Navarro I C, Suen Kin Man, Bensaddek Dalila, Tanpure Arun, Lamond Angus, Balasubramanian Shankar, Miska Eric A
Gurdon Institute, University of Cambridge, Cambridge, CB2 1QN, UK.
Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK.
Wellcome Open Res. 2022 Nov 17;7:282. doi: 10.12688/wellcomeopenres.17893.1. eCollection 2022.
Methylation of carbon-5 of cytosines (m C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield 5-hydroxymethylcytidine (hm C), 5-formylcytidine (f C), 2´-O-methyl-5-hydroxymethylcytidine (hm Cm) and 2´-O-methyl-5-formylcytidine (f Cm). How m C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m C is required for development and fertility under heat stress. m C 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. To contribute to the understanding of how m C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in . Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m C was validated using immunoblotting. Our mass spectrometry analyses revealed unique binding proteins for each of the modifications. analysis for phenotype enrichments suggested that hm Cm unique readers are enriched in proteins involved in RNA processing, while readers for m C, hm C and f C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m C . Finally, sequence alignment analysis showed that several of the putative m C readers contain the conserved RNA recognition motif (RRM), including ALY-1 and ALY-2. The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m C in .
胞嘧啶碳 -5 位的甲基化（mC）是一种保守的RNA转录后核苷酸修饰，广泛分布于各种生物体中。它可以进一步修饰生成5 - 羟甲基胞嘧啶（hmC）、5 - 甲酰基胞嘧啶（fC）、2'-O - 甲基 -5 - 羟甲基胞嘧啶（hmCm）和2'-O - 甲基 -5 - 甲酰基胞嘧啶（fCm）。目前对于mC，尤其是其衍生物在生物学机制上如何发挥作用了解甚少。我们最近发现，在热应激条件下，mC对于发育和生育能力是必需的。mC已被证明分别通过与阅读蛋白ALYREF和YBX1相互作用参与mRNA转运并维持mRNA稳定性。因此，鉴定RNA修饰的阅读蛋白有助于我们深入了解这些修饰的生物学作用。为了有助于理解mC及其氧化衍生物如何介导其功能，我们设计了在不同结构背景下带有修饰胞嘧啶的RNA诱饵，以在体外下拉潜在的阅读蛋白。通过质谱鉴定潜在的阅读蛋白。使用免疫印迹法验证了两个推定的阅读蛋白与mC的相互作用。我们的质谱分析揭示了每种修饰的独特结合蛋白。对表型富集的分析表明，hmCm独特的阅读蛋白在参与RNA加工的蛋白质中富集，而mC、hmC和fC的阅读蛋白则参与生殖系过程。我们通过证明线虫ALYREF同源物ALY - 1和ALY - 2优先结合mC来验证我们的数据集。最后，序列比对分析表明，几个推定的mC阅读蛋白包含保守的RNA识别基序（RRM），包括ALY - 1和ALY - 2。本文提供的数据集是一项重要的科学资源，将有助于发现mC及其衍生物的新功能。此外，我们证明ALY - 1和ALY - 2在体内与mC结合。
