Abstract
Polynucleotide phosphorylase (PNPase), a 3' exoribonuclease that degrades RNA in the 3'-to-5' direction, is the major mRNA decay activity in Bacillus subtilis. PNPase is known to be inhibited in vitro by strong RNA secondary structure, and rapid mRNA turnover in vivo is thought to require an RNA helicase activity working in conjunction with PNPase. The most abundant RNA helicase in B. subtilis is CshA. We found for three small, monocistronic mRNAs that, for some RNA sequences, PNPase processivity was unimpeded even without CshA, whereas others required CshA for efficient degradation. A novel colour screen for decay of mRNA in B. subtilis was created, using mRNA encoded by the slrA gene, which is degraded from its 3' end by PNPase. A significant correlation between the predicted strength of a stem-loop structure, located in the body of the message, and PNPase processivity was observed. Northern blot analysis confirmed that PNPase processivity was greatly hindered by the internal RNA structure, and even more so in the absence of CshA. Three other B. subtilis RNA helicases did not appear to be involved in mRNA decay during vegetative growth. The results confirm the hypothesis that efficient 3' exonucleolytic decay of B. subtilis RNA depends on the combined activity of PNPase and CshA.