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It can be further modified to yield\u00a05-hydroxymethylcytidine (hm\n                    <ns4:sup>5<\/ns4:sup>\n                    C), 5-formylcytidine (f\n                    <ns4:sup>5<\/ns4:sup>\n                    C), 2\u00b4-O-methyl-5-hydroxymethylcytidine (hm\n                    <ns4:sup>5<\/ns4:sup>\n                    Cm) and 2\u00b4-O-methyl-5-formylcytidine (f\n                    <ns4:sup>5<\/ns4:sup>\n                    Cm).\u00a0How m\n                    <ns4:sup>5<\/ns4:sup>\n                    C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m\n                    <ns4:sup>5<\/ns4:sup>\n                    C is required for\n                    <ns4:italic>Caenorhabditis elegans<\/ns4:italic>\n                    development and fertility under heat stress. m\n                    <ns4:sup>5<\/ns4:sup>\n                    C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. Hence, identifying readers for RNA modifications can enhance our understanding in the biological roles of these modifications.\n                  <\/ns4:p>\n                  <ns4:p>\n                    <ns4:bold>Methods:<\/ns4:bold>\n                    To contribute to the understanding of how m\n                    <ns4:sup>5<\/ns4:sup>\n                    C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in\n                    <ns4:italic>C. elegans<\/ns4:italic>\n                    . Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m\n                    <ns4:sup>5<\/ns4:sup>\n                    C was validated using immunoblotting.\n                  <\/ns4:p>\n                  <ns4:p>\n                    <ns4:bold>Results:<\/ns4:bold>\n                    Our mass spectrometry analyses revealed unique binding proteins for each of the modifications.\n                    <ns4:italic>In silico<\/ns4:italic>\n                    analysis for phenotype enrichments suggested that hm\n                    <ns4:sup>5<\/ns4:sup>\n                    Cm unique readers are enriched in proteins involved in RNA processing, while readers for m\n                    <ns4:sup>5<\/ns4:sup>\n                    C, hm\n                    <ns4:sup>5<\/ns4:sup>\n                    C and f\n                    <ns4:sup>5<\/ns4:sup>\n                    C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m\n                    <ns4:sup>5<\/ns4:sup>\n                    C\n                    <ns4:italic>in vitro<\/ns4:italic>\n                    . Finally, sequence alignment analysis showed that several of the putative m\n                    <ns4:sup>5<\/ns4:sup>\n                    C readers contain the conserved RNA recognition motif (RRM), including ALY-1 and ALY-2.\n                  <\/ns4:p>\n                  <ns4:p>\n                    <ns4:bold>Conclusions:<\/ns4:bold>\n                    The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m\n                    <ns4:sup>5<\/ns4:sup>\n                    C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m\n                    <ns4:sup>5<\/ns4:sup>\n                    C in\n                    <ns4:italic>C. elegans<\/ns4:italic>\n                    .\n                  <\/ns4:p>","DOI":"10.12688\/wellcomeopenres.17893.1","type":"journal-article","created":{"date-parts":[[2022,11,17]],"date-time":"2022-11-17T13:05:10Z","timestamp":1668690310000},"page":"282","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA"],"prefix":"10.12688","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6844-2361","authenticated-orcid":false,"given":"IC","family":"Navarro","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9452-2528","authenticated-orcid":false,"given":"Kin Man","family":"Suen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dalila","family":"Bensaddek","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Arun","family":"Tanpure","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6204-6045","authenticated-orcid":false,"given":"Angus","family":"Lamond","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0281-5815","authenticated-orcid":false,"given":"Shankar","family":"Balasubramanian","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4450-576X","authenticated-orcid":false,"given":"Eric 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status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.19826.r53385, Matthias Ronald Schaefer, Centre for Anatomy and Cell Biology, Division of Cell-and Developmental Biology, Medical University of Vienna, Vienna, 09 Dec 2022, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/7-282\/v1#referee-response-53385","order":0,"name":"referee-response-53385","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.19826.r61033, Carolyn Phillips, University of Southern California, Los Angeles, California, 11 Jul 2023, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/7-282\/v1#referee-response-61033","order":1,"name":"referee-response-61033","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.19826.r59586, Anna Y Zinovyeva, Jeffrey Medley, Kansas State University, Manhattan, Kansas, 18 Jul 2023, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/7-282\/v1#referee-response-59586","order":2,"name":"referee-response-59586","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was supported by Wellcome [104640, <a href=https:\/\/doi.org\/10.35802\/104640>https:\/\/doi.org\/10.35802\/104640<\/a>; a Senior Investigator award to E.M.] and [209441, <a href=https:\/\/doi.org\/10.35802\/209441>https:\/\/doi.org\/10.35802\/209441<\/a>; a Senior Investigator award to S.B.]; a Cancer Research UK award (C13474\/A27826) to E.M; Conselho Nacional de Desenvolvimento Cient\u00edfico e Tecnol\u00f3gico doctorate scholarship (CNPq, Brazil - 205589\/2014-6) to I.C.N. The Miska laboratory is supported by core funding from Wellcome [092096; 203144] and Cancer Research UK (C6946\/A24843). The Balasubramanian laboratory is supported by Cancer Research UK core (C9545\/A19836) and programme award funding (C9681\/A29214), and Herchel Smith Funds. Tagged ALY-1 and ALY-2 strains were generated by the TransgeneOme project and provided by the Caenorhabditis Genetics Center \u2013 the latter is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":3,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}