Abstract
BackgroundPost-transcriptional methylation of RNA cytosine residues to 5-methylcytosine (m5C) is an important modification that regulates RNA metabolism and occurs in both eukaryotes and prokaryotes. Yet, to date, no transcriptome-wide identification of m5C sites has been undertaken in plants. Plants provide a unique comparative system for investigating the origin and evolution of m5C as they contain three different genomes, the nucleus, mitochondria and chloroplast. Here we use bisulfite conversion of RNA combined with high-throughput IIlumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites in non-coding ribosomal RNAs and transfer RNAs of all three sub-cellular transcriptomes across six diverse species that included, the single-celled algae Nannochloropsis oculata, the macro algae Caulerpa taxifolia and multi-cellular higher plants Arabidopsis thaliana, Brassica rapa, Triticum durum and Ginkgo biloba.ResultsUsing the plant model Arabidopsis thaliana, we identified a total of 39 highly methylated m5C sites in predicted structural positions of nuclear tRNAs and 7 m5C sites in rRNAs from nuclear, chloroplast and mitochondrial transcriptomes. Both the nucleotide position and percent methylation of tRNAs and rRNAs m5C sites were conserved across all species analysed, from single celled algae N. oculata to multicellular plants. Interestingly the mitochondrial and chloroplast encoded tRNAs were devoid of m5C in A. thaliana and this is generally conserved across Plantae. This suggests independent evolution of organelle methylation in animals and plants, as animal mitochondrial tRNAs have m5C sites. Here we characterize 5 members of the RNA 5-methylcytosine family in Arabidopsis and extend the functional characterization of TRDMT1 and NOP2A/OLI2. We demonstrate that nuclear tRNA methylation requires two evolutionarily conserved methyltransferases, TRDMT1 and TRM4B. trdmt1 trm4b double mutants are hypersensitive to the antibiotic hygromycin B, demonstrating the function of tRNA methylation in regulating translation. Additionally we demonstrate that nuclear large subunit 25S rRNA methylation requires the conserved RNA methyltransferase NSUN5. Our results also suggest functional redundancy of at least two of the NOP2 paralogs in Arabidopsis.ConclusionsOur data demonstrates widespread occurrence and conservation of non-coding RNA methylation in the kingdom Plantae, suggesting important and highly conserved roles of this post-transcriptional modification.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0580-8) contains supplementary material, which is available to authorized users.
Highlights
Post-transcriptional methylation of RNA cytosine residues to 5-methylcytosine (m5C) is an important modification that regulates RNA metabolism and occurs in both eukaryotes and prokaryotes
We determined that NOP2/Sun domain protein 5 (NSUN5) is required for m5C at C2278 in nuclear large subunit (LSU) 25S Ribosomal RNA (rRNA) and uncovered functional redundancy among the Arabidopsis Nucleolar Protein 2 (NOP2) paralogs, NOP2A, NOP2B and NOP2C, as loss of one of these three enzymes is insufficient to remove any rRNA m5C sites, while loss of both NOP2A and NOP2B appears lethal
Arabidopsis RNA methyltransferase (RMTase) enzymes are encoded in the nuclear genome. This suggests movement of either the nuclear encoded RMTase enzymes to organelles, or transport and re-import of organelle rRNAs. We favour the former hypothesis as the Arabidopsis Fmu-like RNMT and NOP2B are predicted to be located in organelles [26], which suggests that they methylate rRNA inside the mitochondria and chloroplast, and that NOP2B acts redundantly with NOP2A and NOP2C
Summary
Post-transcriptional methylation of RNA cytosine residues to 5-methylcytosine (m5C) is an important modification that regulates RNA metabolism and occurs in both eukaryotes and prokaryotes. To date, no transcriptome-wide identification of m5C sites has been undertaken in plants. Plants provide a unique comparative system for investigating the origin and evolution of m5C as they contain three different genomes, the nucleus, mitochondria and chloroplast. We use bisulfite conversion of RNA combined with high-throughput IIlumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites in non-coding ribosomal RNAs and transfer RNAs of all three sub-cellular transcriptomes across six diverse species that included, the single-celled algae Nannochloropsis oculata, the macro algae Caulerpa taxifolia and multi-cellular higher plants Arabidopsis thaliana, Brassica rapa, Triticum durum and Ginkgo biloba
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