Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m5C within Archaeal mRNAs

The presence of 5-methylcytidine (m 5 C) in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m 5 C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive ( B. subtilis ) and gram negative ( E. coli ) bacteria, an archaeon ( S. solfataricus ) and a eukaryote ( S. cerevisiae ), followed by massively parallel sequencing. We were able to recover most previously documented m 5 C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m 5 C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m 5 C was absent were also discovered. Intriguingly, we detected m 5 C sites within archaeal mRNAs, and identified a consensus motif of AU C GANGU that directs methylation in S. solfataricus . Our results, which were validated using m 5 C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.