Bacillus subtilis RNase J1 endonuclease and 5′ exonuclease activities in the turnover of ΔermC mRNA

Abstract

RNase J1, a ribonuclease with 59 exonuclease and endonuclease activities, is an important factor in Bacillus subtilis mRNA decay. A model for RNase J1 endonuclease activity in mRNA turnover has RNase J1 binding to the 59 end and tracking to a target site downstream, where it makes a decay-initiating cleavage. The upstream fragment from this cleavage is degraded by 39 exonucleases; the downstream fragment is degraded by RNase J1 59 exonuclease activity. Previously, DermC mRNA was used to show 59-end dependence of mRNA turnover. Here we used DermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. DermC mRNA showed increased stability in a mutant strain that contained a reduced level of RNase J1. In agreement with the tracking concept, insertion of a strong stem-loop structure at +65 resulted in increased stability. Weakening this stem-loop structure resulted in reversion to wild-type stability. RNA fragments containing the 39 end were detected in a strain with reduced RNase J1 expression, but were undetectable in the wild type. The 59 ends of these fragments mapped to the upstream side of predicted stem-loop structures, consistent with an impediment to RNase J1 59 exonuclease processivity. A DermC mRNA deletion analysis suggested that decay-initiating endonuclease cleavage could occur at several sites near the 39 end. However, even in the absence of these sites, stability was further increased in a strain with reduced RNase J1, suggesting alternate pathways for decay that could include exonucleolytic decay from the 59 end.

What is the role of RNase J in mRNA turnover?

“…This does not rule out the possibility that alternative endonucleasedependent pathways exist for the turnover of at least some portion of these mRNAs, as suggested recently for ∆ermC. 23 Endonucleolytic cleavage by RNase J1. There are a number of clearer examples of a role for RNase J1 endonucleolytic activity in RNA processing and decay.…”

“…However, a model of ribosome-dependent endonucleolytic cleavage by RNase J1 at the upstream boundary of the stalled ribosome is favored by the authors for both ∆ermC and lac-rpsO. 20,22,23 It is not immediately clear to me, however, why these mRNAs would be any different to the cryIIIA and hbs mRNAs. Since the generation of the ribosome-protected species is inhibited by the addition of secondary structures to the 5' end of the ∆ermC and lac-rpsO transcripts, this was interpreted as evidence that the endonucleolytic cleavage by RNase J1 is 5'-end dependent, with the enzyme scanning to the site of ribosome-dependent cutting.…”

Chloroplast RNase J compensates for inefficient transcription termination by removal of antisense RNA

“…It has been implicated in the trimming of 16S rRNA (Britton et al 2007;Mathy et al 2007), and the turnover of small RNAs such as the trp and thrS leaders and DermC (Even et al 2005;Deikus et al 2008;Yao et al 2009). There is also evidence that RNase J has a broader role in determining mRNA lifetime (Mader et al 2008;Bugrysheva and Scott 2010).…”

mRNA degradation and maturation in prokaryotes: the global players

“…For several mRNAs studied (ermC, rpsO, cryIIIA, hbs), the generation of shorter, often more stable mRNA transcripts is thought to be due to RNase J1 exonuclease activity, which trims a longer precursor before it is partially halted, generally by a bound or stalled ribosome (18,(87)(88)(89)(90). In some cases (ermC, rpsO), it has been suggested that RNase J1 might act as an endonuclease, but the corresponding upstream RNA fragments have never been detected (91).…”