Conservation and function of RNA 5-methylcytosine in plants

Abstract

Post-transcriptional methylation of RNA cytosine residues to 5-methylcytosine (m⁵C) is an important modification that regulates RNA metabolism, translation and stress responses and occurs in both eukaryotes and prokaryotes. Yet, to date, no transcriptome-wide identification of m⁵C sites has been undertaken in plants. Here, we present over a thousand m⁵C sites transcriptome-wide in mRNAs and ncRNAs (non-coding RNAs) in three tissue types; siliques, shoots and roots of Arabidopsis thaliana at single nucleotide resolution using high-throughput Illumina sequencing of bisulfite treated RNA (RBS-seq). We show that m⁵C methylation sites can be tissue-specific, or shared among the tissue types investigated. Among the shared m⁵C sites, some are differentially regulated between tissue types, while others are constitutively methylated at the same level across all three tissue types. Within mRNAs, the majority of m⁵C sites are located within coding sequences. A small, significant enrichment of m⁵C sites in 3’UTRs of mRNAs was observed when normalizing for length and sequence coverage. We also investigated ncRNAs and demonstrate conservation of rRNA and tRNA m⁵C sites across six species in the kingdom Plantae, suggesting important and highly conserved roles of this post-transcriptional modification. We identified over 100 m⁵C sites in diverse RNA classes such as mRNAs, lncRNAs (long non-coding RNAs), snoRNAs (small nucleolar RNAs) and tRNAs mediated by Arabidopsis tRNA methyltransferase 4B (TRM4B) in siliques, shoots and roots. TRM4 plays broad roles in many organisms for mediating oxidative stress tolerance and balancing stem cell self-renewal and differentiation. We discovered that these roles are also conserved in plants, as Arabidopsis trm4b mutants have shorter primary roots, which is linked to a reduced capacity for cells to divide in the root meristem. Furthermore, trm4b mutants are also more sensitive to oxidative stress and have reduced stability of non-methylated tRNAs. Here, we extend the known m⁵C sites in tRNAs mediated by Transfer RNA aspartic acid methyltransferase 1 (TRDMT1) and find no evidence of m⁵C sites mediated by TRDMT1 in other RNA classes. Additionally we demonstrate that rRNA methylation requires the conserved RNA methyltransferase (RMTase) NSUN5. Our results also suggest functional redundancy of the three predicted RMTase NOP2 paralogs in Arabidopsis. This thesis provides the first maps of the Arabidopsis m⁵C epitranscriptome and characterization of Arabidopsis genetic mutants needed to further probe functions of this new layer of gene regulation in plants.