Characterization of tRNACys processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control

Abstract

We generated a conditional CCase mutant of Bacillus subtilis to explore the participation in vivo of the tRNA nucleotidyltransferase (CCA transferase or CCase) in the maturation of the singlecopy tRNA Cys , which lacks an encoded CCA 39 end. We observed that shorter tRNA Cys species, presumably lacking CCA, only accumulated when the inducible Pspac : cca was introduced into an rnr mutant strain, but not in combination with pnp. We sequenced the tRNA 39 ends produced in the various mutant tRNA Cys species to detect maturation and decay intermediates and observed that decay of the tRNA Cys occurs through the addition of poly(A) or heteropolymeric tails. A few clones corresponding to full-size tRNAs contained either CCA or other C and/or A sequences, suggesting that these are substrates for repair and/or decay. We also observed editing of tRNA Cys at position 21, which seems to occur preferentially in mature tRNAs. Altogether, our results provide in vivo evidence for the participation of the B. subtilis cca gene product in the maturation of tRNAs lacking CCA. We also suggest that RNase R exoRNase in B. subtilis participates in the quality control of tRNA.

Critical Minireview: The Fate of tRNACys during Oxidative Stress in Bacillus subtilis

“…The smaller tRNA Cys species observed in conditions of oxidative stress had an electrophoretic mobility identical to that previously reported for tRNA Cys lacking the CCA end, as assessed using synthetic CCA-less tRNA Cys as a molecular marker [30]. It must be noted that the purification and sequencing of the smaller tRNA Cys species were not performed in this study.…”

“…In B. subtilis, on the other hand, around 30% of tRNAs lack an encoded CCA at the 3′ end (Table 1). CCA must be added post-transcriptionally to those CCA-less tRNAs [29,30]. …”

“…It must be noted that the purification and sequencing of the smaller tRNA Cys species were not performed in this study. However, the analysis of some fragments of tRNA Cys obtained by reverse transcription polymerase chain reaction (RT-PCR) did demonstrate the presence of tRNA Cys species that had been modified by the addition of poly(A) or heteropolymeric 3′ tails, suggesting that these were immature or damaged species, targeted for degradation [30]. Thus, it seems likely that the smaller tRNA produced by oxidative stress induced by mercury exposure is CCA-less tRNA Cys .…”

Synergies between RNA degradation and trans-translation in Streptococcus pneumoniae: cross regulation and co-transcription of RNase R and SmpB

“…These include Aeromonas hydrophila , Shigella flexneri , enteroinvasive E. coli , and Helicobacter pylori [18-21]. This enzyme has also been involved in the quality control of defective tRNA and rRNA molecules [13,22]. Furthermore, E. coli RNase R was shown to participate in the maturation of the transfer -messenger RNA (tmRNA, also called SsrA) [12], an important small RNA involved in trans -translation.…”

Critical Minireview: The Fate of tRNA^Cysduring Oxidative Stress in <em>Bacillus subtilis</em>

“…In B. subtilis, on the other hand, around 30% of tRNAs lack an encoded CCA at the 3 end (Table 1). CCA must be added post-transcriptionally to those CCA-less tRNAs [29,30]. The presence of cca in the ypjD operon, along with bshA and bshB1 suggests the intriguing possibility that CCAase may be involved in the response of B. subtilis to oxidative stress.…”