Abstract
Background: Methylation of carbon-5 of cytosines (m 5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield 5-hydroxymethylcytidine (hm 5C), 5-formylcytidine (f 5C), 2´-O-methyl-5-hydroxymethylcytidine (hm 5Cm) and 2´-O-methyl-5-formylcytidine (f 5Cm). How m 5C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m 5C is required for Caenorhabditis elegans development and fertility under heat stress. m 5C 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.
Methods: To contribute to the understanding of how m 5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m 5C was validated using immunoblotting.
Results: Our mass spectrometry analyses revealed unique binding proteins for each of the modifications. In silico analysis for phenotype enrichments suggested that hm 5Cm unique readers are enriched in proteins involved in RNA processing, while readers for m 5C, hm 5C and f 5C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m 5C in vitro. Finally, sequence alignment analysis showed that several of the putative m 5C readers contain the conserved RNA recognition motif (RRM), including ALY-1 and ALY-2.
Conclusions: The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m 5C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m 5C in C. elegans.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 282 |
| Journal | Wellcome Open Research |
| Volume | 7 |
| DOIs | |
| State | Published - Nov 17 2022 |
Bibliographical note
KAUST Repository Item: Exported on 2023-09-01Acknowledgements: This work was supported by Wellcome [104640, https://doi.org/10.35802/104640; a Senior Investigator award to E.M.] and [209441, https://doi.org/10.35802/209441; a Senior Investigator award to S.B.]; a Cancer Research UK award (C13474/A27826) to E.M; Conselho Nacional de Desenvolvimento Científico e Tecnológico 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 – the latter is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We would like to thank the Gurdon Institute Media Kitchen for their support providing reagents and media.