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

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Title: Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA.
Authors: Navarro, I. C.1,2, Suen, Kin Man1,3, Bensaddek, Dalila4,5, Tanpure, Arun6, Lamond, Angus4, Balasubramanian, Shankar6,7,8, Miska, Eric A.1,2,9 eam29@cam.ac.uk
Source: Wellcome Open Research. 2022, Vol. 7, p1-19. 19p.
Subject Terms: Methylcytosine, Caenorhabditis elegans, RNA methylation, Germ cells, Post-translational modification
Abstract: 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´-Omethyl-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 for Caenorhabditis 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 in C. 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 m5C in 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 in C. elegans. [ABSTRACT FROM AUTHOR]
Copyright of Wellcome Open Research is the property of Wellcome Trust and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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  Label: Title
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  Data: Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA.
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  Data: <searchLink fieldCode="AR" term="%22Navarro%2C+I%2E+C%2E%22">Navarro, I. C.</searchLink><relatesTo>1,2</relatesTo><br /><searchLink fieldCode="AR" term="%22Suen%2C+Kin+Man%22">Suen, Kin Man</searchLink><relatesTo>1,3</relatesTo><br /><searchLink fieldCode="AR" term="%22Bensaddek%2C+Dalila%22">Bensaddek, Dalila</searchLink><relatesTo>4,5</relatesTo><br /><searchLink fieldCode="AR" term="%22Tanpure%2C+Arun%22">Tanpure, Arun</searchLink><relatesTo>6</relatesTo><br /><searchLink fieldCode="AR" term="%22Lamond%2C+Angus%22">Lamond, Angus</searchLink><relatesTo>4</relatesTo><br /><searchLink fieldCode="AR" term="%22Balasubramanian%2C+Shankar%22">Balasubramanian, Shankar</searchLink><relatesTo>6,7,8</relatesTo><br /><searchLink fieldCode="AR" term="%22Miska%2C+Eric+A%2E%22">Miska, Eric A.</searchLink><relatesTo>1,2,9</relatesTo><i> eam29@cam.ac.uk</i>
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  Data: <searchLink fieldCode="JN" term="%22Wellcome+Open+Research%22">Wellcome Open Research</searchLink>. 2022, Vol. 7, p1-19. 19p.
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  Data: <searchLink fieldCode="DE" term="%22Methylcytosine%22">Methylcytosine</searchLink><br /><searchLink fieldCode="DE" term="%22Caenorhabditis+elegans%22">Caenorhabditis elegans</searchLink><br /><searchLink fieldCode="DE" term="%22RNA+methylation%22">RNA methylation</searchLink><br /><searchLink fieldCode="DE" term="%22Germ+cells%22">Germ cells</searchLink><br /><searchLink fieldCode="DE" term="%22Post-translational+modification%22">Post-translational modification</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: 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´-Omethyl-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 for Caenorhabditis 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 in C. 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 m5C in 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 in C. elegans. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
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  Data: <i>Copyright of Wellcome Open Research is the property of Wellcome Trust and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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        Value: 10.12688/wellcomeopenres.17893.1
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        Text: English
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      – SubjectFull: Methylcytosine
        Type: general
      – SubjectFull: Caenorhabditis elegans
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      – SubjectFull: RNA methylation
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              Text: 2022
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