Transcriptome-wide Mapping of RNA 5-Methylcytosine in Arabidopsis mRNAs and non-coding RNAs

Abstract

Post-transcriptional 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 (bsRNA-seq) for transcriptome-wide quantitative mapping of m5C in the model plant, Arabidopsis thaliana. We discovered more than a thousand m5C sites in Arabidopsis mRNAs, long non-coding RNAs, and other non-coding 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 non-coding RNAs and reduced the stability of tRNAAsp(GTC). We also demonstrate the importance of m5C in plant development, as trm4b mutants have shorter primary roots than wild type due to reduced cell division in the root apical meristem. In addition, trm4b mutants show increased sensitivity to oxidative stress. Finally, we provide insights into the targeting mechanism of TRM4B by demonstrating that a 50 nt sequence flanking m5C C3349 in MAIGO5 mRNA is sufficient to confer methylation of a transgene reporter in Nicotiana benthamiana.