Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA

I C Navarro; Kin Man Suen; Dalila Bensaddek; Arun Tanpure; Angus Lamond; Shankar Balasubramanian; Eric A Miska

doi:10.12688/wellcomeopenres.17893.1

Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA

Wellcome Open Res. 2022 Nov 17:7:282. doi: 10.12688/wellcomeopenres.17893.1. eCollection 2022.

Authors

I C Navarro^#^{1

2}, Kin Man Suen^#^{1

3}, Dalila Bensaddek^{4

5}, Arun Tanpure⁶, Angus Lamond⁴, Shankar Balasubramanian^{6

7

8}, Eric A Miska^{1

2

9}

Affiliations

¹ Gurdon Institute, University of Cambridge, Cambridge, CB2 1QN, UK.
² Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK.
³ School of Molecular and Cellular Biology, University of Leeds, LC Miall Building, Leeds, LS2 9JT, UK.
⁴ Laboratory for Quantitative Proteomics, Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK.
⁵ Bioscience Core Labs, King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia.
⁶ Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
⁷ Cancer Research (UK), Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Robinson Way, Cambridge, CB2 0RE, UK.
⁸ School of Clinical Medicine, University of Cambridge, Cambridge, CB2 0SP, UK.
⁹ Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, CB10 1SA, UK.

^# Contributed equally.

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.

Keywords: 5-methylcytosine; Caenorhabditis elegans; RNA; epitranscriptomics; readers.

Abstract

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