PNPase is a key player in the regulation of small RNAs that control the expression of outer membrane proteins

Abstract

In this report, we demonstrate that exonucleolytic turnover is much more important in the regulation of sRNA levels than was previously recognized. For the first time, PNPase is introduced as a major regulatory feature controlling the levels of the small noncoding RNAs MicA and RybB, which are required for the accurate expression of outer membrane proteins (OMPs). In the absence of PNPase, the pattern of OMPs is changed. In stationary phase, MicA RNA levels are increased in the PNPase mutant, leading to a decrease in the levels of its target ompA mRNA and the respective protein. This growth phase regulation represents a novel pathway of control. We have evaluated other ribonucleases in the control of MicA RNA, and we showed that degradation by PNPase surpasses the effect of endonucleolytic cleavages by RNase E. RybB was also destabilized by PNPase. This work highlights a new role for PNPase in the degradation of small noncoding RNAs and opens the way to evaluate striking similarities between bacteria and eukaryotes.

FIGURE 1.

PNPase regulates ompA and MicA RNA levels. (A) Analysis of ompA mRNA and MicA RNA levels in the wild type and exonuclease mutant strains in stationary phase. ompA mRNA was analyzed by agarose Northern blot. (Upper panel) Reprobing the same membrane with a 23S-RNA probe. (Lower panel) MicA RNA expression was analyzed with the same RNAs in a polyacrylamide Northern. (B) Northern blot showing that overexpression of PNPase from plasmid pKAK7 complements the pnp mutant and changes MicA RNA levels to wild-type amounts. (C) Analysis of PNPase and MicA absence in the levels of ompA mRNA in stationary phase cells. (Top) Loading control with a 23S RNA probe. (Bottom) MicA RNA expression was analyzed as an additional control. Full-length transcripts were quantified using a Molecular Dynamics PhosphorImager. The amount of the RNA found in wild type was set as 1. The ratio between each strain and the wild type is depicted. (ND) Nondetectable.

FIGURE 2.

Protein profile of Escherichia coli MG1693 (wt) and SK5691 (pnp7) strains from stationary phase cultures. (A) Two-dimensional gel electrophoresis (2D-PAGE) analysis of total protein extracts. Proteins with pI values in the range 3.0–10.0 and molecular mass in the range of 10–250 kDa were resolved in 12% SDS-PAGE gels followed by Coomassie G-250 staining; corresponding sections of representative gels show the down-regulation of the OmpA porin in the pnp7 strain compared with wild type. (Arrow) OmpA spot. (B) Outer membrane protein fraction analysis by urea-SDS-polyacrylamyde gel electrophoresis. The positions of the OmpC, OmpF, and OmpA bands are indicated. An ompA mutant was used as control.

FIGURE 3.

Comparison of the decay rates of the MicA RNA in the wild-type and exonuclease mutants in stationary and exponential phase. (A) Northern blot of RNA extracted from stationary phase cultures. (C) Northern blot of RNA extracted from exponential phase cultures. Total RNA was extracted from culture samples withdrawn after inhibition of transcription (timepoints are shown in minutes). (*) Nonspecific band that cross-reacts with the antisense MicA-probe. A more stringent washing eliminates this band, while the signal for MicA RNA remains. (B,D) The band corresponding to the full-length MicA RNA was quantified with a PhosphorImager and plotted versus time of extraction (min).

FIGURE 4.

MicA RNA degradation in the absence of the target ompA mRNA. (A) Northern blot comparing the importance of ompA mRNA in the decay rate with its antisense regulator MicA RNA. Timepoints of sample cultures are shown in minutes after inhibition of transcription. (*) Nonspecific cross-hybridization with the antisense MicA-probe. (B) Full-length MicA RNA stability, after PhosphorImager quantification along time of sample extraction (in min).

FIGURE 5.

The role of the polyadenylation in MicA RNA decay in stationary phase. (A) The MicA RNA stability in the wild type and in the absence of PAP I-dependent polyadenylation (ΔpcnB mutation) was verified by Northern blot analysis. MicA RNA half-lives are shown in minutes (min). (*) Nonspecific band that was used as loading control. (B) Relative amount of the full-length MicA RNA remaining at each timepoint (as determined by PhosphorImager analysis) plotted as function of time.

FIGURE 6.

RNase E is involved in the control of the MicA RNA stability. (A) Cultures were grown at 30°C to an OD₆₀₀ ≈ 2.0 and then shifted to the nonpermissive temperature of 44°C. After 5 min, transcription was blocked with the addition of rifampicin, and samples were withdrawn at the times indicated. Total RNA was extracted as described in the Materials and Methods. MicA RNA stability was analyzed using an antisense RNA probe. (C) RNase E mutant cells impaired in degradosome assembly (rne131 mutation) were analyzed with wild-type and pnp mutant strains. MicA RNA stability is shown in minutes (min). (*) Nonspecific band. (B,D) Full-length MicA RNA was quantified with a PhosphorImager and plotted versus time of extraction (min).

FIGURE 7.

PNPase controls the small RybB RNA. (A) Northern blot analysis of the stability of the RybB RNA in the wt and pnp mutant strains in stationary phase. Full-length RybB and breakdown products of degradation are observable. (Lower panel) The same membrane was stripped and rehybridized with a 5S RNA riboprobe as a loading control. (B) The signal corresponding to the full-length RybB RNA was quantified with a PhosphorImager and plotted versus time of extraction (min).

FIGURE 8.

PNPase-mediated degradation is a major regulatory event controlling the levels of sRNAs (namely MicA and RybB) that are required for the accurate expression of outer membrane proteins. These sRNAs act as antisense RNAs and bind to the 5′ UTR of their target outer membrane mRNAs in an Hfq-dependent mechanism. This process inhibits translation and can help promoting the decay of the target mRNAs. Balanced outer membrane protein composition is essential for survival and affects many cellular processes, such as morphology, permeability, and virulence. PNPase emerges as an important enzyme in the growth phase adaptation to stationary phase.