RNase E, the Major Player in mRNA Degradation, Is Down-Regulated in Escherichia coli during a Transient Growth Retardation (Diauxic Lag)

ABSTRACT The endoribonuclease RNase E participates in mRNA degradation, rRNA processing, and tRNA maturation in Escherichia coli, but the precise reasons for its essentiality are unclear and much debated. The enzyme is most active on RNA substrates with a 5′-terminal monophosphate, which is sensed by a domain in the enzyme that includes residue R169; E. coli also possesses a 5′-pyrophosphohydrolase, RppH, that catalyzes conversion of 5′-terminal triphosphate to 5′-terminal monophosphate on RNAs. Although the C-terminal half (CTH), beyond residue approximately 500, of RNase E is dispensable for viability, deletion of the CTH is lethal when combined with an R169Q mutation or with deletion of rppH. In this work, we show that both these lethalities can be rescued in derivatives in which four or five of the seven rrn operons in the genome have been deleted. We hypothesize that the reduced stable RNA levels under these conditions minimize the need of RNase E to process them, thereby allowing for its diversion for mRNA degradation. In support of this hypothesis, we have found that other conditions that are known to reduce stable RNA levels also suppress one or both lethalities: (i) alterations in relA and spoT, which are expected to lead to increased basal ppGpp levels; (ii) stringent rpoB mutations, which mimic high intracellular ppGpp levels; and (iii) overexpression of DksA. Lethality suppression by these perturbations was RNase R dependent. Our work therefore suggests that its actions on the various substrates (mRNA, rRNA, and tRNA) jointly contribute to the essentiality of RNase E in E. coli. IMPORTANCE The endoribonuclease RNase E is essential for viability in many Gram-negative bacteria, including Escherichia coli. Different explanations have been offered for its essentiality, including its roles in global mRNA degradation or in the processing of several tRNA and rRNA species. Our work suggests that, rather than its role in the processing of any one particular substrate, its distributed functions on all the different substrates (mRNA, rRNA, and tRNA) are responsible for the essentiality of RNase E in E. coli.

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Polyribosome-Dependent Clustering of Membrane-Anchored RNA Degradosomes To Form Sites of mRNA Degradation in Escherichia coli

mBio ◽

10.1128/mbio.01932-21 ◽

2021 ◽

Author(s):

Lina Hamouche ◽

Leonora Poljak ◽

Agamemnon J. Carpousis

Keyword(s):

Escherichia Coli ◽

Cytoplasmic Membrane ◽

Mrna Degradation ◽

Rnase E ◽

Rna Degradosome

Here, we show that RNase E, RhlB, and PNPase act together as components of the multienzyme RNA degradosome in polyribosome-dependent clustering to form puncta on the inner cytoplasmic membrane. Our results support the hypothesis that RNA degradosome puncta are sites of mRNA degradation.

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RNase III-Independent Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase via Translational Repression

Journal of Bacteriology ◽

10.1128/jb.00105-15 ◽

2015 ◽

Vol 197 (11) ◽

pp. 1931-1938 ◽

Cited By ~ 10

Author(s):

Thomas Carzaniga ◽

Gianni Dehò ◽

Federica Briani

Keyword(s):

Escherichia Coli ◽

Posttranscriptional Regulation ◽

Mrna Degradation ◽

Translational Repression ◽

Rnase E ◽

Polynucleotide Phosphorylase ◽

Rnase Iii ◽

Primary Transcript ◽

Content Type ◽

Autogenous Regulation

ABSTRACTThe complex posttranscriptional regulation mechanism of theEscherichia colipnpgene, which encodes the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), involves two endoribonucleases, namely, RNase III and RNase E, and PNPase itself, which thus autoregulates its own expression. The models proposed forpnpautoregulation posit that the target of PNPase is a maturepnpmRNA previously processed at its 5′ end by RNase III, rather than the primarypnptranscript (RNase III-dependent models), and that PNPase activity eventually leads topnpmRNA degradation by RNase E. However, some published data suggest thatpnpexpression may also be regulated through a PNPase-dependent, RNase III-independent mechanism. To address this issue, we constructed isogenic Δpnp rnc+and ΔpnpΔrncstrains with a chromosomalpnp-lacZtranslational fusion and measured β-galactosidase activity in the absence and presence of PNPase expressed by a plasmid. Our results show that PNPase also regulates its own expression via a reversible RNase III-independent pathway acting upstream from the RNase III-dependent branch. This pathway requires the PNPase RNA binding domains KH and S1 but not its phosphorolytic activity. We suggest that the RNase III-independent autoregulation of PNPase occurs at the level of translational repression, possibly by competition forpnpprimary transcript between PNPase and the ribosomal protein S1.IMPORTANCEInEscherichia coli, polynucleotide phosphorylase (PNPase, encoded bypnp) posttranscriptionally regulates its own expression. The two models proposed so far posit a two-step mechanism in which RNase III, by cutting the leader region of thepnpprimary transcript, creates the substrate for PNPase regulatory activity, eventually leading topnpmRNA degradation by RNase E. In this work, we provide evidence supporting an additional pathway for PNPase autogenous regulation in which PNPase acts as a translational repressor independently of RNase III cleavage. Our data make a new contribution to the understanding of the regulatory mechanism ofpnpmRNA, a process long since considered a paradigmatic example of posttranscriptional regulation at the level of mRNA stability.

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