David_et_al_2016.pdf

This paper describes the first transcrriptome-wide identification of 5-methylcytosine modifications in plants.<br>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 use RNA bisulfite sequencing (bsRNA-seq) for transcriptome-wide quantitative mapping of m5C in the model plant, <i>Arabidopsis thaliana</i>. We discover 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 validate 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 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 <i>trm4b</i> mutants have shorter primary roots 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 the first insights into the targeting mechanism of TRM4B by demonstrating that a 50 nt sequence flanking m5C C3349 in MAG5 mRNA is sufficient to confer methylation of a transgene reporter in <i>Nicotiana benthamiana.</i><br>