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      N 6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions

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          Abstract

          RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBM) 1- 3 . However, RBMs can be buried within their local RNA structures 4- 7 , thus inhibiting RNA-protein interactions. N 6-methyladenosine (m 6A), the most abundant and dynamic internal modification in eukaryotic messenger RNA 8- 19 , can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs 15 , but how m 6A achieves wide-ranging physiological significance needs further exploration. Here we show that m 6A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism “m 6A-switch”. We found that m 6A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (hnRNP C), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing 20- 24 . Combining PAR-CLIP and m 6A/MeRIP approaches enabled us to identify 39,060 m 6A-switches among hnRNP C binding sites; and global m 6A reduction decreased hnRNP C binding at 2,798 high confidence m 6A-switches. We determined that these m 6A-switch-regulated hnRNP C binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m 6A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m 6A-dependent RNA structural remodeling, and provide a new direction for investigating RNA-modification-coded cellular biology.

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          Most cited references 16

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          Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing.

          N(6)-methyladenosine-sequencing (m(6)A-seq) is an immunocapturing approach for the unbiased transcriptome-wide localization of m(6)A in high resolution. To our knowledge, this is the first protocol to allow a global view of this ubiquitous RNA modification, and it is based on antibody-mediated enrichment of methylated RNA fragments followed by massively parallel sequencing. Building on principles of chromatin immunoprecipitation-sequencing (ChIP-seq) and methylated DNA immunoprecipitation (MeDIP), read densities of immunoprecipitated RNA relative to untreated input control are used to identify methylated sites. A consensus motif is deduced, and its distance to the point of maximal enrichment is assessed; these measures further corroborate the success of the protocol. Identified locations are intersected in turn with gene architecture to draw conclusions regarding the distribution of m(6)A between and within gene transcripts. When applied to human and mouse transcriptomes, m(6)A-seq generated comprehensive methylation profiles revealing, for the first time, tenets governing the nonrandom distribution of m(6)A. The protocol can be completed within ~9 d for four different sample pairs (each consists of an immunoprecipitation and corresponding input).
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            A universal framework for regulatory element discovery across all genomes and data types.

            Deciphering the noncoding regulatory genome has proved a formidable challenge. Despite the wealth of available gene expression data, there currently exists no broadly applicable method for characterizing the regulatory elements that shape the rich underlying dynamics. We present a general framework for detecting such regulatory DNA and RNA motifs that relies on directly assessing the mutual information between sequence and gene expression measurements. Our approach makes minimal assumptions about the background sequence model and the mechanisms by which elements affect gene expression. This provides a versatile motif discovery framework, across all data types and genomes, with exceptional sensitivity and near-zero false-positive rates. Applications from yeast to human uncover putative and established transcription-factor binding and miRNA target sites, revealing rich diversity in their spatial configurations, pervasive co-occurrences of DNA and RNA motifs, context-dependent selection for motif avoidance, and the strong impact of posttranscriptional processes on eukaryotic transcriptomes.
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              Probing the structure of RNAs in solution.

              During these last years, a powerful methodology has been developed to study the secondary and tertiary structure of RNA molecules either free or engaged in complex with proteins. This method allows to test the reactivity of every nucleotide towards chemical or enzymatic probes. The detection of the modified nucleotides and RNase cleavages can be conducted by two different paths which are oriented both by the length of the studied RNA and by the nature of the probes used. The first one uses end-labeled RNA molecule and allows to detect only scissions in the RNA chain. The second approach is based on primer extension by reverse transcriptase and detects stops of transcription at modified or cleaved nucleotides. The synthesized cDNA fragments are then sized by electrophoresis on polyacrylamide:urea gels. In this paper, the various structure probes used so far are described, and their utilization is discussed.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                19 February 2015
                26 February 2015
                26 August 2015
                : 518
                : 7540
                : 560-564
                Affiliations
                [1 ]Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA.
                [2 ]Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
                [3 ]Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637, USA.
                Author notes
                [4]

                Present address: Faculty of Dentistry, McGill University, Montreal, Quebec H3A 0G1, Canada

                Article
                NIHMS656223
                10.1038/nature14234
                4355918
                25719671
                3f778d02-5869-4253-809f-e00e39ba3958

                Reprints and permissions information is available at www.nature.com/reprints.

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