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.

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