New Twists in Detecting mRNA Modification Dynamics
2020; Elsevier BV; Volume: 39; Issue: 1 Linguagem: Inglês
10.1016/j.tibtech.2020.06.002
ISSN0167-9430
AutoresIna Anreiter, Quoseena Mir, Jared T. Simpson, Sarath Chandra Janga, Matthias Soller,
Tópico(s)RNA and protein synthesis mechanisms
ResumoWriters, readers, and erasers have now been discovered for many mRNA modifications.Global topographic candidate maps have been generated for many modifications, but high error rates need to be addressed by technical improvements in detection and validation using orthogonal methods that apply rigid selection criteria.Nanopore single-molecule direct RNA sequencing is progressing towards reliable detection of modified nucleotides in mRNA. Modified nucleotides in mRNA are an essential addition to the standard genetic code of four nucleotides in animals, plants, and their viruses. The emerging field of epitranscriptomics examines nucleotide modifications in mRNA and their impact on gene expression. The low abundance of nucleotide modifications and technical limitations, however, have hampered systematic analysis of their occurrence and functions. Selective chemical and immunological identification of modified nucleotides has revealed global candidate topology maps for many modifications in mRNA, but further technical advances to increase confidence will be necessary. Single-molecule sequencing introduced by Oxford Nanopore now promises to overcome such limitations, and we summarize current progress with a particular focus on the bioinformatic challenges of this novel sequencing technology. Modified nucleotides in mRNA are an essential addition to the standard genetic code of four nucleotides in animals, plants, and their viruses. The emerging field of epitranscriptomics examines nucleotide modifications in mRNA and their impact on gene expression. The low abundance of nucleotide modifications and technical limitations, however, have hampered systematic analysis of their occurrence and functions. Selective chemical and immunological identification of modified nucleotides has revealed global candidate topology maps for many modifications in mRNA, but further technical advances to increase confidence will be necessary. Single-molecule sequencing introduced by Oxford Nanopore now promises to overcome such limitations, and we summarize current progress with a particular focus on the bioinformatic challenges of this novel sequencing technology. Chemical modifications on RNA are well-established and evolutionarily conserved features of structural RNAs such as rRNA and tRNA [1.Machnicka M.A. et al.Distribution and frequencies of post-transcriptional modifications in tRNAs.RNA Biol. 2014; 11: 1619-1629Crossref PubMed Scopus (95) Google Scholar, 2.Sloan K.E. et al.Tuning the ribosome: the influence of rRNA modification on eukaryotic ribosome biogenesis and function.RNA Biol. 2017; 14: 1138-1152Crossref PubMed Scopus (224) Google Scholar, 3.Soller M. Fray R. RNA modifications in gene expression control.Biochim. Biophys. Acta Gene Regul. Mech. 2019; 1862: 219-221Crossref PubMed Scopus (0) Google Scholar]. In the past few years the occurrence of these modifications on protein-coding mRNAs, long noncoding RNAs (lncRNAs), and small regulatory RNAs (srRNAs) has received renewed attention to determine their role in regulating gene expression, development, and health and disease (Box 1). The rapid evolution of transcriptome sequencing technologies has made it possible to develop methodologies that interrogate the topography of RNA modifications transcriptome-wide. This new field, termed epitranscriptomics (see Glossary), seeks to elucidate the role of RNA modifications in regulating gene expression, with a special focus on their biological functions in mRNA.Box 1Discovery of RNA Modifications and Their Biological FunctionsIn 1957, Davis and Allen discovered pseudouridine as the first known chemically modified RNA nucleotide [121.Davis F.F. Allen F.W. Ribonucleic acids from yeast which contain a fifth nucleotide.J. Biol. Chem. 1957; 227: 907-915Abstract Full Text PDF PubMed Google Scholar]. Since then, 162 more RNA modifications have been described and catalogued (RNA Modification Databases: http://mods.rna.albany.edu and http://modomics.genesilico.pl [115.Boccaletto P. et al.MODOMICS: a database of RNA modification pathways. 2017 update.Nucleic Acids Res. 2018; 46: D303-D307Crossref PubMed Scopus (749) Google Scholar]). RNA modifications occur in all domains of life and in all types of RNA (tRNA, rRNA, mRNA, srRNA, snoRNA, snRNA, and lncRNA), and have been extensively linked to development, health, and disease. The high copy number of rRNAs and tRNAs in cells has greatly facilitated the study of modifications in these RNA species. rRNAs and tRNAs have complex 3D structures, which mediate ribosome function and protein translation. Modified nucleotides in rRNAs and tRNAs play essential roles in ribosome assembly and dynamics, and disruption of these modifications has been associated with lethality, severe growth defects, intellectual disability, diabetes, and cancer [32.Dezi V. et al.Nucleotide modifications in messenger RNA and their role in development and disease.Biochem. Soc. Trans. 2016; 44: 1385-1393Crossref PubMed Scopus (22) Google Scholar,33.Frye M. et al.RNA modifications modulate gene expression during development.Science. 2018; 361: 1346-1349Crossref PubMed Scopus (272) Google Scholar,122.Dimitrova D.G. et al.RNA 2'-O-methylation (Nm) modification in human diseases.Genes (Basel). 2019; 10: 117Crossref Scopus (6) Google Scholar]. Although nucleotide modifications in mRNA have been known for >40 years, their functional interrogation has only become possible in the past decade owing to high-throughput sequencing-based mapping methodologies that overcome the low abundance of these modifications, as well as to the discovery of writer, reader, and eraser proteins (Box 2). Since then, may biological functions in development and disease have been associated to mRNA modifications, including a variety of cancers, mental disorders, and fertility and metabolic phenotypes [4.Galloway A. Cowling V.H. mRNA cap regulation in mammalian cell function and fate.Biochim. Biophys. Acta Gene. Regul. Mech. 2019; 1862: 270-279Crossref PubMed Scopus (45) Google Scholar,30.Roignant J.Y. Soller M. m6A in mRNA: an ancient mechanism for fine-tuning gene expression.Trends Genet. 2017; 33: 380-390Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar,32.Dezi V. et al.Nucleotide modifications in messenger RNA and their role in development and disease.Biochem. Soc. Trans. 2016; 44: 1385-1393Crossref PubMed Scopus (22) Google Scholar,33.Frye M. et al.RNA modifications modulate gene expression during development.Science. 2018; 361: 1346-1349Crossref PubMed Scopus (272) Google Scholar]. In 1957, Davis and Allen discovered pseudouridine as the first known chemically modified RNA nucleotide [121.Davis F.F. Allen F.W. Ribonucleic acids from yeast which contain a fifth nucleotide.J. Biol. Chem. 1957; 227: 907-915Abstract Full Text PDF PubMed Google Scholar]. Since then, 162 more RNA modifications have been described and catalogued (RNA Modification Databases: http://mods.rna.albany.edu and http://modomics.genesilico.pl [115.Boccaletto P. et al.MODOMICS: a database of RNA modification pathways. 2017 update.Nucleic Acids Res. 2018; 46: D303-D307Crossref PubMed Scopus (749) Google Scholar]). RNA modifications occur in all domains of life and in all types of RNA (tRNA, rRNA, mRNA, srRNA, snoRNA, snRNA, and lncRNA), and have been extensively linked to development, health, and disease. The high copy number of rRNAs and tRNAs in cells has greatly facilitated the study of modifications in these RNA species. rRNAs and tRNAs have complex 3D structures, which mediate ribosome function and protein translation. Modified nucleotides in rRNAs and tRNAs play essential roles in ribosome assembly and dynamics, and disruption of these modifications has been associated with lethality, severe growth defects, intellectual disability, diabetes, and cancer [32.Dezi V. et al.Nucleotide modifications in messenger RNA and their role in development and disease.Biochem. Soc. Trans. 2016; 44: 1385-1393Crossref PubMed Scopus (22) Google Scholar,33.Frye M. et al.RNA modifications modulate gene expression during development.Science. 2018; 361: 1346-1349Crossref PubMed Scopus (272) Google Scholar,122.Dimitrova D.G. et al.RNA 2'-O-methylation (Nm) modification in human diseases.Genes (Basel). 2019; 10: 117Crossref Scopus (6) Google Scholar]. Although nucleotide modifications in mRNA have been known for >40 years, their functional interrogation has only become possible in the past decade owing to high-throughput sequencing-based mapping methodologies that overcome the low abundance of these modifications, as well as to the discovery of writer, reader, and eraser proteins (Box 2). Since then, may biological functions in development and disease have been associated to mRNA modifications, including a variety of cancers, mental disorders, and fertility and metabolic phenotypes [4.Galloway A. Cowling V.H. mRNA cap regulation in mammalian cell function and fate.Biochim. Biophys. Acta Gene. Regul. Mech. 2019; 1862: 270-279Crossref PubMed Scopus (45) Google Scholar,30.Roignant J.Y. Soller M. m6A in mRNA: an ancient mechanism for fine-tuning gene expression.Trends Genet. 2017; 33: 380-390Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar,32.Dezi V. et al.Nucleotide modifications in messenger RNA and their role in development and disease.Biochem. Soc. Trans. 2016; 44: 1385-1393Crossref PubMed Scopus (22) Google Scholar,33.Frye M. et al.RNA modifications modulate gene expression during development.Science. 2018; 361: 1346-1349Crossref PubMed Scopus (272) Google Scholar]. Major recent methodological advances in mass spectrometry (MS) and next-generation sequencing combined with immunoprecipitation and chemical or enzymatic conversion methods have increased the catalog of known mRNA modifications, and have led to new insights into the role of these modifications in regulating gene expression in humans and model organisms such as yeast, plants, Drosophila, and mice. However, the field still faces considerable methodological challenges because modifications in mRNA generally are not abundant. Moreover, many current methods for probing RNA modifications are hampered by high error rates, low specificity, and poor reproducibility. We summarize below current approaches and emerging technologies for assessing mRNA modifications. We aim to highlight the strengths and limitations of current methods regarding specificity, sensitivity, and reproducibility, with a particular focus on emerging single-molecule direct RNA sequencing by Nanopore. To date, 13 different chemical modifications have been identified in mRNA transcripts, and these can be divided into modifications of cap-adjacent nucleotides and internal modifications (Figure 1). These modifications are added by a variety of dedicated enzymes (Box 2 and Table 1). Modifications of cap-adjacent nucleotides are added to the 5′-ends of RNAs transcribed by RNA polymerase II [mRNA, primary (pri-)miRNA transcript, lncRNA, small nucleolar (sno)RNA, and small nuclear (sn)RNA] [4.Galloway A. Cowling V.H. mRNA cap regulation in mammalian cell function and fate.Biochim. Biophys. Acta Gene. Regul. Mech. 2019; 1862: 270-279Crossref PubMed Scopus (45) Google Scholar]. The composition of the cap varies with the type of RNA molecule, and typically consists of a 7-methylguanosine (m7G) moiety added in a characteristic 5′–5′ triphosphate linkage to the first transcribed nucleotide (Figure 1A). In some snRNAs and snoRNAs the cap guanosine is trimethylated to m2′2′7G, and further alternative cap structures including NAD+ have recently been identified [4.Galloway A. Cowling V.H. mRNA cap regulation in mammalian cell function and fate.Biochim. Biophys. Acta Gene. Regul. Mech. 2019; 1862: 270-279Crossref PubMed Scopus (45) Google Scholar, 5.Wang J. et al.Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA.Nucleic Acids Res. 2019; 47e130Crossref PubMed Scopus (48) Google Scholar, 6.Walters R.W. et al.Identification of NAD+ capped mRNAs in Saccharomyces cerevisiae.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 480-485Crossref PubMed Scopus (63) Google Scholar]. The first and second nucleotides adjacent to the cap can be 2′-O-methylated at the ribose (cOMe) in animals, viruses, and protists. If the cap-adjacent nucleotide is an adenosine, it can be further methylated to N6,2′-O-dimethyladenosine (m6Am, Figure 1A) [4.Galloway A. Cowling V.H. mRNA cap regulation in mammalian cell function and fate.Biochim. Biophys. Acta Gene. Regul. Mech. 2019; 1862: 270-279Crossref PubMed Scopus (45) Google Scholar,7.Sun H. et al.Cap-specific, terminal N6-methylation by a mammalian m6Am methyltransferase.Cell Res. 2019; 29: 80-82Crossref PubMed Scopus (75) Google Scholar, 8.Kruse S. et al.A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA.Sci. Rep. 2011; 1: 126Crossref PubMed Scopus (33) Google Scholar, 9.Akichika S. et al.Cap-specific terminal N6-methylation of RNA by an RNA polymerase II-associated methyltransferase.Science. 2019; 363eaav0080Crossref PubMed Scopus (135) Google Scholar, 10.Sendinc E. et al.PCIF1 catalyzes m6Am mRNA methylation to regulate gene expression.Mol. Cell. 2019; 75: 620-630Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 11.Boulias K. et al.Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome.Mol. Cell. 2019; 75: 631-643Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]. Of further special note are the extensive modifications in trypanosomes of cap-adjacent nucleotides in the splice leader RNAs that are trans-spliced onto the body of the mRNAs (Figure 1A) [12.Bangs J.D. et al.Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides.J. Biol. Chem. 1992; 267: 9805-9815Abstract Full Text PDF PubMed Google Scholar]. Modifications of cap-adjacent nucleotides are common, differ among tissues and transcripts, and regulate mRNA stability and translation [4.Galloway A. Cowling V.H. mRNA cap regulation in mammalian cell function and fate.Biochim. Biophys. Acta Gene. Regul. Mech. 2019; 1862: 270-279Crossref PubMed Scopus (45) Google Scholar,5.Wang J. et al.Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA.Nucleic Acids Res. 2019; 47e130Crossref PubMed Scopus (48) Google Scholar,7.Sun H. et al.Cap-specific, terminal N6-methylation by a mammalian m6Am methyltransferase.Cell Res. 2019; 29: 80-82Crossref PubMed Scopus (75) Google Scholar,8.Kruse S. et al.A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA.Sci. Rep. 2011; 1: 126Crossref PubMed Scopus (33) Google Scholar,10.Sendinc E. et al.PCIF1 catalyzes m6Am mRNA methylation to regulate gene expression.Mol. Cell. 2019; 75: 620-630Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar,11.Boulias K. et al.Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome.Mol. Cell. 2019; 75: 631-643Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,13.Mauer J. et al.Reversible methylation of m6Am in the 5′ cap controls mRNA stability.Nature. 2017; 541: 371-375Crossref PubMed Scopus (464) Google Scholar].Box 2Writers, Readers, and Erasers: The Complex Machinery Underlying mRNA ModificationsThe enzymes that deposit, remove, and bind to mRNA modifications have been termed 'writers', 'erasers', and 'readers', respectively [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar,34.Shi H. et al.Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers.Mol. Cell. 2019; 74: 640-650Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar,35.Zaccara S. et al.Reading, writing and erasing mRNA methylation.Nat. Rev. Mol. Cell Biol. 2019; 20: 608-624Crossref PubMed Scopus (350) Google Scholar] (see Table 1 in main text). Writers bind to short consensus sequence motifs in target mRNAs; however, these motifs are far more common than the modifications themselves, suggesting that additional factors determine writer binding. Other writers, such as CMTrs, PUS1, and METTL16, bind to mRNA based on mRNA structure motifs [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar,123.Carlile T.M. et al.mRNA structure determines modification by pseudouridine synthase 1.Nat. Chem. Biol. 2019; 15: 966-974Crossref PubMed Scopus (7) Google Scholar]. Erasers and readers recognize the modifications themselves; however, given the large variety of different reader proteins, additional factors are probably involved in targeting readers to their mRNA targets. The machinery of writers, erasers, and readers is complex, often consisting of large protein complexes with a variety of cofactors. The writer complex for m6A, for instance, consists of a ~900 kDa complex with two core methyltransferases (METTL3 and METTL14) and several auxiliary cofactors [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar]. Although loss of the methyltransferases does not result in lethality in Drosophila, loss of several of the cofactors does, suggesting that these enzymes exercise alternative functions unrelated to m6A-methylation. Some writer proteins can also act as reader proteins, as is the case for METTL16 under particular cellular conditions [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar]. In addition, some modifications have alternative writers, erasers, and readers. METTL3/METTL14, METTL4, and METTL16 writers for m6A act in different complexes, and both FTO and ALKBH5 have been shown to erase m6A [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar]. A varying number of m6A readers have been identified across organisms, with a record of 13 different readers in plants [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar]. There is also crosstalk between modifications, and some enzymes target more than one type of modification. For instance, FTO was first described as an eraser for internal m6A [124.Jia G. et al.N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.Nat. Chem. Biol. 2011; 7: 885-887Crossref PubMed Scopus (1676) Google Scholar], but was later shown to also demethylate m6Am at the cap [125.Mauer J. et al.FTO controls reversible m6Am RNA methylation during snRNA biogenesis.Nat. Chem. Biol. 2019; 15: 340-347Crossref PubMed Scopus (101) Google Scholar]. Furthermore, mRNA modifications might influence the ability of mRNA binding proteins to bind to mRNA targets. For instance, interferon-induced proteins with tetratricopeptide repeats (IFITs), mRNA binding proteins that are involved in innate immunity and viral response, show low affinity for mRNA transcripts with ribose methylation at the cap [47.Devarkar S.C. et al.Structural basis for m7G recognition and 2'-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 596-601Crossref PubMed Scopus (140) Google Scholar, 48.Abbas Y.M. et al.Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2'-O methylations.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 2106-2115Crossref PubMed Scopus (0) Google Scholar, 49.Johnson B. et al.Human IFIT3 modulates IFIT1 RNA binding specificity and protein stability.Immunity. 2018; 48: 487-499Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. Whether alternative writers, erasers, and readers of mRNA modifications act in different tissues, in different cellular/physiological contexts, or on different mRNA targets remains mostly an open question. Furthermore, although the machinery acting on m6A has been well characterized in recent years, the factors acting on other modifications are less well explored, and new enzymes or functions could be discovered in years to come. Further identification of these enzymes will aid our understanding of the biological functions of mRNA modifications and yield prime new targets for pharmaceutical targeting of mRNA modification-associated diseases such as cancer and obesity.Table 1Mammalian Writers, Readers, and Erasers of mRNA ModificationsmRNA modificationWriterEraserReaderCap modificationsm7GRNMT [4.Galloway A. Cowling V.H. mRNA cap regulation in mammalian cell function and fate.Biochim. Biophys. Acta Gene. Regul. Mech. 2019; 1862: 270-279Crossref PubMed Scopus (45) Google Scholar]cOMeCMTr1, CMTr2 [4.Galloway A. Cowling V.H. mRNA cap regulation in mammalian cell function and fate.Biochim. Biophys. Acta Gene. Regul. Mech. 2019; 1862: 270-279Crossref PubMed Scopus (45) Google Scholar,122.Dimitrova D.G. et al.RNA 2'-O-methylation (Nm) modification in human diseases.Genes (Basel). 2019; 10: 117Crossref Scopus (6) Google Scholar]m6AmPCIF1aRequires ribose methylation introduced by CMTr to methylate adenosine the at N6 position. [7.Sun H. et al.Cap-specific, terminal N6-methylation by a mammalian m6Am methyltransferase.Cell Res. 2019; 29: 80-82Crossref PubMed Scopus (75) Google Scholar,10.Sendinc E. et al.PCIF1 catalyzes m6Am mRNA methylation to regulate gene expression.Mol. Cell. 2019; 75: 620-630Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar,11.Boulias K. et al.Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome.Mol. Cell. 2019; 75: 631-643Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]FTO [125.Mauer J. et al.FTO controls reversible m6Am RNA methylation during snRNA biogenesis.Nat. Chem. Biol. 2019; 15: 340-347Crossref PubMed Scopus (101) Google Scholar]Internal modificationsm6AMETTL3bPart of a 900 kDa holoenzyme., METTL14bPart of a 900 kDa holoenzyme.METTL4cRemains to be confirmed for mRNA., METTL16 [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar]FTO, ALKBH5 [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar]YTHDF1–3, YTHDC1–2 [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar]m1ATRMT6/61A, TRMT61B [37.Safra M. et al.The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution.Nature. 2017; 551: 251-255Crossref PubMed Scopus (227) Google Scholar,63.Li X. et al.Base-resolution mapping reveals distinct m1A methylome in nuclear- and mitochondrial-encoded transcripts.Mol. Cell. 2017; 68: 993-1005Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar]ALKBH1/3 [37.Safra M. et al.The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution.Nature. 2017; 551: 251-255Crossref PubMed Scopus (227) Google Scholar]YTHDF1–3 [139.Dai X. et al.Identification of YTH domain-containing proteins as the readers for N1-methyladenosine in RNA.Anal. Chem. 2018; 90: 6380-6384Crossref PubMed Scopus (0) Google Scholar]m5CNSUN2 [73.Yang X. et al.5-methylcytosine promotes mRNA export – NSUN2 as the methyltransferase and ALYREF as an m5C reader.Cell Res. 2017; 27: 606-625Crossref PubMed Scopus (287) Google Scholar], DNMT2cRemains to be confirmed for mRNA. [140.Genenncher B. et al.Mutations in cytosine-5 tRNA methyltransferases impact mobile element expression and genome stability at specific DNA repeats.Cell Rep. 2018; 22: 1861-1874Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar]TET (through conversion to hm5C) [141.Fu L. et al.Tet-mediated formation of 5-hydroxymethylcytosine in RNA.J. Am. Chem. Soc. 2014; 136: 11582-11585Crossref PubMed Scopus (181) Google Scholar]YBX1 [142.Yang Y. et al.RNA 5-methylcytosine facilitates the maternal-to-zygotic transition by preventing maternal mRNA decay.Mol. Cell. 2019; 75: 1188-1202Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 143.Chen X. et al.5-methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs.Nat. Cell Biol. 2019; 21: 978-990Crossref PubMed Scopus (122) Google Scholar, 144.Zou F. et al.Drosophila YBX1 homolog YPS promotes ovarian germ line stem cell development by preferentially recognizing 5-methylcytosine RNAs.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 3603-3609Crossref PubMed Scopus (18) Google Scholar, 145.Yang L. et al.m5C methylation guides systemic transport of messenger RNA over graft junctions in plants.Curr. Biol. 2019; 29: 2465-2476Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]hm5CTET1–3cRemains to be confirmed for mRNA., dRequires m5C. [141.Fu L. et al.Tet-mediated formation of 5-hydroxymethylcytosine in RNA.J. Am. Chem. Soc. 2014; 136: 11582-11585Crossref PubMed Scopus (181) Google Scholar]TET (through further oxidation)m3CMETTL8 [24.Xu L. et al.Three distinct 3-methylcytidine (m3C) methyltransferases modify tRNA and mRNA in mice and humans.J. Biol. Chem. 2017; 292: 14695-14703Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar]ALKBH1 [146.Ma C.J. et al.AlkB homologue 1 demethylates N3-methylcytidine in mRNA of mammals.ACS Chem. Biol. 2019; 14: 1418-1425Crossref PubMed Scopus (9) Google Scholar]ac4CNAT10 [22.Arango D. et al.Acetylation of cytidine in mRNA promotes translation efficiency.Cell. 2018; 175: 1872-1886Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar]ΨDKC1, TRUB1, PUS7 [32.Dezi V. et al.Nucleotide modifications in messenger RNA and their role in development and disease.Biochem. Soc. Trans. 2016; 44: 1385-1393Crossref PubMed Scopus (22) Google Scholar,81.Safra M. et al.TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code.Genome Res. 2017; 27: 393-406Crossref PubMed Scopus (51) Google Scholar]m7GMETTL1 [26.Zhang L.S. et al.Transcriptome-wide mapping of internal N7-methylguanosine methylome in mammalian mRNA.Mol. Cell. 2019; 74: 1304-1316Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar,59.Pandolfini L. et al.METTL1 promotes let-7 microRNA processing via m7G methylation.Mol. Cell. 2019; 74: 1278-1290Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar]8-OxoGNonenzymatic [29.Dai D.-P. et al.Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells.Proc. Natl. Acad. Sci. 2018; 115: 4218-4222Crossref PubMed Scopus (11) Google Scholar]NmFTSJ3cRemains to be confirmed for mRNA. [147.Ringeard M. et al.FTSJ3 is an RNA 2'-O-methyltransferase recruited by HIV to avoid innate immune sensing.Nature. 2019; 565: 500-504Crossref PubMed Scopus (0) Google Scholar]A to IADAR 1–3 [148.Nishikura K. A-to-I editing of coding and non-coding RNAs by ADARs.Nat. Rev. Mol. Cell Biol. 2016; 17: 83-96Crossref PubMed Scopus (401) Google Scholar]C to UAPOBEC family [25.Salter J.D. et al.The APOBEC protein family: united by structure, divergent in function.Trends Biochem. Sci. 2016; 41: 578-594Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar]a Requires ribose methylation introduced by CMTr to methylate adenosine the at N6 position.b Part of a 900 kDa holoenzyme.c Remains to be confirmed for mRNA.d Requires m5C. Open table in a new tab The enzymes that deposit, remove, and bind to mRNA modifications have been termed 'writers', 'erasers', and 'readers', respectively [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar,34.Shi H. et al.Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers.Mol. Cell. 2019; 74: 640-650Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar,35.Zaccara S. et al.Reading, writing and erasing mRNA methylation.Nat. Rev. Mol. Cell Biol. 2019; 20: 608-624Crossref PubMed Scopus (350) Google Scholar] (see Table 1 in main text). Writers bind to short consensus sequence motifs in target mRNAs; however, these motifs are far more common than the modifications themselves, suggesting that additional factors determine writer binding. Other writers, such as CMTrs, PUS1, and METTL16, bind to mRNA based on mRNA structure motifs [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar,123.Carlile T.M. et al.mRNA structure determines modification by pseudouridine synthase 1.Nat. Chem. Biol. 2019; 15: 966-974Crossref PubMed Scopus (7) Google Scholar]. Erasers and readers recognize the modifications themselves; however, given the large variety of different reader proteins, additional factors are probably involved in targeting readers to their mRNA targets. The machinery of writers, erasers, and readers is complex, often consisting of large protein complexes with a variety of cofactors. The writer complex for m6A, for instance, consists of a ~900 kDa complex with two core methyltransferases (METTL3 and METTL14) and several auxiliary cofactors [31.Balacco D.L. Soller M. The m6A writer: rise of a machine for growing tasks.Biochemistry. 2019; 58: 363-378Crossref PubMed Scopus (61) Google Scholar]. Although loss of the methyltransferases does not result in lethality in Drosophila, loss of se
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