to be detected.
Further, the identification method comprises the following steps:
1) Extracting RNA;
2) Incubating Prrc2b protein with RNA to be detected;
3) Detecting the interaction between the Prrc2b protein and the RNA to be detected;
4) If Prrc2b is combined with the RNA to be detected, the RNA to be detected is judged to contain m 6 And (C) modifying.
Still further, the identification method further comprises: sequencing RNA bound to Prrc2b to determine m 6 A modification site.
Further, the method for detecting the interaction between the Prrc2b protein and the RNA to be detected comprises RIP and RNA pull-down.
According to yet another aspect of the invention, there is provided a method of repairing myelin damage, promoting myelination, or remyelination, the method comprising administering an agent that promotes Prrc2b.
According to yet another aspect of the invention, there is provided a method of treating a demyelinating disease, the method comprising administering to a subject in need thereof an agent that promotes Prrc2b.
The agent promoting Prrc2b of the present invention is not limited as long as the agent can promote the expression or activity of Prrc2b or a substance involved in the pathway upstream or downstream of Prrc2b, and is an effective drug for repairing myelin damage, promoting myelination or remyelination, or treating a demyelinating disease.
The drug of the present invention may include a naked gene sequence, an expression vector containing a gene sequence, a promoting miRNA, a transcription regulatory factor or a targeted small molecule compound, and the like.
The medicaments of the present invention may be used by formulating pharmaceutical compositions in any manner known in the art. Such compositions comprise the active ingredient, together with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form envisaged. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, ethanol, glycerol and the like, various preservatives, lubricants, dispersants, flavoring agents. Moisturizers, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration, formulations which may be used in such compositions may be in the form of their original compounds as such, or optionally in the form of their pharmaceutically acceptable salts, and the agents of the present invention may be administered alone, in various combinations, and in combination with other therapeutic agents. The compositions so formulated may be administered in any suitable manner known to those skilled in the art, as desired. When using pharmaceutical compositions, a safe and effective amount of the drug of the invention is administered to a human, and the specific dosage will take into account such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The medicine of the present invention may be prepared into various preparation forms. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.
The route of administration of the drug of the present invention is not limited as long as it exerts the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, transmucosal, dermal, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral. In some cases, the administration may be systemic. In some cases topical administration.
The dose of the drug of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained. The dose of the therapeutic agent or prophylactic agent of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.
Other therapeutic agents that may be administered with the agents of the present invention include therapeutic agents for the treatment and/or prevention of demyelinating diseases.
Further, the other therapeutic agents include, but are not limited to, corticosteroid hormones, immunosuppressive agents, immunoglobulins, antibiotics, vitamin B, glatiramer acetate, ocrelizumab or a pharmaceutically acceptable salt thereof, natalizumab or a pharmaceutically acceptable salt thereof.
Examples of corticosteroid hormones include dexamethasone, methylprednisolone.
Examples of immunosuppressive agents include cyclophosphamide, azathioprine, methotrexate, and cyclosporine a.
The term "demyelinating disease" as used herein includes central nervous system demyelinating diseases and peripheral nervous system demyelinating diseases.
Preferably, the demyelinating disease comprises multiple sclerosis, neuromyelitis optica, amyotrophic lateral sclerosis, inflammatory demyelinating disease, central nervous system neuropathy, central pontine myelination, myelopathy, tabes spinosus, syphilitic myelopathy, leukoencephalopathy, alzheimer's disease, guillain-barre syndrome, chronic inflammatory demyelinating polyneuropathy, anti-MAG peripheral neuropathy, charcot-elli-39340li-dus disease, genetic neuropathy susceptible to compression paralysis, peripheral neuropathy, optic neuropathy, progressive inflammatory neuropathy, barlow concentric sclerosis, scheld's disease, chronic inflammatory demyelinating polyneuropathy, progressive inflammatory neuropathy, acute disseminated encephalomyelitis, adrenomyeloneuropathy, progressive multifocal leukoencephalopathy, parkinson's disease, stroke, diabetic complications, small brain vasculopathy.
The term "in need thereof in the present invention includes mammals, including, but not limited to, humans, cows, horses, cats, dogs, rodents or primates. In some embodiments, the individual is a human.
The term "demyelination", also known as "demyelinating disease" in the present invention is a pathological process in which myelin is damaged that occurs in the nervous system. Myelin is a fatty substance formed in the CNS by glial cells called oligodendrocytes and in the PNS by schwann cells. Damage to myelin impairs signal conduction in the affected nerves. Thus, the reduction in conduction capacity leads to a deficiency in sensation, movement, cognition, and/or other functions depending on the nerves involved. The demyelination process may be due to genetics, infectious agents, autoimmune reactions, and unknown factors. These diseases are classified into central demyelination involving the CNS and peripheral demyelination affecting the PNS, based on the major site of demyelination in the nervous system. Demyelinating diseases can also be classified as inflammatory and non-inflammatory diseases based on the presence or absence of inflammation, and can be further classified as myelino-demyelinating diseases (myelino-demyelinating) in which normal and healthy myelin sheaths are destroyed by toxic, chemical or autoimmune substances; and demyelinating leukodystrophy, in which the myelin sheath is abnormal and degenerates.
The term "treatment" of the present invention with respect to a disease, disorder or condition characterized by or associated with demyelination refers to the administration of a therapeutically effective amount of a combination of drugs as described above, which is effective to alleviate the adverse symptoms associated with the disease, disorder or condition; preventing the appearance of such symptoms before they appear; slowing the progression of the disease, disorder or condition; slowing the worsening of symptoms; enhancing the onset of remission; slowing irreversible damage caused in the progressive chronic stage of the disease, disorder or condition; delaying the start of the stage of progress; lessening the severity of or curing the disease, disorder or condition; increased survival or faster recovery; and/or preventing the occurrence of said disease, disorder or condition.
Drawings
FIG. 1 shows a graph of immunoblot results of in vitro RNA pull-down experiments;
FIG. 2 is a graph showing the results of LC-MS/MS detection;
FIG. 3 shows a graph of mouse genotype identification results, in which A: prrc2b primer profile; b: identifying graph of Olig2-Cre primer;
fig. 4 shows a graph of the results of immunofluorescence staining of oligodendrocyte precursor cells, wherein a: an immunofluorescence profile; b: a histogram;
fig. 5 shows a graph of oligodendrocyte immunofluorescent staining results, wherein a: an immunofluorescence profile; b: a histogram;
FIG. 6 shows a graph of Black-gold myelin staining results, where A: prrc2b f/f Mouse myelin staining pattern; b: prrc2b f/+ (ii) a Olig2-Cre mouse myelin staining pattern; c: prrc2b f/f (ii) a Olig2-Cre mouse myelin staining pattern;
FIG. 7 is a graph showing the results of the balance beam and rotating rod test, wherein A: counting the passing time of the balance beam experimental mice; b: counting the sliding times of the balance beam experimental mice; c: and (5) counting the dropping time of the mice in the rod rotating experiment.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.
Example 1m 6 A binding protein assay
1. Main experimental materials
C57BL/6J mouse, HA-prrc2b transgenic mouse (provided by China academy of sciences and molecular cell sciences prominent innovation center), protein A/G magnetic bead (Bimake), pierce TM Anti-HA-tag magnetic beads (Thermo Scientific), TRIzol (Invitrogen), BCA kit (Bycnanthus), RNase (Promega), proteinase K (Roche), MNase (Biolabs).
2. Experimental method
1) In vitro RNA pull-down
Brain tissue of embryonic stage E16 mice (C57 BL/6J mice) was isolated, protein was extracted and BCA quantification was performed. Pretreatment of protein lysate: incubating the magnetic beads and the lysis solution at 4 ℃ for 1h to remove non-specific binding; magnetic (Thermo Scientific) pretreatment of streptomycin ligation: the beads were blocked at non-specific sites on the beads by incubating with 0.2mg/ml tRNA (Sigma) and 0.2mg/ml BSA at 4 ℃ for 1 h. And (3) incubating the pretreated protein lysate and magnetic beads with an RNA oligonucleotide probe labeled by Biotin for 2h at 4 ℃, washing the magnetic beads for 5 times, and detecting the protein.
2) High performance liquid chromatography
Firstly, separating the brain tissue of HA-prrc2b transgenic mice at 6 days (P6) after birth, extracting protein, and carrying out BCA quantification after the protein concentration is determined; a volume of 250. Mu.l of lysate was retained as an Input sample and RNA was extracted by Trizol lysis. Pretreating the tissue lysate with Protein A/G (4 ℃) for 2 hours, removing magnetic beads, and addingAdding HA-tag magnetic beads, mixing and incubating overnight at 4 ℃. The following day, the magnetic beads were collected, washed 8 times with NT2 buffer (200mM NaCl,50mM HEPES pH7.6,2mM EDTA,0.05% NP-40,0.5mM DTT,400U/ml RNase), and 1 XMNase buffer (50 mM Tris-HCl pH7.9,5mM CaCl) containing MNase was added 2 ) Incubate at 37 ℃ for 10min with rotation. The collected magnetic beads were washed 2 times with 1XPNK + EGTA buffer (50 mM Tris-HCl pH 7.5, 2mM EGTA pH 8.0,0.5% NP-40), then washed 2 times with NT2 buffer, then washed 1 time with 1 XPK buffer (100 mM Tris-HCl pH 7.5, 50mM NaCl,1 mM EDTA,0.2% SDS), collected magnetic beads were added to 200. Mu.l 1 XPK buffer containing proteinase K and incubated at 50 ℃ for 40 minutes. Collecting supernatant, extracting RNA with phenol chloroform, and performing on-machine detection by LC-MS/MS.
3)RNA m 6 A methylation sequencing (MeRIP-seq)
Oligodendrocyte precursor cells are separated and purified from cerebral cortex of HA-prrc2b transgenic mice at 3 days after birth (P3), RNA is extracted by Trizol lysis, and mRNA and binding m are fragmented and purified 6 A magnetic bead of antibody A (202003, synthetic Systems) is incubated, the obtained IP product is reverse transcribed to synthesize cDNA, then a linker is added to synthesize double-stranded DNA, and the synthesized DNA library is subjected to two rounds of amplification after purification and removal of ribosome, and then is tested on a computer.
3. Results of the experiment
1) In vitro RNA pull-down experiments and LC-MS/MS detection
By containing m 6 The results of detection after incubation of the A-modified RNA probe and the unmodified RNA probe are shown in FIG. 1, and compared with the unmodified RNA probe, the probe contains m 6 The a modified probe binds to Prrc2b protein.
The RNA bound to Prrc2b was isolated and quantitatively analyzed by chromatography and mass spectrometry, and the results are shown in FIG. 2, where Prrc2b protein was enriched to contain m more than the control 6 A modifies RNA.
2)m 6 A methylation sequencing
RNAm on oligodendrocyte precursor cells 6 A methylation sequencing, finding m on mRNA 6 The a modification may be recognized by Prrc2b protein.
The above data indicate that Prrc2b is a new m 6 A binding protein.
Example 2 physiological function study of Prrc2b
1. Experimental Material
C57BL/6J mice, prrc2b conditional knockout mice, MBP antibody (Abcam), olig2 antibody (Millipore), pdgfr alpha antibody (BD Bioscience), trizol (Invitrogen), BCA kit (Biyunnan biosciences), black-Gold II myelin staining kit (Millipore), CC1 antibody (Millipore).
2. Experimental methods
1) Breeding Prrc2b conditional knockout mice
Prrc2b is reacted f/f The mouse (constructed by doctor King Fengji of Beijing Life sciences research institute, awarded by doctor Zhang Jinhua, beijing university of traffic) was hybridized with Olig2-Cre mouse (awarded by professor Xiaobo, sichuan university, stock No. 011103, jackson Laboratory) to obtain Prrc2b f/+ (ii) a Olig2-Cre mice. Prrc2b f/f Mouse and Prrc2b f/+ (ii) a Olig2-Cre mouse hybridization to obtain progeny Prrc2b f/f 、Prrc2b f/+ (ii) a Olig2-Cre and knockout of Prrc2b gene Prrc2b in Olig 2-expressing oligodendrocyte lineage f/f ;Olig2-Cre。
2) Frozen sections and immunofluorescence staining
Mouse brain tissue was fixed with 4% paraformaldehyde, then dehydrated with 10%, 20% and 30% sucrose solutions in a gradient manner, and then brain tissue was embedded by OCT and then microtomed. After washing brain slices for 3 times by PBS, adding a blocking solution, blocking for 1h at room temperature, and then adding primary antibody for overnight shaking incubation at 4 ℃. Washing with PBS for 3 times the next day, adding fluorescent secondary antibody, and incubating for 1h at room temperature in a dark place; then, the nuclei were stained with hochests for 10min after 3 washes with PBS. After being washed by PBS, the brain slice is attached to a glass slide, and the slice is sealed by an anti-fluorescence quencher and analyzed by a laser confocal microscope.
3) Black-Gold II myelin staining
According to the kit instruction. 0.3% Black-Gold II staining solution and 1% sodium thiosulfate solution were preheated at 60 ℃ in advance. Mouse brain slices prepared by using a freezing microtome are washed 3 times by PBS and hydrated for 5min, then preheated Black-Gold II staining solution is added for incubation for 15-20min at 60 ℃, the staining degree of the brain slices is monitored every 3-5min, and the staining is stopped when the myelinated fibers are stained to dark red. The staining solution was washed off by adding water and incubated with 1% sodium thiosulfate at 60 ℃ for 3min. After washing with water for 3 times, the brain slices are spread on a glass slide and sealed by neutral resin.
4) Balance beam experiment
One end of a balance beam with the diameter of 0.5cm and the length of 1m is suspended, and the other end of the balance beam is hidden in a box; a mouse 3 months old is placed at the suspended end, and is stimulated by a strong light source to move towards the end of a dark box. Training for 3 times every day, wherein the interval is 1h every time, testing is carried out after the mouse can smoothly pass through the balance beam, and the time when the mouse reaches a dark box and the sliding frequency are recorded.
5) Rod rotation experiment
Setting parameters such as time, rotating speed and the like by the bar rotating instrument; the mice 3 months old are trained for two days in advance, and are trained for 3 times under a constant speed model of 10rpm/min every day, the interval is 1h every time, and the mice can be tested after being continuously on a rotating rod for 80-90 s. The mouse is placed on the channel for testing, after the device is started, the device starts to accelerate uniformly to a set rotating speed and then rotates uniformly, namely the rotating speed is accelerated from 4rpm/min to 40rpm/min within 5min, the falling time of the mouse is recorded, and the average value is taken after 3 times of testing.
3. Results
1) Propagation of Prrc2b tissue-specific knockout mice
The mouse genotype identification results are shown in fig. 3: prrc2b f/f Breeding the mouse and Olig2-Cre mouse to obtain offspring Prrc2b f/f 、Prrc2b f/+ (ii) a Olig2-Cre and knockout of Prrc2b gene Prrc2b in Olig 2-expressing oligodendrocyte lineage f/f (ii) a Olig2-Cre. Note: in FIG. 3: CKO is No. 1 mouse, prrc2b f/f (ii) a Olig2-Cre mouse; WT is No. 2 mouse, prrc2b f/f A mouse; HE is No. 3 mouse, prrc2b f/+ (ii) a Olig2-Cre mice.
2) Prrc2b deletion results in a reduction in oligodendrocyte precursor cell numbers
The immunofluorescent staining result of brain tissues is shown in figure 4, and Olig2-cre mediated Prrc2b knockout remarkably reduces the number of oligodendrocyte precursor cells marked by Pdgfr alpha/Olig 2 double positive markers.
3) Prrc2b deficiency reduces oligodendrocyte numbers
The immunofluorescent staining result of brain tissue is shown in fig. 5, and the oligodendrocyte cell lineage specific knockout of Prrc2b significantly reduces the number of oligodendrocytes marked by CC1/Olig2 double positive markers.
4) Knockout of Prrc2b by the oligodendrocyte lineage results in hypomyelination
The results of Black-gold II myelin staining are shown in FIG. 6, prrc2b compared to control mice f/f (ii) a Olig2-Cre mice exhibited a marked hypomyelination profile.
5) Olig2-Cre mediated Prrc2b knockout results in dyskinesia
The results of the balance bar and rotating rod experiments are shown in FIG. 7, where Prrc2b is compared with wild-type mice f/f (ii) a Olig2-Cre mice passed through the balance bar for an increased period of time (FIG. 7A), slipped more frequently (FIG. 7B), moved on the rotarod for a decreased period of time (FIG. 7C), developed dyskinesias, and produced behavioral effects.
The above data demonstrate that Prrc2b affects oligodendrocyte lineage development and myelination, resulting in dyskinesias, pathological injury and behavioral deficits.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that it would be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit of the invention, and these modifications and variations also fall within the scope of the claims of the present invention.
Claims (3)
1.m 6 Use of A binding protein for the preparation of a medicament for repairing myelin damage or promoting remyelination, said m 6 The a binding protein is Prrc2b.
2. M on RNA 6 A modified in vitro identification method, the in vitro identification method comprises the steps of detecting the interaction of Prrc2b protein and RNA to be detected;
the in vitro identification method comprises the following steps:
1) Extracting RNA;
2) Incubating Prrc2b protein with RNA to be detected;
3) Detecting the interaction between the Prrc2b protein and the RNA to be detected;
4) If Prrc2b is combined with the RNA to be detected, m is determined to be contained in the RNA to be detected 6 A modification;
the method for detecting the interaction between the Prrc2b protein and the RNA to be detected comprises RIP and RNA pull-down.
3. The in vitro identification method according to claim 2, further comprising: sequencing RNA bound to Prrc2b to determine m 6 A modification site.
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