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      Arabidopsis m 6A demethylase activity modulates viral infection of a plant virus and the m 6A abundance in its genomic RNAs

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          Significance

          N 6-methyladenosine (m 6A) modification has been found to constitute an important regulatory mechanism in RNA biology. Unlike mammals and yeast, no component of the m 6A cellular machinery has been described in plants at present. Although the influence of the m6A cellular machinery has been suspected to occur in the plant virus cycle, it has never been proved. Here we have identified a plant protein with m 6A demethylase activity (atALKBH9B) and demonstrate that this protein removes m 6A modification from RNA in vitro. Remarkably, we found that m 6A abundance on the viral genome of alfalfa mosaic virus is influenced by atALKBH9B activity and regulates viral infection. This study extends the vast repertoire that plants exploit to control cytoplasmic-replicating RNA viruses.

          Abstract

          N 6-methyladenosine (m 6A) is an internal, reversible nucleotide modification that constitutes an important regulatory mechanism in RNA biology. Unlike mammals and yeast, no component of the m 6A cellular machinery has been described in plants at present. m 6A has been identified in the genomic RNAs of diverse mammalian viruses and, additionally, viral infection was found to be modulated by the abundance of m 6A in viral RNAs. Here we show that the Arabidopsis thaliana protein atALKBH9B (At2g17970) is a demethylase that removes m 6A from single-stranded RNA molecules in vitro. atALKBH9B accumulates in cytoplasmic granules, which colocalize with siRNA bodies and associate with P bodies, suggesting that atALKBH9B m 6A demethylase activity could be linked to mRNA silencing and/or mRNA decay processes. Moreover, we identified the presence of m 6A in the genomes of two members of the Bromoviridae family, alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV). The demethylation activity of atALKBH9B affected the infectivity of AMV but not of CMV, correlating with the ability of atALKBH9B to interact (or not) with their coat proteins. Suppression of atALKBH9B increased the relative abundance of m 6A in the AMV genome, impairing the systemic invasion of the plant, while not having any effect on CMV infection. Our findings suggest that, as recently found in animal viruses, m 6A modification may represent a plant regulatory strategy to control cytoplasmic-replicating RNA viruses.

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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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            MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor.

            N6-Methyladenosine is a ubiquitous modification identified in the mRNA of numerous eukaryotes, where it is present within both coding and noncoding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We show that Arabidopsis thaliana mRNA contains N6-methyladenosine at levels similar to those previously reported for animal cells. We further show that inactivation of the Arabidopsis ortholog of the yeast and human mRNA adenosine methylase (MTA) results in failure of the developing embryo to progress past the globular stage. We also demonstrate that the arrested seeds are deficient in mRNAs containing N6-methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems, and emerging lateral roots. Finally, we show that MTA interacts in vitro and in vivo with At FIP37, a homolog of the Drosophila protein FEMALE LETHAL2D and of human WILMS' TUMOUR1-ASSOCIATING PROTEIN. The results reported here provide direct evidence for an essential function for N6-methyladenosine in a multicellular eukaryote, and the interaction with At FIP37 suggests possible RNA processing events that might be regulated or altered by this base modification.
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              N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection

              Summary The RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove this modification, as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A methyltransferases or an m6A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determined that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Altogether, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.
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                Author and article information

                Journal
                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                pnas
                pnas
                PNAS
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                0027-8424
                1091-6490
                3 October 2017
                18 September 2017
                : 114
                : 40
                : 10755-10760
                Affiliations
                [1] aInstituto de Biología Molecular y Celular de Plantas, Universitat Politécnica de Valencia–Consejo Superior de Investigaciones Científicas , 46022 Valencia, Spain
                Author notes
                2To whom correspondence may be addressed. Email: faparici@ 123456ibmcp.upv.es or vpallas@ 123456ibmcp.upv.es .

                Edited by George E. Bruening, University of California, Davis, CA, and approved August 22, 2017 (received for review February 23, 2017)

                Author contributions: F.A. and V.P. designed research; M.M.-P., F.A., and M.P.L.-G. performed research; M.M.-P., F.A., M.P.L.-G., J.M.B., and J.A.S.-N. contributed new reagents/analytic tools; M.M.-P., F.A., J.M.B., and J.A.S.-N. analyzed data; and M.M.-P., F.A., and V.P. wrote the paper.

                1M.M.-P. and F.A. contributed equally to this work.

                Author information
                http://orcid.org/0000-0003-4954-989X
                Article
                PMC5635872 PMC5635872 5635872 201703139
                10.1073/pnas.1703139114
                5635872
                28923956
                6fd1be96-a136-4e95-873f-f99bceaaa009
                History
                Page count
                Pages: 6
                Funding
                Funded by: Ministerio de Economía y Competitividad (MINECO) 501100003329
                Award ID: BIO2014-54862-R
                Funded by: Generalitat Valenciana (Regional Government of Valencia) 501100003359
                Award ID: GV2015/010
                Categories
                Biological Sciences
                Microbiology

                coat protein,plant virus,ALKBH9B,demethylase,m6A
                coat protein, plant virus, ALKBH9B, demethylase, m6A

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