Processing and decay of 6S-1 and 6S-2 RNAs in Bacillus subtilis

Abstract

Noncoding 6S RNAs regulate transcription by binding to the active site of bacterial RNA polymerase holoenzymes. Processing and decay of 6S-1 and 6S-2 RNA were investigated in Bacillus subtilis by northern blot and RNA-seq analyses using different RNase knockout strains, as well as by in vitro processing assays. For both 6S RNA paralogs, we identified a key—but mechanistically different—role of RNase J1. RNase J1 catalyzes 5′-end maturation of 6S-1 RNA, yet relatively inefficient and possibly via the enzyme's “sliding endonuclease” activity. 5′-end maturation has no detectable effect on 6S-1 RNA function, but rather regulates its decay: The generated 5′-monophosphate on matured 6S-1 RNA propels endonucleolytic cleavage in its apical loop region. The major 6S-2 RNA degradation pathway is initiated by endonucleolytic cleavage in the 5′-central bubble to trigger 5′-to-3′-exoribonucleolytic degradation of the downstream fragment by RNase J1. The four 3′-exonucleases of B. subtilis—RNase R, PNPase, YhaM, and particularly RNase PH—are involved in 3′-end trimming of both 6S RNAs, degradation of 6S-1 RNA fragments, and decay of abortive transcripts (so-called product RNAs, ∼14 nt in length) synthesized on 6S-1 RNA during outgrowth from stationary phase. In the case of the growth-retarded RNase Y deletion strain, we were unable to infer a specific role of RNase Y in 6S RNA decay. Yet, a participation of RNase Y in 6S RNA decay still remains possible, as evidence for such a function may have been obscured by overlapping substrate specificities of RNase Y, RNase J1, and RNase J2.