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

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.

Keywords: 3ʹ exoribonuclease; Bacillus subtilis; CshA; PNPase; RNA helicase; mRNA decay.

Figure 1.

(A-C) Northern blot analysis of rpsO (A), rapA (B), and cggR (C) RNA in wild-type (lanes 1), pnpA (lanes 2), cshA (lanes 3), and pnpA cshA (lanes 4) strains. Migration of full-length RNA is indicated by ‘FL.’ Marker lane in (A) contained RNA Century-Plus marker (Ambion), with the indicated RNA sizes in nucleotides. Values to the left in (B) and (C) indicate migration of 5ʹ-end-labelled fragments (sizes in nucleotides) of a TaqI digest of plasmid pSE420 [49]. Arrows point to RNA decay intermediates that accumulate in the cshA mutant strain. (D) Determination of relative amounts of FLAG-tagged PNPase and CshA. The four lanes are from independent protein isolations, grown under identical growth conditions

Figure 2.

(A) Diagram of the mRNA decay screen. The top line is a schematic of the slrA transcription unit, showing the CDS and 5ʹ and 3ʹ UTRs (not drawn to scale). The structured 38-nt variable sequence is shown schematically, as well as the 3ʹ-proximal predicted secondary structure in a region that is putatively required for Rho-dependent termination. The characteristics of a Rho-dependent terminator in B. subtilis are still not well-defined [50]. (B and C) Structure of the 38-nt variable sequence in constructs 12 and 14, and colony colour on S-gal plates in the wild-type background. (D) Correlation of mean grey value with predicted strength of secondary structure for 40 clones in the wild-type background

Figure 3.

Phenotypes of five clones selected for Northern blot analysis, containing the indicated slrA constructs. (A) Colony colour of wild-type strains. Predicted ΔG₀ (kcal/mol) of the 38-nt sequence and mean grey (MG) value of the colony are shown. (B) Colony colour of cshA deletion mutant strains. (C) Overnight cultures of cshA deletion mutant strains. (D) Phase microscopy (20X) of c shA deletion mutant strains. Bar, 10 µM

Figure 4.

Northern blot analysis of five selected clones. (A) Steady-state slrA RNA in wild-type (left) and cshA mutant (right) strains. Above each lane is the number of the slrA construct. Migration of prominent slrA RNAs indicated on the right: FL, full-length RNA; diamond, 520-nt decay intermediate; DI, decay intermediate with a 3ʹ end at the downstream edge of the 38-nt variable sequence. The blot was stripped and reprobed with the veg probe. Marker lane contained RNA Century-Plus markers, with the indicated RNA sizes in nucleotides. (B) Quantitation of results of three independent Northern blot experiments for steady-state slrA RNA. (C) Northern blot analysis of slrA RNA decay in strains with constructs 12 and 14, after addition of rifampicin at time zero. Aliquots were taken after rifampicin addition at times (min) indicated above each lane. Marker lane contained RNA Century-Plus markers, with the indicated RNA sizes in nucleotides. (D) Half-life determination of full-length slrA RNA in strain with construct 12 (circles, solid line), full-length slrA RNA in strain with construct 14 (squares, dashed line) and decay intermediate from construct 14 (triangles, dashed line). (E) Northern blot analysis of slrA RNA in single and triple helicase mutants. Above each lane is the number of the slrA construct. Migration of RNA Century Plus marker RNAs indicated on the left