m6A 甲基化研究方法

./RNA甲基化修飾〔m6A〕研究思路與方案設(shè)計(jì)RNA甲基化修飾約占所有RNA修飾的60%以上,而N6-甲基腺嘌呤〔N6-methyladenosine,m6A〕是高等生物mRNA和lncRNAs上最為普遍的修飾。目前發(fā)現(xiàn)microRNA,circRNA,rRNA,tRNA和snoRNA上都有發(fā)生m6A修飾。m6A修飾主要發(fā)生在RRACH序列中的腺嘌呤上,其功能由"編碼器<Writer>"、"消碼器<Eraser>"和"讀碼器<Reader>"決定ADDINEN.CITEADDINEN.CITE.DATA[\o"Fu,2014#8522"1]。"編碼器<Writer>"即甲基轉(zhuǎn)移酶,目前已知這個(gè)復(fù)合物的成分有METTL3,METTL14,WTAP和KIAA1429；而ALKBH5和FTO作為去甲基酶〔消碼器〕可逆轉(zhuǎn)甲基化；m6A由m6A結(jié)合蛋白識別,目前發(fā)現(xiàn)m6A結(jié)合蛋白〔讀碼器〕有YTH結(jié)構(gòu)域蛋白〔包括YTHDF1,YTHDF2,YTHDF3,YTHDC1和YTHDC2〕和核不均一蛋白HNRNP家族〔HNRNPA2B1和HNRNPC〕。m6A酶系統(tǒng)METTL3是早先被鑒定為結(jié)合SAM的組件,其缺失引起小鼠胚胎干細(xì)胞、Hela細(xì)胞和HepG2細(xì)胞中m6Apeaks的減少。METTL3與其同源蛋白METTL14定位在富含剪切因子的細(xì)胞核內(nèi)亞細(xì)胞器－核小斑〔Nuclearspeckle〕上,顯示ｍ６Ａ修飾可能和ＲＮＡ的剪切加工相關(guān)。WTAP與METTL3–METTL14二聚體相互作用,并共定位于核小斑,影響甲基化效率,參與mRNA剪。而KIAA1429作為候選的甲基轉(zhuǎn)移酶復(fù)合體的新亞基,是整體甲基化進(jìn)程所必須的ADDINEN.CITEADDINEN.CITE.DATA[\o"Schwartz,2014#8496"2]。FTO是ALKB家族的成員,作為第一個(gè)被發(fā)現(xiàn)的去甲基酶,可影響剪切因子SRSF2的RNA結(jié)合能力,進(jìn)而調(diào)控pre-mRNA的剪切加工過程ADDINEN.CITEADDINEN.CITE.DATA[\o"Zhao,2014#8497"3]。目前已發(fā)現(xiàn)FTO調(diào)節(jié)異常與肥胖、大腦畸形和生長遲緩相關(guān),揭示m6A可能對這些疾病具有重要的調(diào)節(jié)功能ADDINEN.CITEADDINEN.CITE.DATA[\o"Fu,2013#8498"4-6]。ALKBH5是ALKB家族中被發(fā)現(xiàn)具有去甲基作用的另一個(gè)成員,以RNaseA敏感的方式與核小斑共定位,它可直接催化m6A-甲基化腺苷去除甲基而不同于FTO的氧化去甲基化ADDINEN.CITEADDINEN.CITE.DATA[\o"Zheng,2013#8501"7].此外,ALKBH5和它的去甲基化活性影響新生mRNA合的成和剪切效率ADDINEN.CITEADDINEN.CITE.DATA[\o"Zheng,2013#8501"7],且ALKBH5敲除雄性小鼠表現(xiàn)出精子發(fā)生異常,這可能是精子發(fā)生相關(guān)基因表達(dá)改變的結(jié)果ADDINEN.CITEADDINEN.CITE.DATA[\o"Zheng,2013#8501"7]。m6AmRNA修飾執(zhí)行其功能主要通過兩個(gè)途徑：精細(xì)調(diào)控甲基化轉(zhuǎn)錄本的結(jié)構(gòu),以阻止或誘使蛋白-RNA相互作用；或被直接由m6A結(jié)合蛋白識別,誘發(fā)后續(xù)反應(yīng)。目前一類含有YTH功能結(jié)構(gòu)域的蛋白被鑒定為m6A修飾的結(jié)合蛋白。其中YTHDF1,YTHDF2,YTHDF3,YTHDC1和YTHDC2己被證實(shí)是ｍ６Ａ的結(jié)合蛋白.YTHDF1主要影響ｍ６Ａ修飾基因的翻譯,YTHDF2主要影響ｍ６Ａ修飾基因的降解,而YTHDC1結(jié)合ｍ６Ａ修飾的基因后影響其剪接。HNRNPC是一種豐富的核RNA結(jié)合蛋白,參與pre-mRNA的加工ADDINEN.CITE<EndNote><Cite><Author>Cienikova</Author><Year>2014</Year><RecNum>8503</RecNum><DisplayText>[8]</DisplayText><record><rec-number>8503</rec-number><foreign-keys><keyapp="EN"db-id="f2s2fdzw5a5wp6epsfu5dtfpwwfa0p2vw9ev">8503</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Cienikova,Z.</author><author>Damberger,F.F.</author><author>Hall,J.</author><author>Allain,F.H.</author><author>Maris,C.</author></authors></contributors><auth-address>DepartmentofBiology,InstituteofMolecularBiologyandBiophysics,ETHZurich,8093Zurich,Switzerland.</auth-address><titles><title>Structuralandmechanisticinsightsintopoly<uridine>tractrecognitionbythehnRNPCRNArecognitionmotif</title><secondary-title>JAmChemSoc</secondary-title><alt-title>JournaloftheAmericanChemicalSociety</alt-title></titles><pages>14536-44</pages><volume>136</volume><number>41</number><edition>2014/09/13</edition><keywords><keyword>Heterogeneous-NuclearRibonucleoproteinGroupC/*chemistry</keyword><keyword>Humans</keyword><keyword>Kinetics</keyword><keyword>Models,Molecular</keyword><keyword>MolecularStructure</keyword><keyword>PolyU/*chemistry</keyword><keyword>RNA/*chemistry</keyword><keyword>Thermodynamics</keyword></keywords><dates><year>2014</year><pub-dates><date>Oct15</date></pub-dates></dates><isbn>1520-5126<Electronic> 0002-7863<Linking></isbn><accession-num>25216038</accession-num><work-type>ResearchSupport,Non-U.S.Gov't</work-type><urls><related-urls><url>/pubmed/25216038</url></related-urls></urls><electronic-resource-num>10.1021/ja507690d</electronic-resource-num><language>eng</language></record></Cite></EndNote>[\o"Cienikova,2014#8503"8],且研究表明HNRNPC通過m6A與RNA結(jié)合調(diào)控目標(biāo)轉(zhuǎn)錄本的豐度和選擇性剪切ADDINEN.CITEADDINEN.CITE.DATA[\o"Liu,2015#8504"9].圖1m6A修飾的酶系統(tǒng)ADDINEN.CITE<EndNote><Cite><Author>Zhao</Author><Year>2017</Year><RecNum>8521</RecNum><DisplayText>[10]</DisplayText><record><rec-number>8521</rec-number><foreign-keys><keyapp="EN"db-id="f2s2fdzw5a5wp6epsfu5dtfpwwfa0p2vw9ev">8521</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Zhao,B.S.</author><author>Roundtree,I.A.</author><author>He,C.</author></authors></contributors><auth-address>DepartmentofChemistry,DepartmentofBiochemistryandMolecularBiology,andInstituteforBiophysicalDynamics,HowardHughesMedicalInstitute,TheUniversityofChicago,929East57thStreet,Chicago,Illinois60637,USA.</auth-address><titles><title>Post-transcriptionalgeneregulationbymRNAmodifications</title><secondary-title>NatRevMolCellBiol</secondary-title><alt-title>Naturereviews.Molecularcellbiology</alt-title></titles><pages>31-42</pages><volume>18</volume><number>1</number><edition>2016/11/04</edition><keywords><keyword>5-Methylcytosine/metabolism</keyword><keyword>Adenosine/analogs&derivatives/metabolism</keyword><keyword>Animals</keyword><keyword>CellCycle/genetics</keyword><keyword>CellDifferentiation/genetics</keyword><keyword>CircadianRhythm/genetics</keyword><keyword>GeneExpressionRegulation</keyword><keyword>Humans</keyword><keyword>Methylation</keyword><keyword>NucleicAcidConformation</keyword><keyword>ProteinBiosynthesis</keyword><keyword>*RNAProcessing,Post-Transcriptional</keyword><keyword>RNAStability</keyword><keyword>RNA,Messenger/chemistry/genetics/*metabolism</keyword></keywords><dates><year>2017</year><pub-dates><date>Jan</date></pub-dates></dates><isbn>1471-0080<Electronic> 1471-0072<Linking></isbn><accession-num>27808276</accession-num><work-type>Review</work-type><urls><related-urls><url>/pubmed/27808276</url></related-urls></urls><custom2>5167638</custom2><electronic-resource-num>10.1038/nrm.2016.132</electronic-resource-num><language>eng</language></record></Cite></EndNote>[\o"Zhao,2017#8521"10]m6A生物學(xué)功能越來越多的證據(jù)表明m6A修飾在哺乳動物中發(fā)揮重要的生物功能。例如,在轉(zhuǎn)錄后水平上調(diào)控RNA的穩(wěn)定性ADDINEN.CITEADDINEN.CITE.DATA[\o"Wang,2014#8489"11]、定位ADDINEN.CITEADDINEN.CITE.DATA[\o"Fustin,2013#8490"12]、運(yùn)輸、剪切ADDINEN.CITEADDINEN.CITE.DATA[\o"Molinie,2016#8494"13]和翻譯ADDINEN.CITEADDINEN.CITE.DATA[\o"Meyer,2015#8491"14]。ClaudioR.等發(fā)現(xiàn)依賴METTL3的pri-miRNA甲基化,會促進(jìn)DGCR8識別和加工,從而促進(jìn)microRNA的成熟ADDINEN.CITE<EndNote><Cite><Author>Alarcon</Author><Year>2015</Year><RecNum>8493</RecNum><DisplayText>[15]</DisplayText><record><rec-number>8493</rec-number><foreign-keys><keyapp="EN"db-id="f2s2fdzw5a5wp6epsfu5dtfpwwfa0p2vw9ev">8493</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Alarcon,C.R.</author><author>Lee,H.</author><author>Goodarzi,H.</author><author>Halberg,N.</author><author>Tavazoie,S.F.</author></authors></contributors><auth-address>LaboratoryofSystemsCancerBiology,RockefellerUniversity,1230YorkAvenue,NewYork,NewYork10065,USA.</auth-address><titles><title>N6-methyladenosinemarksprimarymicroRNAsforprocessing</title><secondary-title>Nature</secondary-title><alt-title>Nature</alt-title></titles><pages>482-5</pages><volume>519</volume><number>7544</number><edition>2015/03/25</edition><keywords><keyword>Adenosine/*analogs&derivatives/metabolism</keyword><keyword>BaseSequence</keyword><keyword>CellLine</keyword><keyword>GeneExpressionRegulation</keyword><keyword>Humans</keyword><keyword>Methylation</keyword><keyword>Methyltransferases/deficiency/metabolism</keyword><keyword>MicroRNAs/*chemistry/*metabolism</keyword><keyword>MolecularSequenceData</keyword><keyword>NucleicAcidConformation</keyword><keyword>*RNAProcessing,Post-Transcriptional</keyword><keyword>RNA-BindingProteins/metabolism</keyword><keyword>SubstrateSpecificity</keyword></keywords><dates><year>2015</year><pub-dates><date>Mar26</date></pub-dates></dates><isbn>1476-4687<Electronic> 0028-0836<Linking></isbn><accession-num>25799998</accession-num><work-type>ResearchSupport,U.S.Gov't,Non-P.H.S.</work-type><urls><related-urls><url>/pubmed/25799998</url></related-urls></urls><custom2>4475635</custom2><electronic-resource-num>10.1038/nature14281</electronic-resource-num><language>eng</language></record></Cite></EndNote>[\o"Alarcon,2015#8493"15]。此外,m6A識別蛋白HNRNPA2B1促進(jìn)pri-miRNA加工成pre-miRNAADDINEN.CITEADDINEN.CITE.DATA[\o"Alarcon,2015#8495"16]。另外,環(huán)狀RNA上m6A的修飾能促進(jìn)環(huán)狀RNA的翻譯ADDINEN.CITEADDINEN.CITE.DATA[\o"Yang,2017#8505"17]。m6A修飾在基因表達(dá)調(diào)控中起著重要的作用,其調(diào)控機(jī)制的異常可能與人類疾病或癌癥相關(guān)。目前發(fā)現(xiàn)m6A可能會影響精子發(fā)育〔ALKBH5,METTL3,Ythdc2〕、發(fā)育〔METTL3、FTO、ALKBH5〕、免疫〔METTL3〕、UV誘導(dǎo)的DNA損傷反應(yīng)〔METTL3,FTO〕、腫瘤生成〔YTHDF2〕或轉(zhuǎn)移〔METTL14〕、干細(xì)胞更新〔METTL14〕、脂肪分化〔FTO〕、生物節(jié)律、細(xì)胞發(fā)育分化、細(xì)胞分裂與其它的一些生命過程。例如,ALKBH5敲除的雄性小鼠增加了mRNA中的m<6>A修飾,其特點(diǎn)是凋亡影響減數(shù)分裂中期的精子細(xì)胞,引起生育能力受損ADDINEN.CITEADDINEN.CITE.DATA[\o"Zheng,2013#8501"7]。METTL3和METTL14增加弱精癥精子的m6A水平ADDINEN.CITEADDINEN.CITE.DATA[\o"Yang,2016#8506"18],在生殖細(xì)胞中,METTL3的敲除嚴(yán)重抑制精子分化和減數(shù)分裂的發(fā)生,轉(zhuǎn)錄組和m6A分析顯示精子發(fā)生相關(guān)基因的表達(dá)和選擇性剪接發(fā)生了改變ADDINEN.CITEADDINEN.CITE.DATA[\o"Xu,2017#8509"19]。YTHDC2可促進(jìn)靶基因的翻譯效率,并降低其mRNA的豐度,在精子發(fā)生過程中起關(guān)鍵作用。當(dāng)減數(shù)分裂開始時(shí)YTHDC2表達(dá)上調(diào),YTHDC2敲除小鼠的生殖細(xì)胞沒有經(jīng)過偶線期的發(fā)育導(dǎo)致小鼠不育ADDINEN.CITE<EndNote><Cite><Author>Hsu</Author><Year>2017</Year><RecNum>8510</RecNum><DisplayText>[20]</DisplayText><record><rec-number>8510</rec-number><foreign-keys><keyapp="EN"db-id="f2s2fdzw5a5wp6epsfu5dtfpwwfa0p2vw9ev">8510</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Hsu,P.J.</author><author>Zhu,Y.</author><author>Ma,H.</author><author>Guo,Y.</author><author>Shi,X.</author><author>Liu,Y.</author><author>Qi,M.</author><author>Lu,Z.</author><author>Shi,H.</author><author>Wang,J.</author><author>Cheng,Y.</author><author>Luo,G.</author><author>Dai,Q.</author><author>Liu,M.</author><author>Guo,X.</author><author>Sha,J.</author><author>Shen,B.</author><author>He,C.</author></authors></contributors><auth-address>DepartmentofChemistryandInstituteforBiophysicalDynamics,TheUniversityofChicago,Chicago,IL60637,USA. HowardHughesMedicalInstitute,TheUniversityofChicago,Chicago,IL60637,USA. CommitteeonImmunology,TheUniversityofChicago,Chicago,IL60637,USA. StateKeyLaboratoryofReproductiveMedicine,DepartmentofHistologyandEmbryology,NanjingMedicalUniversity,Nanjing211166,China. DepartmentofBiochemistryandMolecularBiology,TheUniversityofChicago,Chicago,IL60637,USA.</auth-address><titles><title>Ythdc2isanN6-methyladenosinebindingproteinthatregulatesmammalianspermatogenesis</title><secondary-title>CellRes</secondary-title><alt-title>Cellresearch</alt-title></titles><edition>2017/08/16</edition><dates><year>2017</year><pub-dates><date>Aug15</date></pub-dates></dates><isbn>1748-7838<Electronic> 1001-0602<Linking></isbn><accession-num>28809393</accession-num><urls><related-urls><url>/pubmed/28809393</url></related-urls></urls><electronic-resource-num>10.1038/cr.2017.99</electronic-resource-num><language>eng</language></record></Cite></EndNote>[\o"Hsu,2017#8510"20]。在DNA損傷反應(yīng)中,METTL3可促進(jìn)DNA聚合酶κ〔Polκ〕與核酸剪切修復(fù)途徑快速定位到UV引起的DNA損傷位點(diǎn),當(dāng)缺失METTL3時(shí),細(xì)胞無法迅速修復(fù)UV照射引起的突變,并且對UV照射更加敏感[25]。在淋巴細(xì)胞性小鼠過繼轉(zhuǎn)移模型中,Mettl3缺陷通過影響mRNAm6A修飾,降低SOCS家族mRNA衰減,增加mRNA和蛋白表達(dá)水平,從而抑制IL-7介導(dǎo)的STAT5活性和T細(xì)胞內(nèi)穩(wěn)態(tài)增殖和分化,進(jìn)而抑制腸炎的發(fā)生ADDINEN.CITEADDINEN.CITE.DATA[\o"Li,2017#8518"21]。在肝癌中,METTL14通過調(diào)控pri-miRNA的m6A修飾,影響MiR-126的生成加工,從而抑制肝癌的轉(zhuǎn)移ADDINEN.CITEADDINEN.CITE.DATA[\o"Ma,2017#8511"22]。在乳腺癌細(xì)胞中,低氧刺激能促進(jìn)依賴低氧誘導(dǎo)因子HIF的ALKBH5的表達(dá),而ALKBH5過表達(dá)降低了NANOGmRNA的m6A修飾,從而穩(wěn)定mRNA提高NANOG的表達(dá)水平,最終增加乳腺癌干細(xì)胞所占的比例ADDINEN.CITEADDINEN.CITE.DATA[\o"Zhang,2016#8512"23]。此外,低氧誘導(dǎo)乳腺癌細(xì)胞中依賴ZNF217的NANOG和KLF4的mRNAm6A甲基化抑制,且ALKBH5敲除顯著降低免疫缺陷小鼠乳腺癌的肺轉(zhuǎn)移ADDINEN.CITEADDINEN.CITE.DATA[\o"Zhang,2016#8513"24]。在肺癌中,METTL3能夠促進(jìn)肺腺癌細(xì)胞的生長、生存和侵襲,但還不清楚它是否作為m6A調(diào)節(jié)器或效應(yīng)器發(fā)揮作用ADDINEN.CITEADDINEN.CITE.DATA[\o"Lin,2016#8514"25]。在急性髓細(xì)胞白血病〔AML〕患者中,m<6>A調(diào)控基因的突變或拷貝數(shù)變化與TP53突變存在密切聯(lián)系,且m<6>A調(diào)控基因的改變與AML不良預(yù)后相關(guān)ADDINEN.CITE<EndNote><Cite><Author>Kwok</Author><Year>2017</Year><RecNum>8515</RecNum><DisplayText>[26]</DisplayText><record><rec-number>8515</rec-number><foreign-keys><keyapp="EN"db-id="f2s2fdzw5a5wp6epsfu5dtfpwwfa0p2vw9ev">8515</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Kwok,C.T.</author><author>Marshall,A.D.</author><author>Rasko,J.E.</author><author>Wong,J.J.</author></authors></contributors><auth-address>Gene&StemCellTherapyProgram,CentenaryInstitute,UniversityofSydney,Camperdown,2050,Australia. GeneRegulationinCancerLaboratory,CentenaryInstitute,UniversityofSydney,Camperdown,2050,Australia. SydneyMedicalSchool,UniversityofSydney,Camperdown,NSW,2006,Australia. CellandMolecularTherapies,RoyalPrinceAlfredHospital,Camperdown,2050,Australia. Gene&StemCellTherapyProgram,CentenaryInstitute,UniversityofSydney,Camperdown,2050,Australia..au. GeneRegulationinCancerLaboratory,CentenaryInstitute,UniversityofSydney,Camperdown,2050,Australia..au. SydneyMedicalSchool,UniversityofSydney,Camperdown,NSW,2006,Australia..au.</auth-address><titles><title>Geneticalterationsofm6Aregulatorspredictpoorersurvivalinacutemyeloidleukemia</title><secondary-title>JHematolOncol</secondary-title><alt-title>Journalofhematology&oncology</alt-title></titles><pages>39</pages><volume>10</volume><number>1</number><edition>2017/02/06</edition><dates><year>2017</year><pub-dates><date>Feb02</date></pub-dates></dates><isbn>1756-8722<Electronic> 1756-8722<Linking></isbn><accession-num>28153030</accession-num><work-type>Letter</work-type><urls><related-urls><url>/pubmed/28153030</url></related-urls></urls><custom2>5290707</custom2><electronic-resource-num>10.1186/s13045-017-0410-6</electronic-resource-num><language>eng</language></record></Cite></EndNote>[\o"Kwok,2017#8515"26]。此外,FTO在AML中高表達(dá),它通過降低mRNA轉(zhuǎn)錄本中的m<6>水平,調(diào)節(jié)ASB2和RARA等靶點(diǎn)的表達(dá),增強(qiáng)了白血病癌基因介導(dǎo)的細(xì)胞轉(zhuǎn)化和白血病形成,并抑制全反式維甲酸<ATRA>誘導(dǎo)的AML細(xì)胞分化ADDINEN.CITEADDINEN.CITE.DATA[\o"Li,2017#8516"27]。在脂肪形成過程中,FTO表達(dá)與m6A水平成負(fù)相關(guān),促進(jìn)脂肪形成ADDINEN.CITEADDINEN.CITE.DATA[\o"Zhao,2014#8497"3]。在膠質(zhì)細(xì)胞瘤樣細(xì)胞中,ALKBH5通過lncRNAFOXM1介導(dǎo)FOXM1基因pre-mRNA上的m6A修飾維持膠質(zhì)瘤細(xì)胞的成瘤性ADDINEN.CITEADDINEN.CITE.DATA[\o"Zhang,2017#8507"28]。此外,甲基轉(zhuǎn)移酶METTL3或METTL14的敲除,能夠改變m6A的富集和ADAM19的表達(dá),極大地促進(jìn)了膠質(zhì)瘤細(xì)胞的生長、自我更新和腫瘤形成ADDINEN.CITEADDINEN.CITE.DATA[\o"Cui,2017#8508"29]。圖2m6ARNA修飾和介導(dǎo)的功能ADDINEN.CITEADDINEN.CITE.DATA[\o"Cao,2016#8520"30]m6A的研究方向主要是通過研究m6A修飾相關(guān)的甲基化、去甲基化酶和識別蛋白的功能,進(jìn)而研究m6A修飾的生物學(xué)功能和作用機(jī)制：一般通過敲除m6A酶分子,研究下游功能基因分子的表達(dá)和m6A甲基化情況,通過介導(dǎo)相關(guān)基因異?！部勺兗羟?、穩(wěn)定性、翻譯、miRNA調(diào)控〕影響細(xì)胞表型和功能特征。m6A修飾圖譜構(gòu)建與作用機(jī)制：通過m6A甲基化測序〔MeRIP-Seq,miCLIP〕構(gòu)建疾病細(xì)胞模型或者發(fā)病組織的m6A修飾譜,分析m6A的motif,peaks數(shù)量與分布,Peak關(guān)聯(lián)基因的特征,聯(lián)合RNA-seq研究m6A甲基化與表達(dá)的關(guān)系。m6A研究思路m6A研究方案疾病樣本疾病樣本VS正常樣本RNA-seqMeRIP-seqm6A修飾圖譜分析m6A修飾差異基因分析差異表達(dá)基因m6A修飾特征分析差異表達(dá)基因關(guān)聯(lián)分析MeRIP-PCR、qPCR驗(yàn)證方案一TCGA等數(shù)據(jù)庫篩選異常表達(dá)的m6A相關(guān)基因〔疾病vs正?！撑R床樣本qPCR驗(yàn)證目標(biāo)TCGA等數(shù)據(jù)庫篩選異常表達(dá)的m6A相關(guān)基因〔疾病vs正?！撑R床樣本qPCR驗(yàn)證目標(biāo)基因腫瘤細(xì)胞中干擾目標(biāo)基因MTT、流式、transwell等檢測細(xì)胞增殖、凋亡、侵襲和遷移MeRIP-SeqRNA-seq篩選下游基因IP/pulldown驗(yàn)證目標(biāo)基因通過m6A調(diào)控下游基因研究案例研究案例一〔m6A修飾圖譜分析〕BerulavaT,RahmannS,RademacherK,Klein-HitpassL,HorsthemkeB:N6-adenosinemethylationinMiRNAs.PLoSOne2015,10<2>:e0118438.在許多不同種類的RNA中,都已觀察到N6-腺苷<m6A>的甲基化,但其在microRNAs中還沒有被研究。研究者在FTO1C1,FTO2D4和FTO3C3細(xì)胞系中,通過敲除m6A甲基轉(zhuǎn)移酶FTO篩選到表達(dá)差異的microRNA,說明miRNA受m6A甲基化的調(diào)控。進(jìn)一步通過MeRIP-Seq發(fā)現(xiàn)相當(dāng)一部分的microRNA具有m6A修飾。通過motif分析,他們發(fā)現(xiàn)了區(qū)分甲基化和非甲基化microRNA的一致序列。該文章所述的表觀遺傳修飾在基因表達(dá)的轉(zhuǎn)錄后調(diào)控的復(fù)雜性上增加了一個(gè)新的層次。圖1FTO敲除對甲基化的miRNAs的穩(wěn)定狀態(tài)的影響。研究案例二〔機(jī)制研究〕IF=13.2MaJZ,YangF,ZhouCC,LiuF,YuanJH,WangF,WangTT,XuQG,ZhouWP,SunSH:METTL14suppressesthemetastaticpotentialofhepatocellularcarcinomabymodulatingN6-methyladenosine-dependentprimaryMicroRNAprocessing.Hepatology2017,65<2>:529-543.m6A修飾已被證明具有重要的生物學(xué)功能,但其在癌癥上的作用還未得到較好的研究。為探索m6A修飾是否參與肝癌的調(diào)控,作者利用試劑盒檢測發(fā)現(xiàn)m6A整體甲基化在肝癌下調(diào),分離RNA做m6A免疫印跡驗(yàn)證m6A水平在肝癌中下調(diào)。為研究哪些因子導(dǎo)致m6A在肝癌的下調(diào),作者在20例肝癌與癌旁中檢測m6A甲基化酶和去甲基酶的表達(dá),發(fā)現(xiàn)METTL14在肝癌顯著下調(diào),進(jìn)一步通過130例病人分析發(fā)現(xiàn)METTL14作為肝癌預(yù)后因子,細(xì)胞實(shí)驗(yàn)發(fā)現(xiàn)其敲除可增強(qiáng)肝癌轉(zhuǎn)移。接下來作者研究METTL14抑制肝癌轉(zhuǎn)移的機(jī)制,由于已有研究顯示m6A修飾能夠增強(qiáng)DGCR8蛋白識別pri-miRNAs,促進(jìn)miRNAs的成熟,因此作者在METTL14敲除細(xì)胞中檢測miRNA和pri-miRNAs的表達(dá),發(fā)現(xiàn)miR-126下調(diào)。通過免疫沉淀反應(yīng)發(fā)現(xiàn)METTL14與DGCR8依賴RNA相互作用,且CLIP實(shí)驗(yàn)顯示DGCR8與m6A-RNA相互作用且敲除METTL14后結(jié)合作用降低,說明METTL14通過m6A修飾促進(jìn)DGCR8識別pri-miR-126.最后細(xì)胞實(shí)驗(yàn)證明MiR-126能夠回復(fù)METTL14的肝癌細(xì)胞轉(zhuǎn)移抑制功能,證明了METTL14通過m6A修飾促進(jìn)miR-126加工,從而抑制肝癌細(xì)胞轉(zhuǎn)移。圖1METTL14在肝癌中下調(diào)圖2METTL14依賴的m6A通過DGCR8調(diào)控miR-126加工研究案例三〔機(jī)制研究〕IF=27.4ZhangS,ZhaoBS,ZhouA,LinK,ZhengS,LuZ,ChenY,SulmanEP,XieK,BoglerOetal:m6ADemethylaseALKBH5MaintainsTumorigenicityofGlioblastomaStem-likeCellsbySustainingFOXM1ExpressionandCellProliferationProgram.CancerCell2017,31<4>:591-606e596.DNA甲基化異常是膠質(zhì)瘤的表觀遺傳調(diào)控因子,但RNA甲基化在腫瘤包括惡性膠質(zhì)瘤〔GBM〕中的調(diào)控還尚未清楚。為研究m6A調(diào)節(jié)可能導(dǎo)致GBM患者臨床療效不佳,作者通過TCGA數(shù)據(jù)庫,發(fā)現(xiàn)ALKBH5在惡性膠質(zhì)瘤樣干細(xì)胞〔GSCs〕中高表達(dá)且與GBM病人不良預(yù)后相關(guān),干擾ALKBH5降低GSCs細(xì)胞的自我更新能力且抑制GSCs增殖,進(jìn)一步體內(nèi)驗(yàn)證敲除ALKBH5可抑制腫瘤生長。為研究ALKBH5的m6A作用機(jī)制,作者利用芯片和m6A-seq篩選到膠質(zhì)瘤增殖相關(guān)的FOXM1,最后通過qPCR、WB、免疫熒光、核質(zhì)分離WB/qPCR、RIP和MeRIP等實(shí)驗(yàn)證明ALKBH5通過去甲基化初期轉(zhuǎn)錄本調(diào)節(jié)FOXM1在GSCs中的表達(dá)。為研究ALKBH5對FOXM1的作用是否受其他因子的調(diào)節(jié),作者研究了FOXM1的鄰近基因,發(fā)現(xiàn)lncRNAFOXM1-AS與FOXM1序列互補(bǔ),且共表達(dá)、共定位,進(jìn)一步通過RIP,RNApulldown等實(shí)驗(yàn)證明lncRNAFOXM1-AS促進(jìn)ALKBH5和FOXM1初級轉(zhuǎn)錄本的相互作用。最后通過細(xì)胞實(shí)驗(yàn)進(jìn)一步驗(yàn)證ALKBH5在lncRNAFOXM1-AS的作用下維持FOXM1的表達(dá)和細(xì)胞增殖程序,從而維持GSCs的干性。圖1ALKBH5敲除細(xì)胞中m6A修飾的特征和基因表達(dá)的變化ADDINEN.REFLIST1. FuY,DominissiniD,RechaviG,HeC:Geneexpressionregulationmediatedthroughreversiblem<6>ARNAmethylation.NatRevGenet2014,15<5>:293-306.2. SchwartzS,MumbachMR,JovanovicM,WangT,MaciagK,BushkinGG,MertinsP,Ter-OvanesyanD,HabibN,CacchiarelliDetal:Perturbationofm6AwritersrevealstwodistinctclassesofmRNAmethylationatinternaland5'sites.CellRep2014,8<1>:284-296.3. ZhaoX,YangY,SunBF,ShiY,YangX,XiaoW,HaoYJ,PingXL,ChenYS,WangWJetal:FTO-dependentdemethylationofN6-methyladenosineregulatesmRNAsplicingandisrequiredforadipogenesis.CellRes2014,24<12>:1403-1419.4. FuY,JiaG,PangX,WangRN,WangX,LiCJ,SmemoS,DaiQ,BaileyKA,NobregaMAetal:FTO-mediatedformationofN6-hydroxymethyladenosineandN6-formyladenosineinmammalianRNA.NatCommun2013,4:1798.5. DinaC,MeyreD,GallinaS,DurandE,KornerA,JacobsonP,CarlssonLM,KiessW,VatinV,LecoeurCetal:VariationinFTOcontributestochildhoodobesityandsevereadultobesity.NatGenet2007,39<6>:724-726.6. BoisselS,ReishO,ProulxK,Kawagoe-TakakiH,SedgwickB,YeoGS,MeyreD,GolzioC,MolinariF,KadhomNetal:Loss-of-functionmutationinthedioxygenase-encodingFTOgenecausesseveregrowthretardationandmultiplemalformations.AmJHumGenet2009,85<1>:106-111.7. ZhengG,DahlJA,NiuY,FedorcsakP,HuangCM,LiCJ,VagboCB,ShiY,WangWL,SongSHetal:ALKBH5isamammalianRNAdemethylasethatimpactsRNAmetabolismandmousefertility.MolCell2013,49<1>:18-29.8. CienikovaZ,DambergerFF,HallJ,AllainFH,MarisC:Structuralandmechanisticinsightsintopoly<uridine>tractrecognitionbythehnRNPCRNArecognitionmotif.JAmChemSoc2014,136<41>:14536-14544.9. LiuN,DaiQ,ZhengG,HeC,ParisienM,PanT:N<6>-methyladenosine-dependentRNAstructuralswitchesregulateRNA-proteininteractions.Nature2015,518<7540>:560-564.10. ZhaoBS,RoundtreeIA,HeC:Post-transcriptionalgeneregulationbymRNAmodifications.NatRevMolCellBiol2017,18<1>:31-42.11. WangX,LuZ,GomezA,HonGC,YueY,HanD,FuY,ParisienM,DaiQ,JiaGetal:N6-methyladenosine-dependentregulationofmessengerRNAstability.Nature2014,505<7481>:117-120.12. FustinJM,DoiM,YamaguchiY,HidaH,NishimuraS,YoshidaM,IsagawaT,MoriokaMS,KakeyaH,ManabeIetal:RNA-methylation-dependentRNAprocessingcontrolsthespeedofthecircadianclock.Cell2013,155<4>:793-806.13. MolinieB,WangJ,LimKS,HillebrandR,LuZX,VanWittenbergheN,HowardBD,DaneshvarK,MullenAC,DedonPetal:m<6>A-LAIC-seqrevealsthecensusandcomplexityofthem<6>Aepitranscriptome.NatMethods2016,13<8>:692-698.14. MeyerKD,PatilDP,ZhouJ,ZinovievA,SkabkinMA,ElementoO,PestovaTV,QianSB,JaffreySR:5'UTRm<6>APromotesCap-IndependentTranslation.Cell2015,163<4>:999-1010.15. AlarconCR,LeeH,GoodarziH,HalbergN,TavazoieSF:N6-methyladenosinemarksprimarymicroRNAsforprocessing.Nature2015,519<7544>:482-485.16. AlarconCR,GoodarziH,LeeH,LiuX,TavazoieS,TavazoieSF:HNRNPA2B1IsaMediatorofm<6>A-DependentNuclearRNAProcessingEvents.Cell2015,162<6>:1299-1308.17. YangY,FanX,MaoM,SongX,WuP,ZhangY,JinY,ChenLL,WangY,WongCCetal:ExtensivetranslationofcircularRNAsdrivenbyN6-methyladenosine.CellRes2017,27<5>:626-641.18. YangY,HuangW,HuangJT,ShenF,XiongJ,YuanEF,QinSS,ZhangM,FengYQ,YuanBFetal:IncreasedN6-methyladenosineinHumanSpermRNAasaRiskFactorforAsthenozoospermia.SciRep2016,6:24345.19. XuK,YangY,FengGH,SunBF,ChenJQ,LiYF,ChenYS,ZhangXX,WangCX,JiangLYetal:Mettl3-mediatedm6Aregulatesspermatogonialdifferentiationandmeiosisinitiation.CellRes2017