1、A D VA N C E D R E V I E WRNA methylation in nuclear pre-mRNA processingHelena Covelo-Molares|Marek Bartosovic|Stepanka VanacovaCEITEC,Masaryk University,Brno,CzechRepublicCorrespondenceStepanka Vanacova,CEITEC,MasarykUniversity,Brno,Czech Republic.Email:stepanka.vanacovaceitec.muni.czPresent addres
2、sDepartment of Medical Biochemistry andBiophysics,Karolinska Institutet,Solna,Sweden.Funding informationBrno City Municipality Scholarship;Ministry ofEducation,Youth and Sports,Grant/AwardNumber:LQ1601;Czech Science Foundation,Grant/Award Numbers:305/11/1095,14-25884S;Wellcome Trust,Grant/Award Numb
3、er:084316/B/07/ZEukaryotic RNA can carry more than 100 different types of chemical modifica-tions.Early studies have been focused on modifications of highly abundant RNA,such as ribosomal RNA and transfer RNA,but recent technical advances havemade it possible to also study messenger RNA(mRNA).Subseq
4、uently,mRNAmodifications,namely methylation,have emerged as key players in eukaryoticgene expression regulation.The most abundant and widely studied internal mRNAmodification is N6-methyladenosine(m6A),but the list of mRNA chemical modifi-cations continues to grow as fast as interest in this field.O
5、ver the past decade,transcriptome-wide studies combined with advanced biochemistry and the discov-ery of methylation writers,readers,and erasers revealed roles for mRNA methyla-tion in the regulation of nearly every aspect of the mRNA life cycle and in diversecellular,developmental,and disease proce
6、sses.Although large parts of mRNAfunction are linked to its cytoplasmic stability and regulation of its translation,anumber of studies have begun to provide evidence for methylation-regulatednuclear processes.In this review,we summarize the recent advances in RNA meth-ylation research and highlight
7、how these new findings have contributed to ourunderstanding of methylation-dependent RNA processing in the nucleus.This article is categorized under:RNA Processing RNA Editing and ModificationRNA Processing Splicing Regulation/Alternative SplicingRNA Interactions with Proteins and Other Molecules Pr
8、oteinRNA Interac-tions:Functional ImplicationsKEYWORDSRNA demethylase,RNA methylase,RNA processing1|INTRODUCTIONPosttranscriptional processing of messenger RNA(mRNA)is a common feature of eukaryotes.The 50capping and 30polyade-nylation modifications were discovered almost 50 years ago and have remai
9、ned as one of the hallmarks of eukaryotic mRNAprocessing(Edmonds,Vaughan,&Nakazato,1971;Muthukrishnan,Both,Furuichi,&Shatkin,1975;C.M.Wei,Gersho-witz,&Moss,1975b).However,the recent discovery of additional internal,functional mRNA modifications has introduced apreviously unappreciated level of mRNA
10、metabolism regulation.This review will discuss the latest development in the fieldof mRNA modifications from the perspective of nuclear mRNA processing.The pioneering analysis of methylated nucleotides in mammalian mRNA in the 1970s revealed the presence of a7-methylguanosine(m7G)cap at the 50end.It
11、 also unexpectedly detected internal methylated nucleotides(Desrosiers,Received:14 February 2018Revised:14 May 2018Accepted:15 May 2018DOI:10.1002/wrna.1489This is an open access article under the terms of the Creative Commons Attribution License,which permits use,distribution and reproduction in an
12、y medium,provided the original work is properly cited.2018 The Authors.WIREs RNA published by Wiley Periodicals,Inc.WIREs RNA.2018;9: of 17https:/doi.org/10.1002/wrna.1489Friderici,&Rottman,1974;Perry&Kelley,1974);N6-methyladenosine(m6A)was the most abundant modification(Dubin&Taylor,1975;Salditt-Ge
13、orgieff et al.,1976).These studies provided valuable insights into the composition and pro-portions of various internal mRNA modifications.However,they lacked thorough biochemical and functional analyses due totechnical limitations.The big boom in the field came with the development of high-throughp
14、ut sequencing methods combinedwith biochemistry and/or specific antibodies,which provided tools to obtain a whole-transcriptome view of RNA modifica-tions.Individual RNA modifications can be detected by specific approaches.Some may be directly detected as mismatchmutations(e.g.,A to I or C to U edit
15、ing)or they must first be chemically modified to induce a mutation(e.g.,bisulfite-basedsequencing of m5C).In other cases,such as N1-methyladenosine(m1A)or m6A,the detection methods either rely on interfer-ence with reverse transcriptase or utilization of specific antibodies(Dominissini et al.,2012;M
16、eyer et al.,2012;Tserovskiet al.,2016).The recognition of the biological importance of RNA modifications,along with methodical developments,hasled to“epitranscriptomics.”This field,analogous to epigenetics,studies functionally relevant chemical RNA modificationsthat do not alter the genomic sequence
17、.Thanks to methodological and technical developments,several major discoveries have changed our view on the impor-tance of RNA modifications.In addition to reversible chemical modifications of DNA and proteins,RNA marks(mainly aden-osine methylations)constitute yet another layer of gene expression r
18、egulation.In diverse eukaryotic lineages,dynamic RNAmethylation plays a key role in such elementary processes as germline maturation(Batista et al.,2014;Geula et al.,2015;Y.Wang et al.,2014),early embryogenesis(Ivanova et al.,2017;Zhao et al.,2017),host defense against pathogens(Martinez-Perez et al
19、.,2017;Tirumuru et al.,2016),and cancer self-renewal and tumorigenesis(Cui et al.,2017;Z.Li et al.,2017;Zhang et al.,2017)(Box 1).On the cellular level,methylation regulates a wide range of RNA-related processes:RNAprocessing,stability,and translation.The aim of this review is to discuss the functio
20、n of RNA methylation in nuclear pre-mRNA processing.Most of the recent discoveries have been made in mammalian systems,but there is also new data fromother eukaryotes,such as yeast,flies,and plants(Schwartz et al.,2013;Zhang et al.,2015).The spectrum of newly identifiedmRNA modifications grows every
21、 year;however,only a few have been studied in such detail as to be able to draw conclu-sions about their function.We will first discuss the growing spectrum of mRNA methylation marks,describe the knownmachinery involved in writing,erasing,and reading methylated mRNA,and finally we will discuss how t
22、hese marks areinvolved in the early steps of mRNA processing in the nucleus.2|THE SPECTRUM OF MRNA METHYL MARKSThe spectrum of mRNA methyl marks includes,so far,six different modifications.RNA methylation may occur at the N1 andN6 atoms in adenosine,N3 and C5 in cytidine,N7 in guanosine,and at the 2
23、0-OH of ribose(Figure 1).m7G,at the 50end of mRNA,marks the beginning of nearly all cellular mRNA transcribed by RNA polymerase II(RNAPII)(C.M.Wei et al.,1975b).In higher eukaryotes,the two nucleotides immediately adjacent to the cap show aBOX 1m6A IN DEVELOPMENT AND CANCERSeveral studies have indic
24、ated that m6A is a key regulator during early development and gametogenesis in different spe-cies.In mammals,m6A controls the stability of pluripotency regulators and thus allows embryonic stem cell(ESC)dif-ferentiation into specific cell types(Batista et al.,2014;Geula et al.,2015;Y.Wang,Li,et al.,
25、2014).Deletion of them6A writer,the Methyltransferase-Like 3(METTL3)in(KO)mouse ESCs is viable,but they failed to differentiate intospecific lineages.Mettl3 KO in mouse is embryonically lethal(Geula et al.,2015).Failure of embryonic developmentwas also observed in Arabidopsis thaliana upon depletion
26、 of the METTL3 ortholog(Zhong et al.,2008).Drosophilaembryos were viable,but they suffered from severe neurological defects and reduced fertility(Haussmann et al.,2016;Lence et al.,2016).Moreover,the m6A eraser AlkB Homolog 5(ALKBH5)was required for mouse spermatogenesis(Zheng et al.,2013),and the m
27、6A readers YTHD Domain Family 2(YTHDF2)and YTH Domain Containing 2(YTHDC2)were needed for early zygote development in mammals and zebrafish and successful meiotic program in themammalian germline,respectively(Hsu et al.,2017;Ivanova et al.,2017;Wojtas et al.,2017;Zhao et al.,2017;Box 2).Emerging evi
28、dence also suggests that m6A could play a role in cancer.For instance,the m6A methylation/demethylationpathway regulated glioblastoma stem cell self-renewal and tumorigenesis(Cui et al.,2017;Zhang et al.,2017),andMETTL3,METTL14,and Wilms Tumor 1-Associating Protein(WTAP)were highly expressed in myel
29、oid leukemia(Jaffrey&Kharas,2017).These pioneering and encouraging discoveries require further investigation to discover themechanistic details underlying the observed phenotypes.2 of 17COVELO-MOLARESET AL.complex methylation pattern(Figure 2).The characteristics and roles of the 20-O-methylation(Nm
30、)and N6,20-O-methyaldenosine(m6Am)found next to the cap will be discussed later in this review.Most modifications found in mRNA,such as m1A,N3-methylcytosine(m3C),N5-methylcytosine(m5C),and m6A,were first observed in noncoding RNA(ncRNA),mainly in the abundant and stable transfer RNA(tRNA)and riboso
31、mal RNA(rRNA).Both adenosine modifications,m1A and m6A,were identified by the 1970s;however,more detailed molecular and bio-chemical studies began only recently.Both modifications are reversible,meaning that they can be added and subsequentlyerased from mRNA by specific enzymes.m1A was first identif
32、ied on rRNA and tRNA,where it is important for the formation of tertiary structure conformations(Helm,Giege,&Florentz,1999),and in 2016 it was also reported in mRNA(Dominissini et al.,2016).It is written byTRMT6/TRM61A tRNA methyltransferase(X.Li et al.,2017;Safra et al.,2017),and it can be erased f
33、rom tRNA and mRNAby the DNA/RNA demethylase ALKBH3(Aas et al.,2003;Li,Xiong,Wang,et al.,2016;Ougland et al.,2004;Sundheimet al.,2006).In 2016 and 2017,several groups employed a method for site-specific detection of m1A in mRNA,but differentmethodological approaches and data analyses led to controver
34、sial findings and conclusions.By using a combination of Dim-roth rearrangement,antibody enrichment,and stalled reverse transcription,m1A was located at the 50untranslated region(UTR)and around start codons of several hundreds to several thousands of different mRNA transcripts(Dominissini et al.,2016
35、;Li,Xiong,Wang,et al.,2016).These studies also revealed that m1A was dynamically regulated by diverse stress condi-tions,such as starvation or heat shock(Li,Xiong,Wang,et al.,2016),and its 50location was proposed to positively regulateOHOHOHOHOONNON+CH3CH3CH3CH3CH3CH3CH3m3Cm1Am5Cm6Am6Amm7Gm7Gm6Amm1A
36、m6Am6Am5Cm1Am6ApA(n)AUGSTOPNmNmNH2NH2NH2CH3NHNH2OHONNNHNOHOHOHOHOHOHOHOHOHOHOHOONOHOHOHNOONNNNNNNNNNN+HNOOOOBaseFIGURE 1A schematic view of the structure and localization of methylated nucleosides in eukaryotic mRNA.The bold line represents the coding sequence,and the thin lines are 50and 30untransl
37、ated regions(UTRs).Abbreviations:m7G,7-methylguanosine;m6Am,N6,20-O-methyaldenosine;Nm,20-O-ribosemethylation;m1A,N1-methyladenosine;m6A,N6-methyladenosine;m5C,5-methylcytosine;m3C,3-methylcytosineNHOCap0Cap1Cap2RNMT:RAMCH3HNNNOOOPPPOOOHOHOOOOOOOOCH3CH3OOOOORNAOPOBaseOOPNCMTR1CMTR2NNNCH3?FTOON+H2NFI
38、GURE 2The chemical formula of the eukaryotic mRNA 50-m7G cap structure with the two downstream nucleotides included.Red:stable methyl groupthat forms the“Cap 0”structure(m7GpppN).Green:stable methyl groups that form the“Cap 1”(m7GpppNm)and“Cap 2”(m7GpppNmNm)structures.Blue:if the first nucleotide of
39、 the mRNA is adenosine,it can be further methylated at the N6position of the base(m7Gpppm6Am).The enzymes responsiblefor methylation/demethylation of the specific groups are depictedCOVELO-MOLARESET AL.3 of 17translation initiation(Dominissini et al.,2016).However,by using reverse transcription-base
40、d misincorporation and truncationat modified sites as well as more strict bioinformatics analysis,Safra et al.(2017)found m1A only in a handful of cytosolicmRNAs and a few mitochondrial mRNAs(in strong stem-loop structures)and linked m1A to translational repression anddevelopmental regulation.Since
41、this field is very young,it needs further biochemical and chemical validations to accompanythe high-throughput sequencing studies.For instance,it is important to test the relevance of 50terminal m1As as well as tostudy the molecular mechanism that underlies the role of m1A at structured elements.Not
42、ably,m1A has been detected onMALAT1-associated small cytoplasmic RNA(mascRNA),which is a small tRNA-like RNA processed from MALAT1 longnoncoding RNA(lncRNA)by an unusual mechanism that involves RNase P and RNase Z(Wilusz,Freier,&Spector,2008).It is possible that m1A,like in some tRNA,stabilizes the
43、tertiary conformation of mascRNA and thus promotes its yetunknown function in the cytoplasm.The additional methylation modifications m3C and m5C,which are also typically found in ncRNA(such as tRNA andrRNA)were recently also reported in mRNA(Clark,Evans,Dominissini,Zheng,&Pan,2016;Cozen et al.,2015;
44、Iwanami&Brown,1968a,1968b;Squires et al.,2012;Xu et al.,2017).Two different enzymes are responsible for m3C deposition in tRNA,METTL2,and METTL6(Xu et al.,2017),whereasMETTL8 modifies mRNA(Xu et al.,2017).The presence of m3C on mRNA was indicated by high performance liquid chro-matography coupled to
45、 mass spectrometry,but the specific locations are not yet known(Xu et al.,2017).Neither erasers norreaders have been identified for m3C so far.Regarding m5C,its presence in mRNA remains debated.Several methods based on indirect m5C detection have beenused to localize m5C in coding and ncRNA.These me
46、thods include m5C-RNA immunoprecipitation(RIP),5-azacytidine-mediated RIP(Aza-IP),and methylation at individual nucleotide resolution crosslinking and immunoprecipitation(miCLIP)(Hussain,Aleksic,Blanco,Dietmann,&Frye,2013;Li,Xiong,&Yi,2016).For direct mapping of m5C in native RNA,bisulfite sequencin
47、g,a technique widely used to study DNA methylation,was adopted(Amort et al.,2017;David et al.,2017;Khoddami&Cairns,2013;Legrand et al.,2017;Squires et al.,2012).Several of these studies reported thousands ofm5C positions in poly(A)+RNA,with even higher m5C levels in nuclear fractions(Amort et al.,20
48、17;Khoddami&Cairns,2013;Squires et al.,2012).Thousands of m5C marks have been reported in plants.In Arabidopsis,the tRNA m5Cmethyltransferase TRM4B was linked to the methylation of thousands of sites in mRNA coding sequences,and m5C wassuggested to play a role in mRNA stability and root development(
49、David et al.,2017).The presence of m5C in eukaryoticmRNA has,however,been strongly questioned by the work of Legrand et al.(2017),who developed a stringent and statisti-cally robust pipeline for whole-transcriptome bisulfite sequencing data analysis and performed a comprehensive methyla-tion analysi
50、s of mouse coding and ncRNA.Whereas their results revealed highly reproducible and robust detection of m5Cin tRNA and rRNA,they did not show any significant m5C in mRNA(Legrand et al.,2017).However,some studies havelinked m5C in mRNA and ncRNA with specific functions in mammals.For instance,the huma