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

  • Anal Chem. 2020 Jan 7;92(1):1346-1354. doi: 10.1021/acs.analchem.9b04505.
Xiaoxia Dai  1  2 ,  Gwendolyn Gonzalez  3 ,  Lin Li  1 ,  Jie Li  4  5 ,  Changjun You  1  2 ,  Weili Miao  1 ,  Junchi Hu  6 ,  Lijuan Fu  3 ,  Yonghui Zhao  7 ,  Ruidong Li  8 ,  Lichao Li  8 ,  Xuemei Chen  7 ,  Yanhui Xu  4  5 ,  Weifeng Gu  8 ,  Yinsheng Wang  1  3
Affiliations
  • 1. Department of Chemistry , University of California , Riverside , California 92521-0403 , United States.
  • 2. State Key Laboratory of Chemo/Bio-sensing and Chemometrics, College of Chemistry and Chemical Engineering , Hunan University , Changsha , Hunan 410082 , China.
  • 3. Environmental Toxicology Graduate Program , University of California , Riverside , California 92521-0403 , United States.
  • 4. Fudan University Shanghai Cancer Center, Department of Oncology; and Institutes of Biomedical Sciences and School of Basic Medical Sciences , Shanghai Medical College of Fudan University , Shanghai 200032 , China.
  • 5. State Key Laboratory of Genetic Engineering, School of Life Sciences , Fudan University , Shanghai 200433 , China.
  • 6. Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica , Chinese Academy of Sciences , Shanghai 201203 , China.
  • 7. Department of Botany and Plant Sciences, Institute of Integrative Genome Biology , University of California , Riverside , California 92521-0403 , United States.
  • 8. Department of Cell Biology and Neuroscience , University of California , Riverside , California 92521-0403 , United States.
PMID: 31815440 DOI: 10.1021/acs.analchem.9b04505
Abstract

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.