Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA
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TLDR
A method that accurately determines m(6)A status at any site in mRNA/lncRNA, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET), which determines the precise location of the m( 6)A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m(7)A modification.Abstract:
N(6)-methyladenosine (m(6)A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m(6)A demethylases and cell-type and cell-state-dependent m(6)A patterns indicate that m(6)A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m(6)A modification include mRNA splicing, export, stability, and immune tolerance; but m(6)A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m(6)A status at any site in mRNA/lncRNA, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET). The method determines the precise location of the m(6)A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m(6)A modification. We applied the method to determine the m(6)A status at several sites in two human lncRNAs and three human mRNAs and found that m(6)A fraction varies between 6% and 80% among these sites. We also found that many m(6)A candidate sites in these RNAs are however not modified. The precise determination of m(6)A status in a long noncoding RNA also enables the identification of an m(6)A-containing RNA structural motif.read more
Citations
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Journal ArticleDOI
Dynamic RNA Modifications in Gene Expression Regulation
TL;DR: Roles for mRNA modification in nearly every aspect of the mRNA life cycle, as well as in various cellular, developmental, and disease processes are revealed.
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Post-transcriptional gene regulation by mRNA modifications
TL;DR: N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses.
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N 6 -methyladenosine-dependent RNA structural switches regulate RNA–protein interactions
TL;DR: It is found that m6A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing.
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Gene expression regulation mediated through reversible m 6 A RNA methylation
TL;DR: This Review focuses on reversible methylation through the most prevalent mammalian mRNA internal modification, N6-methyladenosine (m6A), and indicates dynamic regulatory roles that are analogous to the well-known reversible epigenetic modifications of DNA and histone proteins.
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Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome
Bastian Linder,Anya V. Grozhik,Anthony O. Olarerin-George,Cem Meydan,Christopher E. Mason,Samie R. Jaffrey +5 more
TL;DR: m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) is developed and used to demonstrate that antibodies to m6A can induce specific mutational signatures at m 6A residues after ultraviolet light–induced antibody-RNA cross- linking and reverse transcription.
References
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Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq
Dan Dominissini,Sharon Moshitch-Moshkovitz,Schraga Schwartz,Schraga Schwartz,Mali Salmon-Divon,Lior Ungar,Sivan Osenberg,Sivan Osenberg,Karen Cesarkas,Jasmine Jacob-Hirsch,Ninette Amariglio,Martin Kupiec,Rotem Sorek,Gideon Rechavi,Gideon Rechavi +14 more
TL;DR: The findings suggest that RNA decoration by m6A has a fundamental role in regulation of gene expression, and a subset of stimulus-dependent, dynamically modulated sites is identified.
Journal ArticleDOI
Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and near Stop Codons
Kate D. Meyer,Yogesh Saletore,Paul Zumbo,Olivier Elemento,Christopher E. Mason,Samie R. Jaffrey +5 more
TL;DR: A method is presented for transcriptome-wide m(6)A localization, which combines m( 6)A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq) and reveals insights into the epigenetic regulation of the mammalian transcriptome.
Journal ArticleDOI
N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.
Guifang Jia,Ye Fu,Xu Zhao,Xu Zhao,Qing Dai,Guanqun Zheng,Ying Yang,Ying Yang,Chengqi Yi,Tomas Lindahl,Tao Pan,Yun-Gui Yang,Yun-Gui Yang,Chuan He +13 more
TL;DR: FTO exhibits efficient oxidative demethylation activity of abundant N6-methyladenosine (m6A) residues in RNA in vitro, and it is shown that FTO partially colocalizes with nuclear speckles, supporting m6A in nuclear RNA as a physiological substrate of FTO.
Journal ArticleDOI
ALKBH5 Is a Mammalian RNA Demethylase that Impacts RNA Metabolism and Mouse Fertility
Guanqun Zheng,John Arne Dahl,Yamei Niu,Peter Fedorcsak,Chun-Min Huang,Charles J. Li,Cathrine Broberg Vågbø,Yue Shi,Yue Shi,Wen-Ling Wang,Wen-Ling Wang,Shuhui Song,Zhike Lu,Ralph P. G. Bosmans,Qing Dai,Ya-Juan Hao,Ya-Juan Hao,Xin Yang,Xin Yang,Wenming Zhao,Wei-Min Tong,Xiu-Jie Wang,Florian Bogdan,Kari Furu,Ye Fu,Guifang Jia,Xu Zhao,Xu Zhao,Jun Liu,Hans E. Krokan,Arne Klungland,Yun-Gui Yang,Yun-Gui Yang,Chuan He +33 more
TL;DR: The discovery of ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo strongly suggests that the reversible m( 6)A modification has fundamental and broad functions in mammalian cells.
Journal ArticleDOI
The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation.
Vidisha Tripathi,Jonathan D. Ellis,Zhen Shen,David Y. Song,Qun Pan,Andrew T. Watt,Susan M. Freier,C. Frank Bennett,Alok Sharma,Paula A. Bubulya,Benjamin J. Blencowe,Supriya G. Prasanth,Kannanganattu V. Prasanth +12 more
TL;DR: Evidence is provided for a role for the long nuclear-retained regulatory RNA, MALAT1 in AS regulation and for the role for an nrRNA in the regulation of gene expression, which suggests that MALat1 regulates AS by modulating the levels of active SR proteins.
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