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      m 6A-dependent regulation of messenger RNA stability

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          Abstract

          N 6 -methyladenosine (m 6A) is the most prevalent internal (non-cap) modification present in the messenger RNA (mRNA) of all higher eukaryotes 1, 2 . Although essential to cell viability and development 35 , the exact role of m 6A modification remains to be determined. The recent discovery of two m 6A demethylases in mammalian cells highlighted the importance of m 6A in basic biological functions and disease 68 . Here we show that m 6A is selectively recognized by the human YTH domain family 2 (YTHDF2) protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m 6A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies 9 . The C-terminal domain of YTHDF2 selectively binds to m 6A-containing mRNA whereas the N-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m 6A modification is recognized by selective-binding proteins to affect the translation status and lifetime of mRNA.

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          Most cited references 25

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          Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish.

          MicroRNAs regulate gene expression through deadenylation, repression, and messenger RNA (mRNA) decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild-type and dicer mutant zebrafish embryos and found that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deadenylation with use of an internal polyadenylate tail did not block target repression. Lastly, we observed that ribosome density along the length of the message remains constant, suggesting that translational repression occurs by reducing the rate of initiation rather than affecting elongation or causing ribosomal drop-off. These results show that miR-430 regulates translation initiation before inducing mRNA decay during zebrafish development.
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            mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.

            MicroRNAs (miRNAs) silence the expression of target genes post-transcriptionally. Their function is mediated by the Argonaute proteins (AGOs), which colocalize to P-bodies with mRNA degradation enzymes. Mammalian P-bodies are also marked by the GW182 protein, which interacts with the AGOs and is required for miRNA function. We show that depletion of GW182 leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of the essential Drosophila miRNA effector AGO1, indicating that GW182 functions in the miRNA pathway. When GW182 is bound to a reporter transcript, it silences its expression, bypassing the requirement for AGO1. Silencing by GW182 is effected by changes in protein expression and mRNA stability. Similarly, miRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay, and both mechanisms require GW182. mRNA degradation, but not translational repression, by GW182 or miRNAs is inhibited in cells depleted of CAF1, NOT1, or the decapping DCP1:DCP2 complex. We further show that the N-terminal GW repeats of GW182 interact with the PIWI domain of AGO1. Our findings indicate that GW182 links the miRNA pathway to mRNA degradation by interacting with AGO1 and promoting decay of at least a subset of miRNA targets.
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              A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins.

              Cross-linking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins. We developed a method for CLIP data analysis, and applied it to compare CLIP with photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) and to uncover how differences in cross-linking and ribonuclease digestion affect the identified sites. We found only small differences in accuracies of these methods in identifying binding sites of HuR, which binds low-complexity sequences, and Argonaute 2, which has a complex binding specificity. We found that cross-link-induced mutations led to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites. This bias can be substantially reduced by milder nuclease digestion conditions.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                15 November 2013
                27 November 2013
                2 January 2014
                02 July 2014
                : 505
                : 7481
                Affiliations
                [1 ]Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois, 60637, USA
                [2 ]Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, UCSD Moores Cancer Center and Institute of Genome Medicine, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0653, USA
                [3 ]Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois, 60637, USA
                [4 ]Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
                Author notes
                [* ]To whom correspondence should be addressed. chuanhe@ 123456uchicago.edu
                Article
                NIHMS529324
                10.1038/nature12730
                3877715
                24284625

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                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM088599 || GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM071440 || GM
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