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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, Δ ermC  mRNA was used to show 5′-end dependence of mRNA turnover. Here we used Δ ermC  mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. Δ ermC  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 Δ ermC  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.

Bacillus subtilis RNase J1 endonuclease and 5' exonuclease activities in the turnover of ermC mRNA