Yi Gene｜In-depth review: epigenetic regulation of m6A RNA methylation in brain development and disease

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2020年12月,《J Cereb Blood Flow Metab》杂志发表了“Epitranscriptomic regulation by m6A RNA methylation in brain development and diseases”的综述文章,讨论了m6AThe molecular mechanism of regulation and the developing brain、The role of physiological and pathological process.

期刊：JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM

日期：2020.12

IF：6.96

摘要

细胞RNAThere are a variety of different chemical tag,epitranscriptome（epitranscriptomic）修饰.Adenosine inN6site methylationN6-甲基腺苷（m6A）,Is the most abundant in mammals and reversible apparent transcriptome modification.m6A信号由writers、erasers和 readerscomposed protein-mediated.Continuously new research evidence thatm6AMethylation can be regulatedmRNAMultiple Aspects of Metabolism,For example, splicing、core out、稳定性、Translation and degradation,Leading to gene expression fine-tuning.The brain show show high abundance ratios of developmental change in the bodym6A甲基化.in the brain,m6AMethylation bias neurons transcript,sensitive to neuronal activity.在健康的大脑中,m6AMaintains some growth and physiological process,neurogenesis、axonal growth、突触可塑性、昼夜节律、Cognitive Function and Stress Response.m6AImbalance will lead to acute and chronic central nervous system（CNS）损伤、The pathogenesis of brain and nerve mental illness.本综述讨论了m6AThe molecular mechanism of regulation and the developing brain、The role of physiological and pathological process.

图1：mRNAThe common apparent transcriptome modification.Mature RNA有 172种化学修饰;其中72Methylation is modified variant.Study the most apparent transcriptome modifications haveN6-甲基腺苷（m6A）、假尿苷（ψ）、5-甲基胞苷（m5C）、N1-甲基腺苷（m1A）,核糖2'-O-甲基化（Nm）和N4-乙酰胞苷（ac4C）.

表1：m6AThe role of methylation in the developing brain

表2：m6AThe role of methylation in the brain physiological

表3：m6AThe role of methylation in brain disease

m6A的分子机制

m6Amethylation bym6A甲基化转移酶（m6A writers）、m6A去甲基化酶（m6A erasers）、m6A识别蛋白（m6A readers）调控,其中,m6A readers和反readers（Anti-readers）决定m6AModifies the fate of transcripts.

图2：m6AMolecular Mechanisms of Methylation.m6A修饰主要发生在3'-UTR中的RRACH motifs上（Ris a purine base,A是m6Amodified adenine,Hnon-guanine base）.m6Amainly composed of catalytic subunitsMETTL3and regulatory subunitsMETTL14组成的m6AMethylation transferase complex（writers）沉积.m6AMethylation is reversible,甲基化的RNA被FTO和ALKBH5去甲基化,Have different removal mechanisms.m6A readersby specificityYTHDomain structure combined with methyl to conductionm6A信号.此外,m6AMethylation ism6A反readers G3BP1／2排斥,优先结合RNA未甲基化的m6A motifs.

m6A 的添加（writer）

m6A writersComplex is mainly composed ofMETTL3、METTL14和Wilms肿瘤相关蛋白（WTAP）Of three indispensable sub,Individual loss in mouse embryos with deadly.此外还包括VIRMA、ZC3H13、RBM15/15B、CBLL1Specific functions such as have not been detailed assessment of sub.m6ASedimentary transcription specificity and site specific mechanism is not clear.然而,mRNAs的de novo m6AMethylation level by transcription factors and methylation transferase interactions、RNAPolymerase elongation and epigenetic modifications（such as histonesH3第36lysine methylation）影响.METTL3的SUMOInhibition in vivom6Amethyltransferase activity.大脑m6A writerComplex mainly located in nucleus neurons in.detected in synaptic componentsMETTL3和METTL14,That area outside the cells might happenm6Amethylation dynamics.

m6A的去除（erasers）

m6AMethylation is reversible.在真核生物中,m6A被FTO（fat mass and obesity‐associated protein,）和ALKBH5（alkylation repair homolog 5）两种m6ADemethylase removal.FTOthrough the neuronexportin2Protein interactions between the nucleus and cytoplasm shuttle,同时FTOThere also exist in axons and dendrites shaft cells such as area,揭示了m6AThe dynamic removal and other apparent transcriptome tag.FTO依赖m6AMethylation and dopaminergic neurotransmission to、Adult neurogenesis and axon elongation about.Due to these effects,FTOKnock out can lead to stunted growth after birth of mice and humans、Microcephaly and functional deficits.

ALKBH5Wide express,Highest expression in lung,followed by the testicles.与FTO不同,ALKBH5Located in the nucleus spot area,cytoplasm excludedmRNAdynamic demethylation of.小鼠ALKBH5Knock out lead to highly specific sperm damage phenotypic.Although to the transcription of the selective methylation enzyme is unclear,But a recent study put forwardm6A RNA甲基化与FTO或ALKBH5The interaction depends on the sequence of the background.此外RNA结合蛋白（RBP）通过招募FTOpromote proximalm6ATo methylation and give transcript selective.

m6A The identification of proteins and the identification of protein（readers 和anti-readers）

m6A甲基化RNAThe downstream effects ofRBP介导（m6A readers）,post-transcriptionalRNA的加工.Recognize proteins that bind directlym6A、增加RNA结合motifsaccessibility andRBPexclusion to identifym6A.m6A Identification of proteins by highly conservativeYTH（YT521-B homology）domain directly withm6A甲基化的RNA结合.在哺乳动物中,有YTHDF1、YTHDF2、YTHDF3、YTHDC1、YTHDC2五种YTH蛋白,YTHDC1Identify the nucleus transcription of thism6A,其他YTHProtein identification cytoplasmic transcription of thism6A.YTH蛋白与m6A motifsCombination ratios are similar,可以调节mRNA m6Amodified splicing、nuclear output、Translation and degradation.

IGF2BPs蛋白（Insulin-like growth factor 2 mRNA-binding proteins）and certain neuron-specificRBP一样,也是潜在的m6A 识别蛋白.stress granule protein,例如GTP酶激活蛋白（SH3结构域）结合蛋白1和2（G3BP1和G3BP2）被命名为m6Aanti-recognition protein,The identification of proteins with thousands of transcription in this3’-UTRunmethylated inm6A motifs CAACUCbind and promote its stability.

m6AMethylation Research Technology

Methyl don't change the base pairing of adenosine,且m6AChemically unmodifiable,Therefore standard hybridization and sequencing technology tests.基于m6AAntibody spots and immune, hybridization（immuno-northern blot）Can detect overall fasterm6A水平,But lower sensitivity and half quantitative.液相色谱/Mass spectrometry can high sensitive quantitativem6A水平,But doesn't provide sequence background and site information.m6APrecipitation and the second generation sequencing（MeRIP-seq）可以以50-200The resolution of the nucleotides, analysism6A甲基化.另外m6ASingle nucleotide resolution crosslinking and precipitation can be identified by single nucleotide resolution specificm6A残基,But applies only to verify the specific sitesm6A变化.

m6A敏感性MazFEnzymatic digestion sequencing（RNase MazF-sequencing ,MAZTER-seq）和RNAModified target sequencing（DART-seq）Can to transcriptome range at the same timem6AMethylation of single nucleotide resolution and stoichiometric quantitative.在MAZTER-seq中,未甲基化ACA motifsupstream bacteriaRNase（MazF）selective cleavage,且ACA reads与m6AAbundance correlation,But this method in mammals is16%-25%的m6Asite can be captured.DART-sequsing engineered fusion proteins,m6Abinding domainYTH的Nterminal and cytosine deaminase、载脂蛋白B mRNAEdit the enzyme、catalytic peptide1editing domain fusion of.

Recent studies have usedCRISPR（clustered regularly interspaced short palindromic repeats） myelinationSite specific apparent transcriptome modified transcription editor to analysis of specificm6Arole of site.for a specific areaRNA m6AThe precise effect of integral.

图3：m6AMethylation Research Technology.LC-MS/MSquantifiablem6A修饰（a）.MeRIP-seqCan be identified within the scope of the transcriptomem6A图谱.MAZTER-seqBy single nucleotide resolution displaym6ASite and methylation of stoichiometric（b）.METTL3或FTOFusion to sequence specificitygRNA靶向m6AInactive near the sitedCas9的CRISPR/Cas9Gene editing can adjust site specificm6A甲基化（c）.

m6AIn the brain cell type specific distribution

Clear cell specificity methylation patterns in understanding the physiological and pathological conditions of intercellular communication is important.大脑的MeRIP分析显示m6AModified anomalies in the prevalence of neurons was above the glial transcript,m6AEffect of protein expression in neurons than in glial cells wider.m6AModify the transcription and their interactions group located in axons and dendrites axis,Show that its role in the activity dependency gene regulation.while non-neuronal cellsm6AAstrocytes in synapse formation transcript,For example, to participate in the synapse formationSparcl1和Slc1a2、As well as participation in microglia complement cascade transcriptCX3CR1,have passed the heightm6A修饰.with inflammatory response、proximal tubule development、丝裂原活化蛋白激酶（MAPK）级联和cAMPResponse Element Binds Associated5000A week of spontaneously hypertensive rat microvascular cellsmRNA显示约7500 m6A peaks,表明m6AThe importance of methylation in the neurons function.m6AIn the oligodendrocytes specific transcription（如Olig2）also very extensive.总之,m6ASignal mainly in neurons in,To some extent is also available in otherCNScell types seen in.

m6AMethylation of the transcription regulatory mechanism

m6AMethylation modification affect many downstream function,包括RNA剪接、nuclear output、稳定性、Translation and degradation.m6A RNAThe methylation function results fromm6A readers决定,The specific cellular mechanisms to targetRNA.m6ARegulation is dynamic,up-regulated by heat shock stress5’-UTR区域的Hsp70 mRNA m6A以促进capindependent translation.Hypoxic stress up-regulatedmRNAtargets such asGlut1、c-Myc和Jun的m6A,In order to enhance the stability of efficiency without affecting the translation.

图4：m6APost-transcriptional regulation of methylation

剪接（Splicing）

在METTL3knockout cells,m6AAbundance difference splicing exons related,Show that its role in alternative splicing.对MeRIPexons in the datam6A motifs分析结果显示m6A发生在5'和3'near the splice site.Caused by the difference splicing sites to choose alternative splicing mainly by serine/Arginine enriched splicing factor（SRSF）Combined with splicing site near cis regulation,Resulting in exon is embedded or exclusion.exons in adipocytesm6A增强了SRSF2to promote exon inclusionRNA招募.细胞核m6A readers YTHDC1通过与SRSF3Binding promotes exon inclusion,同时抑制SRSF10Combined with the splicing enhancer element.因此,m6ABy adjusting the splicing regulatory factors of targetRNAClose to regulate alternative splicing.

nuclear output（Export）

ALKBH5Knockout cells show cytoplasmmRNApart significantly increased,ALKBH5overexpression to normalize it,表明m6AMethylation is regulatingmRNArole in nuclear export.mRNAA nuclear protein subunits from more complexTREX（transcription-export）驱动,TREX与剪接的mRNA互作,Induction of the through hole complex nucleus into the cytoplasm.m6Amethyltransferase willTREX招募到m6A修饰的RNAeffective nuclear output.m6Amethyltransferase subunitVIRMA和WTAPKnockout inhibits this process.YTHDC1Knockout increasesm6AModified transcript nuclei to keep,在促进RNAThe nuclear output plays an important role.另一个m6A识别蛋白FMRPalso mediatem6A修饰的mRNAcore out,但在FMRPblocked in knockout mice.FMRP通过促进Notch和Hedgehog信号相关mRNANucleation to regulate neural differentiation.从机制上来说,FMRP与exportin 1protein association mediationm6A修饰RNAnuclear output.因此,m6ABy stimulating way dependence recruit output factor to adjust variable transcript out nuclear.

稳定性（Stability）

约1/10neural transcriptomemRNAStability of decisionmRNA丰度.mRNA稳定性与RBP和目标mRNACompetitive binding correlation.RBP特异性结合m6AMethylation transcript to control the stability of.此外,enriched in certain neuronsm6A识别蛋白,包括FMRP和PRRC2A,与YTHDF2Competitive combination of methylation transcript,thus preventing its degradation.

翻译（Translation）

YTHDF1translation initiation factor3（eIF3）interaction promotesm6AModified transcript translation,而YTHDF1Knockout inhibited the process.YTHDF3与YTHDF1combine to promotem6ATranslation of modified transcripts.m6AIn the translation of the regulation mechanismmRNAsite-specific.YTHDF1Directed translation regulation is mainly inmRNA的3'-UTR区域的m6A位点,而5'-UTR区域的m6Asite directly witheIF3互作,leads to translation initiation withcap无关.约13%endogenous ringRNA被m6A甲基化,YTHDF3Recruit translation initiation factorseIF4G2和eIF3Alead to its translation.

降解（Degradation）

m6A识别蛋白YTHDF2为m6A甲基化RNA招募RNA衰变机制,包括CCR4-NOTdeadenylase complex andRNase P/MRP,to control them inP-bodiesdecay in.YTHDF2Knock out can bem6AModified transcript half-life extend about30%.

转录（Transcription）

RNA m6AMethylation and transcriptional regulation related.lncRNA的XIST转录本（X-inactive specific transcript）比其他RNA有更多m6A位点, METTL3敲除m6A会损害XIST介导的Xrelated gene transcriptional repression.m6ASite in the regulation of transcription terminationR-inside the ring structureRNAvery broad.METTL3Knock out near the end of the transcription sitesRRing reduction,表明m6AIs essential to the effective transcription termination,YTHDF2与m6A修饰的RCombine to make the damage stability and to prevent its accumulation.in stem cellsMETTL3将m6ADeposited on chromosomes to regulateRNA上,而YTHDC1to degrade,Lead to closure of chromatin and transcriptional inactivation.

m6AThe role of methylation in the developing brain

神经发生（Neurogenesis）

Epigenetic modifications can regulate neural stem cells(NSCs)proliferation and differentiation.多项研究表明m6AMethylation regulates neurogenesis.在发育过程中METTL3Expression peaks early（peaks）,FTOExpression in neurogenesis is latepeaks.METTL3和METTL14The knockout extendedNSCs细胞周期进程,Thus delay upper-middle-class postnatal mice cortex neurons produce.METTL14和YTHDF2The knock out resulting in ventricular enlargement and cortical thickness decrease.E13.5forebrain,and cell cycle（Cdk9、Ccnh和Cdkn1C）Neural stem cells and specific transcription factor（Pax6、Sox1和Emx2）Several related transcripts arem6A标记,它们在METTL14敲除NSCsincreased stability.Neurod1和Neurod2transcript inNSCs中被m6A标记,表明m6AThe role of transcription for the methylation patterns.YTHDF2knockoutNSCsReduced proliferation and differentiation,源自YTHDF 2敲除的NSCsWith shorter neurites and influenced by oxidative stress.With the negative control stem cell proliferationJAK–STATThe transcription of pathways related to thisFlrt2和Ptprd,在YTHDF2敲除NSCsBy high methylation and stability.METTL14敲除NSCs中H3K27ac、H3C27me3、H3K4me3Significant increase in protein abundance.而m6ASilence increased proliferation related genes（如Egr2和Egr3）Gene silencing near promotersH3K27me3标记,and differentiation-related genes（如Kif26a、Gas7和Pdgf1b）Activation of genes near promotersH3K27mac标记.Use of histone acetyltransferaseCBP/p300and histone methyltransferasesEzh2Inhibitors regulate these changes can promoteMETTL14knockoutNSCs增殖.表明m6AInduced changes in histone regulationNSCs基因表达.从机制上讲,未去除m6A标记的CBP/p300会破坏METTL14knockout embryosNSCs神经发生.由于Ezh2过表达,METTL3knockout reducedEzh2in transcriptH3K27me3的m6A,减少了NSCsproliferation and differentiation.Taken together, these studies show,during neurogenesis,RNAInteraction between modifications and histone modification.

m6A erasersAlso play a role in neurogenesis,FTOKnock out the brain derived neurotrophic factor（BDNF）Signaling pathways related transcription of thisBDNF、PI3K、Akt1、Akt2、Akt3和S6K1中的m6A水平升高,Transcripts are subsequently degraded,lead to adulthoodNSCsdecreased proliferation.FTOKnockout reduces maturationBDNF蛋白水平,reduced survivalMAPK信号通路的激活,Resulting in adult neurogenesis is damaged.

gliogenesis（Gliogenesis）

In addition to the oligodendrocytes happen,Under the background of glial cell line progressm6ALess researched on signaling.PRRC2A与YTHDF2Competitive binding stabilizationm6AMethylation oligodendrocytes decision factorOlig2 的mRNA,因此PRRC2ADefects in mice can't mature oligodendrocytes and show low.另外,FTOdemethylation and blockingOlig2的PRRC2AStable dependencies,因此FTOOverexpression mouse phenotypingPRRC2A缺陷小鼠.METTL14In less sudden knock out of glial cell line cells leads to several transcription factors（Hey1,Klf19, Zeb2）、histone modifier（Hdac3,Kdm2b,Prdm2）and growth factors（Igf-1,Fgf, Bmp）低甲基化,Promote the oligodendrocytes mature,To reduce the number of mature oligodendrocytes,cause myelin dysplasia.METTL14Knock out also leads to oligodendrocytes specific protein fibers（Nfasc）Splice isoform reduction,随后产生Ranvier形态异常.且METTL14和YTHDF2缺陷的NSCsFailed to differentiate into astrocytes.这些研究表明m6AThe glial cells mature.

cerebellum development（Cerebellar development）

小脑的m6AMethylation is twice of the cerebral cortex.A variety of apoptotic cells（Dapk1,Fadd,Ngfr）and synaptic transcripts（Grin1,Atp2b3,Grm1和Lrp8）在METTL3Knockout mice cerebellum low methylation,Leading to serious cerebellar granule cells apoptosis and dysplasia.during postnatal development,Cerebellum were found in a set of unique stage specificm6A甲基化mRNA,表明m6AMethylation is essential to the cerebellum development.

axonal growth（Axonal growth）

m6AMethylation in the process of regulating the development in the axon guidance and extendedmRNA翻译.dorsal root ganglion（DRG）and dorsal spinal cord（DSC）轴突中的RNAshowing high abundancem6A甲基化和m6A调节蛋白FTO和YTHDF1.在DRGin neuron axons,FTO使Gap43To methylation in order to promote the translation leads to the axon elongation,YTHDF 1促进DSCMethylated axon guidancemRNA Robo3.1的翻译.

m6AThe role of methylation in physiological brain

突触可塑性（Synaptic plasticity）

m6AMethylation in synaptic transcript is very broad(约76%突触后mRNA和30%突触前mRNAMethylated in mice）.研究表明,METTL14、FTO和YTHDF1、2和3Located in the cortex and hippocampus neuron dendritic process,且超过1200synaptic organization、组装、Mature and synaptic transmission regulation of transcription by high methylation.FTOKnockout mice brain synaptic transmission relatedmRNAMethylation increases.in hippocampal neuronsYTHDF1Knock out lead to excitatory synaptic structure and function defects,including narrowing of the head of the spine、Decreased levels of postsynaptic density、Excitatory postsynaptic currents decrease.in striatal neuronsMETTL14Knockout reduces and the excitability of neurons damaged the transcription of synaptic plasticity associatedm6A甲基化.These observations show thatm6AThe key role of methylation in synaptic plasticity.

生物钟（Circadian clock）

用3-Denitrification adenosine inhibit clock transcript asPer1、Per3、Tef、Dbp、Nfil3、Bhlhe41和Nr1d的m6A甲基化,Can prolong the mice body clock oscillation.METTL3Inhibition to methylation clock transcriptPer2和Bmal1nuclear accumulation of.CK1δ激酶（clock protein degradation）的m6A高甲基化,敲除CK1δ基因中的m6A motifsIncreased its translation in the brain,Mice showed longer rhythmic movement activities.因此,m6AMethylation may play an important role in circadian rhythm.

behavioral adaptation（Behavioral adaptation）

by modulating neuroplasticity,m6AMethylation can affect behavior to adapt to.m6AMethylation interference can change the mice cognitive、成瘾、Anxiety and Depressive Behavior.Through the fear conditioning behavior training significant changes in the brains of micem6A甲基化.研究证明,敲除FTO或METTL14或YTHDF1Will damage the mice cognitive.在FTOKnockout mice induced by cocaine was observed in the movement and reward stimulation is damaged,某些FTOVariation is closely related to human drinking.FTOKnockout also inhibit the depression and anxiety behavior of mice,This is due to inflammation of the gut microbes disorders cause to reduce.

应激反应（Stress response）

in non-neuronal cells,various environmental stresses（如热休克、缺氧、Oxidative stress and ultraviolet light）,使m6AEpitranscriptome changes.In mice with acute bondage stress result in the prefrontal cortexm6ALow methylation and the amygdala high methylation.m6AAll the enzymes in the transcription start site10kbThe upstream areas contain sugar cortical hormone component,Verify the stress response of the forecast control.同时,stress-inducedm6AMethylation suppresses the synaptic plasticity and morphogenesis of related transcription of this translation,如Camk2n1、Dusp1和Homer1.METTL3和FTONeuron specific knock out improved clues/situational fear memory.最近研究表明,after fear conditioning,m6A水平升高,prefrontal cortex and hippocampusFTO表达减少.In the hippocampus and the prefrontal cortexFTOInhibition also strengthened the fear memory.These studies showm6AThe role of signal in stress-related illness.

m6AThe role of methylation in brain disease

中风（Stroke）

Secondary brain damage after a stroke by a variety of pathological synergy mediated,including excitotoxicity、水肿、细胞凋亡、线粒体功能障碍、氧化应激、Endoplasmic reticulum stress and inflammation.最近的研究表明,Epigenetic changes regulate dysfunction after stroke.Mannheim–HeidelbergStroke research shows,FTOVariation and genetic correlation between transient ischemic attack.4000个m6A单核苷酸多态性（SNPs）（包括IRF6和NDST1基因）,Proved to be associated with ischemic stroke.Rodent model shows that cut brain ischemic strokeFTO水平,导致m6AMethylation increases.in ischemic brain,several mediated apoptosis（Fas,Trib3和Bcl2a1c）and inflammation（Tnf,IL-6和Sele）transcripts are hypermethylated.Stroke upYTHDF1表达,可能与m6AInflammation and apoptosis in methylation transcript combine to increase the translation.FTOExpression through the stability of antiapoptotic transcriptBcl2To avoid cortical neurons in oxygen-Glucose deprivation of cell apoptosis after.Gerbil forebrain ischemia lower hippocampalFTO蛋白表达,When the hippocampal neurons of oxygen,The phosphate and tension induced cell death protein homologous transcription of thism6A水平升高,While the low temperature back to normal and raise the neurons survival.

最近的一项研究表明,Congenital arteriovenous malformation vascular dysfunction（AVM）in the patient's brainWTAP表达降低.WTAPknockout reducedm6ALevel and cell adhesion moleculesDSP（desmoplakin）表达,Results in the decrease of endothelial cell angiogenic potential.METTL3Expression and brainAVMsNegative correlation of lesion size,Notchpathway regulatorDTX3L与AVM形成有关.在METTL3knockout in endothelial cells,DTX3L mRNAShow the low methylation or silence,表明m6Acritical to its stability.IRF6in the transcriptm6A SNPassociated with intracerebral hemorrhage in humans.这些研究表明m6AMethylation in the role of head injury incidence of stroke and stroke.

周围神经损伤（Peripheral nerve injury）

Peripheral nerve injury causes axon discontinuous、Myelin fiber loss and neurons died.表观遗传修饰（including histone acetylation andDNA羟甲基化）By increasing the renewable related gene（RAG）Transcription to promote axon regeneration after peripheral nerve injury.最近的一项研究表明,DRG中许多RAGs（Atf3,Sox11,Gadd45a和Tet3）以及核糖体（Rps14,Rps20,Rps23,Rps28,Rps29,Eif1a和Eif3b）After sciatic nerve injury in micem6A RNAelevated methylation levels.METTL14或YTHDF1Knockout reduces damageDRG中RAG的翻译,And reduces the axon growth and neural control again,lead to limited functional recovery.

创伤性脑损伤（Traumatic brain injury）

多项研究表明,Epigenetic modifications in traumatic brain injury（TBI）中具有重要作用.adult mouseTBI在受伤后6-24Hours reduced the hippocampus inMETTL3Expression and overallm6A水平,TBIHouhai hippocampus in many differencesm6AMethylation this transcription regulation neurons metabolic process.大鼠TBIalso reduces the cerebral cortexMETTL14和FTO 中的mRNA表达,许多m6Amodified transcript,如CD14（炎症）、Dll4（Notch信号）和Bcl2（凋亡）是已知的TBIPost-pathological promoters.FTOInhibition intensifiedTBIpost neurological deficit,but does not affect cognitive function.

阿尔茨海默病（Alzheimer’s disease）

Epigenetic disorders is alzheimer's disease（AD）Protein aggregation and neuron loss in the driver.double transgenicADThe hippocampus of mice showedMETTL3induce andFTO抑制,lead to involvement in dendritic development、synapse growth、presynaptic and postsynaptic assembly、Glutamate receptor signaling、axon guidance、Long-term enhanced overallm6A甲基化变化.means abnormalm6A甲基化是ADpromoters of pathogenesis.m6A去甲基酶FTOin triple transgenicADIncreased expression in mouse brain,FTOKnockout reduces phosphorylationtauwithout affecting neuronsAβ的积累.Further research proves,This effect is made up of tuberous sclerosis complex1（tuberous sclerosis complex 1）methylation-mediated,The complex is adjusting mammalstauEgg Bai Lei drug upstream kinase targets of inhibitor.与野生型小鼠相比,FTO敲除ADShow better cognitive function in mice.总的来说,FTOModulation may be a reliefADA potential strategy of related defects.

帕金森病（Parkinson’s disease）

Epigenetic changes,如SNCA启动子（转录α-synuclein gene）处的DNAMethylation and histone acetylation with lowerα-Synaptic nucleoprotein aggregation and Parkinson's disease（PD）进展有关.FTOknock outm6Aelevated methylation levels,And reduce the dopaminergic pathway related transcription of this（如Drd3,Girk2）翻译.6-羟基多巴胺（6-OHDA）诱导PDin rat striatum,表现出m6A去甲基化酶ALKBH5和FTO表达增加,以及m6ADecreased methylation levels.表明FTO抑制保护PC12cells are protected from6-OHDA诱导的细胞凋亡.

脑癌（Brain cancer）

多项研究表明,m6A writers, erasers,和readersThe expression in pleomorphic glioblastoma（GBM）significantly disturbed in patients.Glioma stem cells in patients with（GSC）中m6AMechanism of regulation changed its self-renewal and carcinogenicity.GBMThe tumor tissue of patients with showMETTL3高表达,The associated with surial in patients with low.当然,METTL3在调节GSCThe role of carcinogenic remains controversial.Initial research showedMETTL3is a tumor suppressor,Knock out promote deterioration of tumor progression and survival in the body.GBM细胞系U251中METTL3Express inhibits cell migration and proliferation of,Simultaneously induce apoptosis.而METTL3Knock out can interfere with glioma reprogramming factorsSox2的m6A甲基化,导致GSCApoptosis and radiosensitization,Reduced tumor size in mice.此外METTL3Can control many and carcinogenic signal andRNAEdit relevant transcripts,Can also through the regulation of alternative splicing mechanism andSRSFto promote glioma growth.different research pairsMETTL3Filed as the role of tumor suppressor or promoter contradictory to the conclusion that,May be due to the high heterogeneity of the glioma.WTAP、ALKBH5和FTOPredictable rise in levelsGBMThe poor prognosis of patients.总之,研究表明m6Asignaling pathway isGBMcomponents of pathogenesis.

Other diseases of the central nervous system（Other CNS disorders）

m6A RNAMethylation is related to various other neurodevelopmental and nerve mental illness.FTOCertain gene variants vastly increases the major depression（MDD）and attention deficit/Risk of Hyperactivity Disorder.ALKBH5中的SNP与MDDvarious clinical features,包括焦虑、Developmental delay and cognitive dysfunction.Further research proves,ALKBH5Can go to the relevant transcript methylation and stable emotionFAAH,Promotes depressive behavior in mice,ALKBH5是Smith-MagenisSyndrome is a rare neurodevelopmental disorders disease genes.据报道YTHDC2Is the potential risk of autism spectrum disorder among Japanese site.METTL21B基因中的m6A SNP（G-A突变）Associated with multiple sclerosis.总之,m6AMethylation of the key regulatory factors associated with various diseases of the central nervous system.

结论和未来方向

The brain has evolved a variety ofRNARegulatory mechanism to optimize the energy consumption of polarization structure、Plasticity and Spatial Regulation,These mechanisms willRBP和非编码RNA与RNASequence element binding in,To recruit different cellular mechanisms to affect the targetRNA命运.RNAThe fidelity and kinetics of chemical modification to determine the process,因此m6AMethylation is a composed mainly of neurons tightly control gene expression in the process of development and diseases of apparent transcriptome mechanism.同时,m6AReversible makes it become a powerful tool for environment and stimulate the dependency regulation.FTOFrom brain tumors to various acute and chronic brain damageCNSdisorders in disease,ALKBH5Compensatory to methylation showed that each knockouterasersControls the different targets in the brain.

In spite of the various diseases of the nervous system（CNS）表现出m6A失调,But its role in the disease outcome is not yet fully understand.新的研究表明,剂量（m6A共识motifsNumber and methylation at any given time）Is very important to promote the function change.基于CRISPR位点特异性m6AEditing techniques may allow study its role in the disease outcome.目前,几种m6AEdit small molecule inhibitors of enzymes also can be used for the solutionwriters, erasers和 readers的角色.最后,Several studies have shownm6Aand other regulatory factors（Such as epigenetic modifications and microbial groups）crosstalk.

 图5：m6AMethylation in brain development、The role of physiology and disease

关于易基因RNA m6A甲基化测序(MeRIP-seq)技术

易基因MeRIP-seq技术利用m6A特异性抗体富集发生m6A修饰的RNA片段（包括mRNA、lncRNA等rRNA去除所有RNA）,结合高通量测序,可以对RNA上的m6A修饰进行定位与定量,总RNA起始量可降低至10μg,最低仅需1μg总RNA.广泛应用于组织发育、干细胞自我更新和分化、热休克或DNA损伤应答、癌症发生与发展、药物应答等研究领域;可应用于动物、植物、细胞及组织的m6A检测.

大样本量m6A-QTL性状关联分析,传统MeRIP单个样品价格高,通常难以承担.易基因开发建立MeRIP-seq2技术,显著提成IP平行性,实现不同样本间相对定量,降低检测成本.

易基因提供适用于不同科研需求的MeRIP技术：

• m6A甲基化-常量mRNA 甲基化测序（MeRIP-seq）
• m6A甲基化-常量mRNA +lncRNA甲基化测序（lnc-MeRIP-seq）
• m6A甲基化-微量mRNA +lncRNA甲基化测序（Micro-lnc-MeRIP-seq）
• 高通量m6A甲基化-常量mRNA甲基化测序（MeRIP-seq2）

技术优势：

• 起始量低：样本起始量可降低至10-20μg,最低仅需1μg总RNA;
• 转录组范围内：可以同时检测mRNA和lncRNA;
• 样本要求：可用于动物、植物、细胞及组织的m6A检测;
• 重复性高：IP富集重复性高,最大化降低抗体富集偏差;
• 应用范围广：广泛应用于组织发育、干细胞自我更新和分化、热休克或DNA损伤应答、癌症的发生与发展、药物应答等研究领域.

研究方向：

m6A甲基化目前主要运用在分子机制的理论性研究

• 疾病发生发展：肿瘤、代谢疾病（如肥胖/糖尿病）、神经和精神疾病（如阿尔兹海默症/抑郁症）、炎症…
• 发育和分化：早期胚胎发育、个体/组织/器官生长发育、干细胞分化与命运决定、衰老
• 环境暴露与响应：污染、抗逆、生活方式

参考文献：

http://doi.org/10.1177/0271678X20960033

相关阅读：

项目集锦 | 易基因近期m6A甲基化（MeRIP-seq）研究成果

项目文章 | 90天见刊,易基因m6A RNA甲基化(MeRIP)+转录组组学研究

干货：m6A RNA甲基化MeRIP-seq测序分析实验全流程解析

植物中m6A甲基化酶调节机制：组成、功能和进化

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