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5-Methylcytosine RNA Modifications Promote Retrovirus Replication in an ALYREF Reader Protein-Dependent Manner.

ABSTRACT: RNA modifications play diverse roles in regulating RNA function, and viruses co-opt these pathways for their own benefit. While recent studies have highlighted the importance of N ⁶-methyladenosine (m⁶A)-the most abundant mRNA modification-in regulating retrovirus replication, the identification and function of other RNA modifications in viral biology have been largely unexplored. Here, we characterized the RNA modifications present in a model retrovirus, murine leukemia virus (MLV), using mass spectrometry and sequencing. We found that 5-methylcytosine (m⁵C) is highly enriched in viral genomic RNA relative to uninfected cellular mRNAs, and we mapped at single-nucleotide resolution the m⁵C sites, which are located in multiple clusters throughout the MLV genome. Further, we showed that the m⁵C reader protein ALYREF plays an important role in regulating MLV replication. Together, our results provide a complete m⁵C profile in a virus and its function in a eukaryotic mRNA.IMPORTANCE Over 130 modifications have been identified in cellular RNAs, which play critical roles in many cellular processes, from modulating RNA stability to altering translation efficiency. One such modification, 5-methylcytosine, is relatively abundant in mammalian mRNAs, but its precise location and function are not well understood. In this study, we identified unexpectedly high levels of m⁵C in the murine leukemia virus RNA, precisely mapped its location, and showed that ALYREF, a reader protein that specifically recognizes m⁵C, regulates viral production. Together, our findings provide a high-resolution atlas of m⁵C in murine leukemia virus and reveal a functional role of m⁵C in viral replication.

SUBMITTER: Eckwahl M

PROVIDER: S-EPMC7307164 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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5-Methylcytosine RNA Modifications Promote Retrovirus Replication in an ALYREF Reader Protein-Dependent Manner.

Eckwahl Matthew M Xu Ruyi R Michalkiewicz Julia J Zhang Wen W Patel Pooja P Cai Zhen Z Pan Tao T

Journal of virology 20200616 13

RNA modifications play diverse roles in regulating RNA function, and viruses co-opt these pathways for their own benefit. While recent studies have highlighted the importance of N6-methyladenosine (m6A)-the most abundant mRNA modification-in regulating retrovirus replication, the identification and function of other RNA modifications in viral biology have been largely unexplored. Here, we characterized the RNA modifications present in a model retrovirus, murine leuke ...[more]

PMID: 32321818

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Project description: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.

Dataset Information

5-Methylcytosine RNA Modifications Promote Retrovirus Replication in an ALYREF Reader Protein-Dependent Manner.

Publications

5-Methylcytosine RNA Modifications Promote Retrovirus Replication in an ALYREF Reader Protein-Dependent Manner.

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