Philippe Régnier

Degradation of mRNA in bacteria: emergence of ubiquitous features.

The amount of a messenger RNA available for protein synthesis depends on the efficiency of its transcription and stability. The mechanisms of degradation that determine the stability of mRNAs in bacteria have been investigated extensively during the last decade and have begun to be better understood. Several endo‐ and exoribonucleases involved in the mRNA metabolism have been characterized as well as structural features of’mRNA which account for its stability have been determined. The most important recent developments have been the discovery that the degradosome—a multiprotein complex containing an endoribonuclease (RNase E), an exoribonuclease (polynucleotide phosphorylase), and a DEAD box helicase (RhlB)—has a central role in mRNA degradation and that oligo(A) tails synthesized by poly(A) polymerase facilitate the degradation of mRNAs and RNA fragments. Moreover, the phosphorylation status and the base pairing of 5′ extremities, together with 3′ secondary structures of transcriptional terminators, contribute to the stability of primary transcripts. Degradation of mRNAs can follow several independent pathways. Interestingly, poly(A) tails and multienzyme complexes also control the stability and the degradation of eukaryotic mRNAs. These discoveries have led to the development of refined models of mRNA degradation. BioEssays 22:235–244, 2000.

Decay of mRNA encoding ribosomal protein S15 of Escherichia coli is initiated by an RNase E-dependent endonucleolytic cleavage that removes the 3′ stabilizing stem and loop structure

The transcripts of the rpsO-pnp operon of Escherichia coli, coding for ribosomal protein S15 and polynucleotide phosphorylase, are processed at four sites in the 249 nucleotides of the intercistronic region. The initial processing step in the decay of the pnp mRNA is made by RNase III, which cuts at two sites upstream from the pnp gene. The other two cleavages are dependent on the wild-type allele of the rne gene, which encodes the endonucleolytic enzyme RNase E. The cuts are made 37 nucleotides apart at the base of the stem-loop structure of the rho-independent attenuator located downstream from rpsO. The cleavage downstream from the attenuator generates an rpsO mRNA.nearly identical with the monocistronic attenuated transcript, while the cleavage upstream from the transcription attenuator gives rise to an rpsO mesage lacking the terminal 3 hairpin structure. The rapid degradation of the processed mRNA in an rne+ strain, compared to the slow degradation of the transcript that accumulates in an rne- strain, suggests that RNase E initiates the decay of the rpsO message by removing the stabilizing stem-loop at the 3 end of the RNA.

Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA

Hfq protein is vital for the function of many non-coding small (s)RNAs in bacteria but the mechanism by which Hfq facilitates the function of sRNA is still debated. We developed a fluorescence resonance energy transfer assay to probe how Hfq modulates the interaction between a sRNA, DsrA, and its regulatory target mRNA, rpoS. The relevant RNA fragments were labelled so that changes in intra- and intermolecular RNA structures can be monitored in real time. Our data show that Hfq promotes the strand exchange reaction in which the internal structure of rpoS is replaced by pairing with DsrA such that the Shine-Dalgarno sequence of the mRNA becomes exposed. Hfq appears to carry out strand exchange by inducing rapid association of DsrA and a premelted rpoS and by aiding in the slow disruption of the rpoS secondary structure. Unexpectedly, Hfq also disrupts a preformed complex between rpoS and DsrA. While it may not be a frequent event in vivo, this melting activity may have implications in the reversal of sRNA-based regulation. Overall, our data suggests that Hfq not only promotes strand exchange by binding rapidly to both DsrA and rpoS but also possesses RNA chaperoning properties that facilitates dynamic RNA–RNA interactions.

Chapter 4 Poly(A)‐Assisted RNA Decay and Modulators of RNA Stability

In Escherichia coli, RNA degradation is orchestrated by the degradosome with the assistance of complementary pathways and regulatory cofactors described in this chapter. They control the stability of each transcript and regulate the expression of many genes involved in environmental adaptation. The poly(A)-dependent degradation machinery has diverse functions such as the degradation of decay intermediates generated by endoribonucleases, the control of the stability of regulatory non coding RNAs (ncRNAs) and the quality control of stable RNA. The metabolism of poly(A) and mechanism of poly(A)-assisted degradation are beginning to be understood. Regulatory factors, exemplified by RraA and RraB, control the decay rates of subsets of transcripts by binding to RNase E, in contrast to regulatory ncRNAs which, assisted by Hfq, target RNase E to specific transcripts. Destabilization is often consecutive to the translational inactivation of mRNA. However, there are examples where RNA degradation is the primary regulatory step.

Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails

The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3′ end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymeraseu2003I. In contrast, formation of the Hfq–RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).

Polyadenylation of a functional mRNA controls gene expression in Escherichia coli

Although usually implicated in the stabilization of mRNAs in eukaryotes, polyadenylation was initially shown to destabilize RNA in bacteria. All the data are consistent with polyadenylation being part of a quality control process targeting folded RNA fragments and non-functional RNA molecules to degradation. We report here an example in Escherichia coli, where polyadenylation directly controls the level of expression of a gene by modulating the stability of a functional transcript. Inactivation of poly(A)polymerase I causes overexpression of glucosamine–6-phosphate synthase (GlmS) and both the accumulation and stabilization of the glmS transcript. Moreover, we show that the glmS mRNA results from the processing of the glmU-glmS cotranscript by RNase E. Interestingly, the glmU-glmS cotranscript and the mRNA fragment encoding GlmU only slightly accumulated in the absence of poly(A)polymerase, suggesting that the endonucleolytically generated glmS mRNA harbouring a 5′ monophosphate and a 3′ stable hairpin is highly susceptible to poly(A)-dependent degradation.

Hfq affects mRNA levels independently of degradation

BackgroundThe bacterial Lsm protein, Hfq, is an RNA chaperone involved in many reactions related to RNA metabolism, such as replication and stability, control of small RNA activity and polyadenylation. Despite this wide spectrum of known functions, the global role of Hfq is almost certainly undervalued; its capacity to bind DNA and to interact with many other proteins are only now beginning to be taken into account.ResultsThe role of Hfq in the maturation and degradation of the rpsO mRNA of E. coli was investigated in vivo. The data revealed a decrease in rpsO mRNA abundance concomitant to an increase in its stability when Hfq is absent. This indicates that the change in mRNA levels in hfq mutants does not result from its modification of RNA stability. Moreover, a series of independent experiments have revealed that the decrease in mRNA level is not a consequence of a reduction of translation efficiency and that Hfq is not directly implicated in translational control of rpsO expression. Reduced steady-state mRNA levels in the absence of Hfq were also shown for rpsT, rpsB and rpsB-tsf, but not for lpp, pnp or tRNA transcripts. The abundance of chimeric transcripts rpsO-lacZ and rpsB-lacZ, whose expression was driven by rpsO and rpsB promoters, respectively, was also lower in the hfq null-mutants, while the β-galactosidase yield remained about the same as in the parent wild-type strain.ConclusionsThe data obtained suggest that alteration of rpsO, rpsT and rpsB-tsf transcript levels observed under conditions of Hfq deficiency is not caused by the post-transcriptional events, such as mRNA destabilization or changes in translation control, and may rather result from changes in transcriptional activity. So far, how Hfq affects transcription remains unclear. We propose that one of the likely mechanisms of Hfq-mediated modulation of transcription might operate early in the elongation step, when interaction of Hfq with a nascent transcript would help to overcome transcription pauses and to prevent preliminary transcript release.

Inactivation of the decay pathway initiated at an internal site by RNase E promotes poly(A)‐dependent degradation of the rpsO mRNA in Escherichia coli

In Escherichia coli, RNA degradation is mediated by endonucleolytic processes, frequently mediated by RNase E, and also by a poly(A)‐dependent mechanism. The dominant pathway of decay of the rpsO transcripts is initiated by an RNase E cleavage occurring at a preferential site named M2. We demonstrate that mutations which prevent this cleavage slow down degradation by RNase E. All these mutations reduce the single‐stranded character of nucleotides surrounding the cleavage site. Moreover, we identify two other cleavage sites which probably account for the slow RNase E‐mediated degradation of the mutated mRNAs. Failure to stabilize the rpsO transcript by appending a 5′ hairpin indicates that RNase E is not recruited by the 5′ end of mRNA. The fact that nucleotide substitutions which prevent cleavage at M2 facilitate the poly(A)‐dependent degradation of the rpsO transcripts suggest an interplay between the two mechanisms of decay. In the discussion, we speculate u200athat u200aa u200astructural u200afeature u200alocated u200ain u200athe u200avicinity of M2 could be an internal degradosome entry site promoting both RNase E cleavages and poly(A)‐dependent degradation of the rpsO mRNA. We also discuss the role of poly(A)‐dependent decay in mRNA metabolism.