Guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) as a negative modulator of polynucleotide phosphorylase activity in a ‘rare’ actinomycete

With the beginning of the idiophase the highly phosphorylated guanylic nucleotides guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) and guanosine 5′‐triphosphate 3′‐diphosphate (pppGpp), collectively referred to as (p)ppGpp, activate stress survival adaptation programmes and trigger secondary metabolism in actinomycetes. The major target of (p)ppGpp is the RNA polymerase, where it binds altering the enzyme activity. In this study analysis of the polynucleotide phosphorylase (PNPase)‐encoding gene pnp mRNA, in Nonomuraea sp. ATCC 39727 wild‐type, constitutively stringent and relaxed strains, led us to hypothesize that in actinomycetes (p)ppGpp may modulate gene expression at the level of RNA decay also. This hypothesis was supported by: (i) in vitro evidence that ppGpp, at physiological levels, inhibited both polynucleotide polymerase and phosphorolytic activities of PNPase in Nonomuraea sp., but not in Escherichia coli, (ii) in vivo data showing that the pnp mRNA and the A40926 antibiotic cluster‐specific dpgA mRNA were stabilized during the idiophase in the wild‐type strain but not in a relaxed mutant and (iii) measurement of chemical decay of pulse‐labelled bulk mRNA. The results of biochemical tests suggest competitive inhibition of ppGpp with respect to nucleoside diphosphates in polynucleotide polymerase assays and mixed inhibition with respect to inorganic phosphate when the RNA phosphorolytic activity was determined.     

Bacillus subtilis RNase J1 endonuclease and 5′ exonuclease activities in the turnover of ΔermC mRNA

RNase J1, a ribonuclease with 5′ exonuclease and endonuclease activities, is an important factor in Bacillus subtilis mRNA decay. A model for RNase J1 endonuclease activity in mRNA turnover has RNase J1 binding to the 5′ end and tracking to a target site downstream, where it makes a decay-initiating cleavage. The upstream fragment from this cleavage is degraded by 3′ exonucleases; the downstream fragment is degraded by RNase J1 5′ exonuclease activity. Previously, ΔermC mRNA was used to show 5′-end dependence of mRNA turnover. Here we used ΔermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. ΔermC mRNA showed increased stability in a mutant strain that contained a reduced level of RNase J1. In agreement with the tracking concept, insertion of a strong stem–loop structure at +65 resulted in increased stability. Weakening this stem–loop structure resulted in reversion to wild-type stability. RNA fragments containing the 3′ end were detected in a strain with reduced RNase J1 expression, but were undetectable in the wild type. The 5′ ends of these fragments mapped to the upstream side of predicted stem–loop structures, consistent with an impediment to RNase J1 5′ exonuclease processivity. A ΔermC mRNA deletion analysis suggested that decay-initiating endonuclease cleavage could occur at several sites near the 3′ end. However, even in the absence of these sites, stability was further increased in a strain with reduced RNase J1, suggesting alternate pathways for decay that could include exonucleolytic decay from the 5′ end.     

Small stable RNA maturation and turnover in Bacillus subtilis

Stable RNA maturation is a key process in the generation of functional RNAs, and failure to correctly process these RNAs can lead to their elimination through quality control mechanisms. Studies of the maturation pathways of ribosomal RNA and transfer RNA in Bacillus subtilis showed they were radically different from Escherichia coli and led to the identification of new B. subtilis‐specific enzymes. We noticed that, despite their important roles in translation, a number of B. subtilis small stable RNAs still did not have characterised maturation pathways, notably the tmRNA, involved in ribosome rescue, and the RNase P RNA, involved in tRNA maturation. Here, we show that tmRNA is matured by RNase P and RNase Z at its 5′ and 3′ extremities, respectively, whereas the RNase P RNA is matured on its 3′ side by RNase Y. Recent evidence that several RNases are not essential in B. subtilis prompted us to revisit maturation of the scRNA, a component of the signal recognition particle involved in co‐translational insertion of specific proteins into the membrane. We show that RNase Y is also involved in 3′ processing of scRNA. Lastly, we identified some of the enzymes involved in the turnover of these three stable RNAs.     

Exonuclease hDIS3L2 specifies an exosome‐independent 3′‐5′ degradation pathway of human cytoplasmic mRNA

Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5′‐3′ exoribonuclease hXRN1 and the 3′‐5′ exoribonucleolytic RNA exosome complex. However, in addition to the exosome‐associated 3′‐5′ exonucleases hDIS3 and hDIS3L, the human genome encodes another RNase II/R domain protein—hDIS3L2. Here, we show that hDIS3L2 is an exosome‐independent cytoplasmic mRNA 3′‐5′ exonuclease, which exhibits processive activity on structured RNA substrates in vitro. hDIS3L2 associates with hXRN1 in an RNA‐dependent manner and can, like hXRN1, be found on polysomes. The impact of hDIS3L2 on cytoplasmic RNA metabolism is revealed by an increase in levels of cytoplasmic RNA processing bodies (P‐bodies) upon hDIS3L2 depletion, which also increases half‐lives of investigated mRNAs. Consistently, RNA sequencing (RNA‐seq) analyses demonstrate that depletion of hDIS3L2, like downregulation of hXRN1 and hDIS3L, causes changed levels of multiple mRNAs. We suggest that hDIS3L2 is a key exosome‐independent effector of cytoplasmic mRNA metabolism.     

A small RNA activates CFA synthase by isoform‐specific mRNA stabilization

Small RNAs use a diversity of well‐characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq‐associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation‐independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot‐exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E‐mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA‐controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.