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

Methylation of carbon-5 of cytosines (m ⁵ C) is a post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. m ⁵ C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. We recently showed that m ⁵ C is required for Caenorhabditis elegans development and fertility under heat stress. To contribute to the understanding of how m ⁵ C 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 . Our mass spectrometry analyses reveal unique binding proteins for each of the modifications. We validate our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m ⁵ C in vitro . The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m ⁵ C and its derivatives.