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

Wellcome Open Research

article

IC Navarro¹  Kin Man Suen¹  Dalila Bensaddek⁴  Arun Tanpure⁶  Angus Lamond⁴  Shankar Balasubramanian⁶  Eric A Miska¹ 

[1] Gurdon Institute, University of Cambridge;Department of Genetics, University of Cambridge;School of Molecular and Cellular Biology, University of Leeds;Laboratory for Quantitative Proteomics, Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee;Bioscience Core Labs, King Abdullah University of Science and Technology;Department of Chemistry, University of Cambridge;Cancer Research ,(UK), Cambridge Institute, Li Ka Shing Centre, University of Cambridge;School of Clinical Medicine, University of Cambridge;Wellcome Sanger Institute, Wellcome Genome Campus

关键词: RNA;    5-methylcytosine;    Caenorhabditis elegans;    epitranscriptomics;    readers;   

DOI  :  10.12688/wellcomeopenres.17893.1

学科分类：内科医学

来源: Wellcome

【 摘 要 】

Background: Methylation of carbon-5 of cytosines (m5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield 5-hydroxymethylcytidine (hm5C), 5-formylcytidine (f5C), 2´-O-methyl-5-hydroxymethylcytidine (hm5Cm) and 2´-O-methyl-5-formylcytidine (f5Cm). How m5C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m5C is required forCaenorhabditis elegans development and fertility under heat stress. m5C 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 m5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers inC. elegans. Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m5C 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 hm5Cm unique readers are enriched in proteins involved in RNA processing, while readers for m5C, hm5C and f5C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m5Cin vitro. Finally, sequence alignment analysis showed that several of the putative m5C 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 m5C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m5C inC. elegans.

【 授权许可】

CC BY