Polynucleotide phosphorylase and RNA helicase CshA cooperate inBacillus subtilismRNA decay

Analysis of mRNA Decay Intermediates in Bacillus subtilis 3′ Exoribonuclease and RNA Helicase Mutant Strains

“…These data indicated that, in many cases, both PNPase and CshA activities are required for degradation through sequences that otherwise represent hindrances to 3′-to-5′ decay. This is suggestive of a cooperation of the RNA-unwinding activity of CshA working in advance of PNPase processivity to allow efficient mRNA decay, as was also suggested by previous work ( 12 ). On the other hand, ∼60% of the 3′ ends in either of the pnpA mutant strains were not shared with the cshA mutant strain.…”

“…When a secondary structure of considerable strength is encountered, PNPase may stall, giving rise to a 3′ end. We have shown previously that strong secondary structure blocks PNPase processivity, even when CshA is present ( 12 ). In the absence of PNPase activity, another 3′ exonuclease, most likely RNase R, is the primary activity degrading in the 3′-to-5′ direction, and it is not sensitive to secondary structure (see the slrA results below).…”

“…9 ). We found previously that the insertion of a strong stem-loop structure (Δ G = −18.8 kcal/mol) 60 nt downstream of the slrA CDS resulted in the accumulation of an mRNA decay intermediate of ∼440 nt, which was due to hindrance of PNPase-mediated decay by the inserted stem-loop structure ( 12 ).…”

“…We integrated at the amyE locus of each triple RNase deletion strain either a WT copy of the slrA gene or a copy of an slrA gene construct that had the strong stem-loop structure in the 3′ UTR. Since we found previously that the overexpression of slrA causes a severe chaining phenotype ( 12 ), slrA in the amyE locus was under the control of a bacitracin-inducible promoter ( 3 , 35 ). In the absence of bacitracin, the transcription of the slrA construct is completely off, whereas the addition of bacitracin results in the rapid induction of transcription.…”

“…In the current study, a transcriptome sequencing method (called Term-seq) that was first used to map transcription terminators ( 9 , 10 ) was used to obtain a transcriptomic view of 3′ ends of mRNA decay fragments in the wild-type (WT) strain compared to strains that were deleted for the pnpA gene or the cshA gene encoding the DEAD box RNA helicase CshA ( 11 ). Recently, we found evidence that PNPase and CshA cooperate in the turnover of several monocistronic mRNAs ( 12 ). The accumulation of 3′ ends in the Δ cshA strain would identify mRNAs that rely on CshA helicase activity for efficient degradation.…”