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YTHDF2 Binds to 5-Methylcytosine in RNA and Modulates the Maturation of Ribosomal RNA

Dai, Xiaoxia, Gonzalez, Gwendolyn, Li, Lin, Li, Jie, You, Changjun, Miao, Weili, Hu, Junchi, Fu, Lijuan, Zhao, Yonghui, Li, Ruidong, Li, Lichao, Chen, Xuemei, Xu, Yanhui, Gu, Weifeng, Wang, Yinsheng
Analytical chemistry 2019 v.92 no.1 pp. 1346-1354
DNA, DNA methylation, bisulfites, genes, hydrophobicity, proteins, proteomics, ribosomal RNA, sequence analysis
5-Methylcytosine is found in both DNA and RNA; although its functions in DNA are well established, the exact role of 5-methylcytidine (m⁵C) in RNA remains poorly defined. Here we identified, by employing a quantitative proteomics method, multiple candidate recognition proteins of m⁵C in RNA, including several YTH domain-containing family (YTHDF) proteins. We showed that YTHDF2 could bind directly to m⁵C in RNA, albeit at a lower affinity than that toward N⁶-methyladenosine (m⁶A) in RNA, and this binding involves Trp⁴³², a conserved residue located in the hydrophobic pocket of YTHDF2 that is also required for m⁶A recognition. RNA bisulfite sequencing results revealed that, after CRISPR-Cas9-mediated knockout of the YTHDF2 gene, the majority of m⁵C sites in rRNA (rRNA) exhibited substantially augmented levels of methylation. Moreover, we found that YTHDF2 is involved in pre-rRNA processing in cells. Together, our data expanded the functions of the YTHDF2 protein in post-transcriptional regulations of RNA and provided novel insights into the functions of m⁵C in RNA biology.