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

Abstract

RNase J1, a ribonuclease with 5' 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 5' end and tracking to a target site downstream, where it makes a decay-initiating cleavage. The upstream fragment from this cleavage is degraded by 3' exonucleases; the downstream fragment is degraded by RNase J1 5' exonuclease activity. Previously, DeltaermC mRNA was used to show 5'-end dependence of mRNA turnover. Here we used DeltaermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. DeltaermC 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 3' end were detected in a strain with reduced RNase J1 expression, but were undetectable in the wild type. The 5' ends of these fragments mapped to the upstream side of predicted stem-loop structures, consistent with an impediment to RNase J1 5' exonuclease processivity. A DeltaermC mRNA deletion analysis suggested that decay-initiating endonuclease cleavage could occur at several sites near the 3' 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 5' end.