Abstract
The presence of 5-methylcytidine (m5C) 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 m5C 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 m5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m5C was absent were also discovered. Intriguingly, we detected m5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m5C-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.
Highlights
We envision RNA as a mere copy of the DNA four-base code, modification of specific RNA bases can expand the information code. Such modifications are abundant in transfer RNA and ribosomal RNA, where they contribute to translation fidelity and ribosome assembly
Recent studies in eukaryotes have shown that mRNA modifications such as RNA-editing, N6-adenine methylation (m6A), and 5-methylcytidine (m5C) can change the coding sequence, alter splicing patterns, or change RNA stability
No mRNA modifications in bacteria or archaea have been documented to date
Summary
RNA samples preparation Escherichia coli (MG1655) cells were grown in LB medium to log phase at 37uC. 168 cells were grown in LB medium to mid log phase at 37uC. S. solfataricus P2 (DSMZ 1617) cells were grown in defined modified Brock’s mineral medium with final pH 3.5, and S. cerevisiae (BY4741) cells were grown in YPD medium to log phase at 30uC. All cells pellets were suspended in RNAlater (Ambion, AM7022) for 30 min at room temperature. Pre-treatments for RNA isolation included Lysozyme digestion for E. coli cells, glass beads vortex for B. subtilis and Lyticase digestion for S. cerevisiae. Total RNA was extracted using Tri-Reagent (Molecular Research Center Inc.) according to manufacturer’s instructions. RNA samples were treated with Turbo DNA-free kit (Ambion) and rRNA was removed from the E.coli and B.subtilis samples using MICROBExpress mRNA enrichment kit (Ambion)
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