Transcriptome-Wide Mapping of RNA 5-Methylcytosine in Arabidopsis mRNAs and Noncoding RNAs

Abstract

Posttranscriptional methylation of RNA cytosine residues to 5-methylcytosine (m⁵C) is an important modification with diverse roles, such as regulating stress responses, stem cell proliferation, and RNA metabolism. Here, we used RNA bisulfite sequencing for transcriptome-wide quantitative mapping of m⁵C in the model plant <i>Arabidopsis thaliana</i> We discovered more than a thousand m⁵C sites in Arabidopsis mRNAs, long noncoding RNAs, and other noncoding RNAs across three tissue types (siliques, seedling shoots, and roots) and validated a number of these sites. Quantitative differences in methylated sites between these three tissues suggest tissue-specific regulation of m⁵C. Perturbing the RNA m⁵C methyltransferase TRM4B resulted in the loss of m⁵C sites on mRNAs and noncoding RNAs and reduced the stability of tRNA^Asp(GTC) We also demonstrate the importance of m⁵C in plant development, as <i>trm4b</i> mutants have shorter primary roots than the wild type due to reduced cell division in the root apical meristem. In addition, <i>trm4b</i> mutants show increased sensitivity to oxidative stress. Finally, we provide insights into the targeting mechanism of TRM4B by demonstrating that a 50-nucleotide sequence flanking m⁵C C3349 in <i>MAIGO5</i> mRNA is sufficient to confer methylation of a transgene reporter in <i>Nicotiana benthamiana</i>.