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

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.