Polynucleotide phosphorylase and RNA helicase CshA cooperate in Bacillus subtilis mRNA decay

Polynucleotide phosphorylase (PNPase), a 3MODIFIER LETTER PRIME exoribonuclease that degrades RNA in the 3MODIFIER LETTER PRIME-to-5MODIFIER LETTER PRIME 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 3MODIFIER LETTER PRIME 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 3MODIFIER LETTER PRIME exonucleolytic decay of B. subtilis RNA depends on the combined activity of PNPase and CshA.

Automated summary

PNPase is the major mRNA decay activity in Bacillus subtilis, and its processivity can be affected by RNA secondary structure, which is thought to be overcome by the helicase activity of CshA. A color screen for mRNA decay in B. subtilis revealed a significant correlation between internal stem-loop structure strength and PNPase processivity, which is hindered in the absence of CshA. Other RNA helicases in B. subtilis were found not to play a role in mRNA decay.