Bacillus subtilis NrnB is expressed during sporulation and acts as a unique 3'-5' exonuclease.

All cells employ a combination of endo- and exoribonucleases to degrade long RNA polymers to fragments 2-5 nucleotides in length. These short RNA fragments are processed to monoribonucleotides by nanoRNases. Genetic depletion of nanoRNases has been shown to increase abundance of short RNAs. This deleteriously affects viability, virulence, and fitness, indicating that short RNAs are a metabolic burden. Previously, we provided evidence that NrnA is the housekeeping nanoRNase for Bacillus subtilis. Herein, we investigate the biological and biochemical functions of the evolutionarily related protein, B. subtilis NrnB (NrnBBs). These experiments show that NrnB is surprisingly different from NrnA. While NrnA acts at the 5' terminus of RNA substrates, NrnB acts at the 3' terminus. Additionally, NrnA is expressed constitutively under standard growth conditions, yet NrnB is selectively expressed during endospore formation. Furthermore, NrnA processes only short RNAs, while NrnB unexpectedly processes both short RNAs and longer RNAs. Indeed, inducible expression of NrnB can even complement the loss of the known global 3'-5' exoribonucleases, indicating that it acts as a general exonuclease. Together, these data demonstrate that NrnB proteins, which are widely found in Firmicutes, Epsilonproteobacteria and Archaea, are fundamentally different than NrnA proteins and may be used for specialized purposes.

Analysis of mRNA Decay Intermediates in Bacillus subtilis 3' Exoribonuclease and RNA Helicase Mutant Strains.

The Bacillus subtilis genome encodes four 3' exoribonucleases: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Previous work showed that PNPase, encoded by the pnpA gene, is the major 3' exonuclease involved in mRNA turnover; in a pnpA deletion strain, numerous mRNA decay intermediates accumulate. Whether B. subtilis mRNA decay occurs in the context of a degradosome complex is controversial. In this study, global mapping of mRNA decay intermediate 3' ends within coding sequences was performed in strains that were either deleted for or had an inactivating point mutation in the pnpA gene. The patterns of 3'-end accumulation in these strains were highly similar, which may have implications for the role of a degradosome in mRNA decay. A comparison with mapped 3' ends in a strain lacking CshA, the major RNA helicase, indicated that many mRNAs require both PNPase and CshA for efficient decay. Transcriptome sequencing (RNA-seq) analysis of strains lacking RNase R suggested that this enzyme did not play a major role in mRNA turnover in the wild-type strain. Strains were constructed that contained only one of the four known 3' exoribonucleases. When RNase R was the only 3' exonuclease present, it was able to degrade a model mRNA efficiently, showing processive decay even through a strong stem-loop structure that inhibits PNPase processivity. Strains containing only RNase PH or only YhaM were also insensitive to this RNA secondary structure, suggesting the existence of another, as-yet-unidentified, 3' exoribonuclease. IMPORTANCE The ability to rapidly change bacterial gene expression programs in response to environmental conditions is highly dependent on the efficient turnover of mRNA. While much is known about the regulation of gene expression at the transcriptional and translational levels, much less is known about the intermediate step of mRNA decay. Here, we mapped the 3' ends of mRNA decay intermediates in strains that were missing the major 3' exoribonuclease PNPase or the RNA helicase CshA. We also assessed the roles of three other B. subtilis 3' exonucleases in the mRNA decay process. The data confirm the primary role of PNPase in mRNA turnover and suggest the involvement of one or more unknown RNases.

Discovery and initial characterization of YloC, a novel endoribonuclease in Bacillus subtilis.

The Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3' exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3' exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in the identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved "YicC family" of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes the cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.

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

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.

Bacterial ribonucleases and their roles in RNA metabolism.

Ribonucleases (RNases) are mediators in most reactions of RNA metabolism. In recent years, there has been a surge of new information about RNases and the roles they play in cell physiology. In this review, a detailed description of bacterial RNases is presented, focusing primarily on those from Escherichia coli and Bacillus subtilis, the model Gram-negative and Gram-positive organisms, from which most of our current knowledge has been derived. Information from other organisms is also included, where relevant. In an extensive catalog of the known bacterial RNases, their structure, mechanism of action, physiological roles, genetics, and possible regulation are described. The RNase complement of E. coli and B. subtilis is compared, emphasizing the similarities, but especially the differences, between the two. Included are figures showing the three major RNA metabolic pathways in E. coli and B. subtilis and highlighting specific steps in each of the pathways catalyzed by the different RNases. This compilation of the currently available knowledge about bacterial RNases will be a useful tool for workers in the RNA field and for others interested in learning about this area.

Expression of multiple Bacillus subtilis genes is controlled by decay of slrA mRNA from Rho-dependent 3' ends.

Timely turnover of RNA is an important element in the control of bacterial gene expression, but relatively few specific targets of RNA turnover regulation are known. Deletion of the Bacillus subtilis pnpA gene, encoding the major 3' exonuclease turnover enzyme, polynucleotide phosphorylase (PNPase), was shown previously to cause a motility defect correlated with a reduced level of the 32-gene fla/che flagellar biosynthesis operon transcript.fla/che operon transcript abundance has been shown to be inhibited by an excess of the small regulatory protein, SlrA, and here we find that slrA mRNA accumulated in the pnpA-deletion mutant. Mutation of slrA was epistatic to mutation of pnpA for the motility-related phenotype. Further, Rho-dependent termination was required for PNPase turnover of slrA mRNA. When the slrA gene was provided with a Rho-independent transcription terminator, gene regulation was no longer PNPase-dependent. Thus we show that the slrA transcript is a direct target of PNPase and that regulation of RNA turnover is a major determinant of motility gene expression. The interplay of specific transcription termination and mRNA decay mechanisms suggests selection for fine-tuning of gene expression.

Mapping of internal monophosphate 5' ends of Bacillus subtilis messenger RNAs and ribosomal RNAs in wild-type and ribonuclease-mutant strains.

The recent findings that the narrow-specificity endoribonuclease RNase III and the 5' exonuclease RNase J1 are not essential in the Gram-positive model organism,Bacillus subtilis, facilitated a global analysis of internal 5' ends that are generated or acted upon by these enzymes. An RNA-Seq protocol known as PARE (Parallel Analysis of RNA Ends) was used to capture 5' monophosphorylated RNA ends in ribonuclease wild-type and mutant strains. Comparison of PARE peaks in strains with RNase III present or absent showed that, in addition to its well-known role in ribosomal (rRNA) processing, many coding sequences and intergenic regions appeared to be direct targets of RNase III. These target sites were, in most cases, not associated with a known antisense RNA. The PARE analysis also revealed an accumulation of 3'-proximal peaks that correlated with the absence of RNase J1, confirming the importance of RNase J1 in degrading RNA fragments that contain the transcription terminator structure. A significant result from the PARE analysis was the discovery of an endonuclease cleavage just 2 nts downstream of the 16S rRNA 3' end. This latter observation begins to answer, at least for B. subtilis, a long-standing question on the exonucleolytic versus endonucleolytic nature of 16S rRNA maturation.

Interaction of Bacillus subtilis Polynucleotide Phosphorylase and RNase Y: STRUCTURAL MAPPING AND EFFECT ON mRNA TURNOVER.

Polynucleotide phosphorylase (PNPase), a 3'-to-5' phosphorolytic exoribonuclease, is thought to be the primary enzyme responsible for turnover ofBacillus subtilismRNA. The role of PNPase inB. subtilismRNA decay has been analyzed previously by comparison of mRNA profiles in a wild-type strainversusa strain that is deleted forpnpA, the gene encoding PNPase. Recent studies have provided evidence for a degradosome-like complex inB. subtilisthat is built around the major decay-initiating endonuclease, RNase Y, and there is ample evidence for a strong interaction between PNPase and RNase Y. The role of the PNPase-RNase Y interaction in the exonucleolytic function of PNPase needs to be clarified. We sought to construct aB. subtilisstrain containing a catalytically active PNPase that could not interact with RNase Y. Mapping studies of the PNPase-RNase Y interaction were guided by a homology model ofB. subtilisPNPase based on the known structure of theEscherichia coliPNPase in complex with an RNase E peptide. Mutations inB. subtilisresidues predicted to be involved in RNase Y binding showed a loss of PNPase-RNase Y interaction. Two mRNAs whose decay is dependent on RNase Y and PNPase were examined in strains containing full-length PNPase that was either catalytically active but unable to interact with RNase Y, or catalytically inactive but able to interact with RNase Y. At least for these two mRNAs, disruption of the PNPase-RNase Y interaction did not appear to affect mRNA turnover.

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.

Global analysis of mRNA decay intermediates in Bacillus subtilis wild-type and polynucleotide phosphorylase-deletion strains.

Messenger RNA decay in Bacillus subtilis is accomplished by a combination of exoribonucleases and endoribonucleases. Intermediates in the decay process have not been readily detectable, and previous studies on mRNA decay have used a handful of highly expressed transcripts as models. Here, we use RNA-Seq analysis to probe mRNA turnover globally. A significant fraction of messages showed differential accumulation of RNA fragments that mapped near the 5' or 3' end of the coding sequence, consistent with initiation of decay from either the 5' end or from an internal cleavage site. Patterns of mRNA decay in the wild type were compared with patterns in a mutant strain lacking polynucleotide phosphorylase (PNPase), which is considered the major 3' exonuclease activity in mRNA decay and which is one of four known 3' exonucleases in B. subtilis. The results showed a striking dependence on PNPase for mRNA turnover in many cases, suggesting specificity in the ability of 3' exonucleases to degrade from 3'-hydroxyl termini. RNA-Seq data demonstrated a sharp decrease in expression of Sigma D in the PNPase-deletion strain. Reduction in sigD regulon expression explained the chain growth phenotype of the PNPase mutant and also predicted a defect in swarming motility.

Bacillus subtilis mRNA decay: new parts in the toolkit.

Representatives of two new ribonuclease families have recently been discovered in the gram-positive model organism, Bacillus subtilis. The RNase J family founding members, RNase J1 and RNase J2, are highly homologous but show differential activities. Although both are broad-specificity endonucleases, only the essential RNase J1 is a 5' → 3' exonuclease-a type of ribonuclease activity that was previously thought not to exist in bacteria. Current data suggest that RNase J1 is highly involved in the turnover of mRNA decay intermediates and may also be involved in the initiation of mRNA decay. A second family of ribonucleases is represented by RNase Y, an endonuclease that exerts a large effect on global mRNA half-life. The presence of these ribonucleases in B. subtilis predicts significant differences from the well-established model of mRNA decay in Escherichia coli.

An RNA pyrophosphohydrolase triggers 5'-exonucleolytic degradation of mRNA in Bacillus subtilis.

In Escherichia coli, RNA degradation often begins with conversion of the 5'-terminal triphosphate to a monophosphate, creating a better substrate for internal cleavage by RNase E. Remarkably, no homolog of this key endonuclease is present in many bacterial species, such as Bacillus subtilis and various pathogens. Here, we report that the degradation of primary transcripts in B. subtilis can nevertheless be triggered by an analogous process to generate a short-lived, monophosphorylated intermediate. Like its E. coli counterpart, the B. subtilis RNA pyrophosphohydrolase that catalyzes this event is a Nudix protein that prefers unpaired 5' ends. However, in B. subtilis, this modification exposes transcripts to rapid 5' exonucleolytic degradation by RNase J, which is absent in E. coli but present in most bacteria lacking RNase E. This pathway, which closely resembles the mechanism by which deadenylated mRNA is degraded in eukaryotic cells, explains the stabilizing influence of 5'-terminal stem-loops in such bacteria.

Decay of a model mRNA in Bacillus subtilis by a combination of RNase J1 5' exonuclease and RNase Y endonuclease activities.

The involvement of the recently characterized 5' exonuclease activity of RNase J1 and endonuclease activity of RNase Y in the turnover of ΔermC mRNA in Bacillus subtilis was investigated. Evidence is presented that both of these activities determine the half-life of ΔermC mRNA.

5' End-independent RNase J1 endonuclease cleavage of Bacillus subtilis model RNA.

Bacillus subtilis trp leader RNA is a small (140-nucleotide) RNA that results from attenuation of trp operon transcription upon binding of the regulatory TRAP complex. Previously, endonucleolytic cleavage by ribonuclease RNase J1 in a 3'-proximal, single-stranded region was shown to be critical for initiation of trp leader RNA decay. RNase J1 is a dual-specificity enzyme, with both 5' exonucleolytic and endonucleolytic activities. Here, we provide in vivo and in vitro evidence that RNase J1 accesses its internal target site on trp leader RNA in a 5' end-independent manner. This has important implications for the role of RNase J1 in RNA decay. We also tested the involvement in trp leader RNA decay of the more recently discovered endonuclease RNase Y. Half-lives of several trp leader RNA constructs, which were designed to probe pathways of endonucleolytic versus exonucleolytic decay, were measured in an RNase Y-deficient mutant. Remarkably, the half-lives of these constructs were indistinguishable from their half-lives in an RNase J1-deficient mutant. These results suggest that lowering RNase Y concentration may affect RNA decay indirectly via an effect on RNase J1, which is thought to exist with RNase Y in a degradosome complex. To generalize our findings with trp leader RNA to other RNAs, we show that the mechanism of trp leader RNA decay is not dependent on TRAP binding.

Regulated RNA stability in the Gram positives.

Regulation of bacterial gene expression at the post-transcriptional level has emerged as a major control mechanism, although not yet as well recognized as the mechanisms of control at the transcriptional level. In this article, we focus on regulated RNA decay in the control of gene expression in Gram-positive organisms, with an emphasis on Bacillus subtilis. Discovery of new ribonuclease activities in B. subtilis and other Gram-positive species, especially the dual-functioning RNase J1, which specifies both an endonuclease activity and the long-sought bacterial 5'-to-3' exoribonuclease activity, has led to the recognition of intriguing mechanisms of gene regulation at the level of RNA decay.

Initiation of decay of Bacillus subtilis rpsO mRNA by endoribonuclease RNase Y.

rpsO mRNA, a small monocistronic mRNA that encodes ribosomal protein S15, was used to study aspects of mRNA decay initiation in Bacillus subtilis. Decay of rpsO mRNA in a panel of 3'-to-5' exoribonuclease mutants was analyzed using a 5'-proximal oligonucleotide probe and a series of oligonucleotide probes that were complementary to overlapping sequences starting at the 3' end. The results provided strong evidence that endonuclease cleavage in the body of the message, rather than degradation from the native 3' end, is the rate-determining step for mRNA decay. Subsequent to endonuclease cleavage, the upstream products were degraded by polynucleotide phosphorylase (PNPase), and the downstream products were degraded by the 5' exonuclease activity of RNase J1. The rpsO mRNA half-life was unchanged in a strain that had decreased RNase J1 activity and no RNase J2 activity, but it was 2.3-fold higher in a strain with decreased activity of RNase Y, a recently discovered RNase of B. subtilis encoded by the ymdA gene. Accumulation of full-length rpsO mRNA and its decay intermediates was analyzed using a construct in which the rpsO transcription unit was under control of a bacitracin-inducible promoter. The results were consistent with RNase Y-mediated initiation of decay. This is the first report of a specific mRNA whose stability is determined by RNase Y.

Bacillus subtilis RNase J1 endonuclease and 5' exonuclease activities in the turnover of DeltaermC 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, DeltaermC mRNA was used to show 5'-end dependence of mRNA turnover. Here we used DeltaermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. DeltaermC 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 DeltaermC 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.

Bacillus subtilis trp Leader RNA: RNase J1 endonuclease cleavage specificity and PNPase processing.

In the presence of ample tryptophan, transcription from the Bacillus subtilis trp operon promoter terminates to give a 140-nucleotide trp leader RNA. Turnover of trp leader RNA has been shown to depend on RNase J1 cleavage at a single-stranded, AU-rich region just upstream of the 3' transcription terminator. The small size of trp leader RNA and its strong dependence on RNase J1 cleavage for decay make it a suitable substrate for analyzing the requirements for RNase J1 target site specificity. trp leader RNAs with nucleotide changes around the RNase J1 target site were more stable than wild-type trp leader RNA, showing that sequences on either side of the cleavage site contribute to RNase J1 recognition. An analysis of decay intermediates from these mutants suggested limited 3'-to-5' exonuclease processing from the native 3' end. trp leader RNAs were designed that contained wild-type or mutant RNase J1 targets elsewhere on the molecule. The presence of an additional RNase J1 cleavage site resulted in faster RNA decay, depending on its location. Addition of a 5' tail containing 7 A residues caused destabilization of trp leader RNAs. Surprisingly, addition at the 5' end of a strong stem loop structure that is known to stabilize other RNAs did not result in a longer trp leader RNA half-life, suggesting that the RNase J1 cleavage site may be accessed directly. In the course of these experiments, we found evidence that polynucleotide phosphorylase processivity was inhibited by a GCGGCCGC sequence.

Processing and stability of inducibly expressed rpsO mRNA derivatives in Bacillus subtilis.

The Bacillus subtilis rpsO gene specifies a small (388-nucleotide), monocistronic mRNA that encodes ribosomal protein S15. We showed earlier that rpsO mRNA decay intermediates accumulated to a high level in a strain lacking polynucleotide phosphorylase. Here, we used inducibly expressed derivatives of rpsO, encoding smaller RNAs that had the complex 5' region deleted, to study aspects of mRNA processing in B. subtilis. An IPTG (isopropyl-beta-d-thiogalactopyranoside)-inducible rpsO transcript that contained lac sequences at the 5' end, called lac-rpsO RNA, was shown to undergo processing to result in an RNA that was 24 nucleotides shorter than full length. Such processing was dependent on the presence of an accessible 5' terminus; a lac-rpsO RNA that contained a strong stem-loop at the 5' end was not processed and was extremely stable. Interestingly, this stability depended also on ribosome binding to a nearby Shine-Dalgarno sequence but was independent of downstream translation. Either RNase J1 or RNase J2 was capable of processing lac-rpsO RNA, demonstrating for the first time a particular in vivo processing event that could be catalyzed by both enzymes. Decay intermediates were detected in the pnpA strain only for a lac-rpsO RNA that was untranslated. Analysis of processing of an untranslated lac-rpsO RNA in the pnpA strain shortly after induction of transcription suggested that endonuclease cleavage at 3'-proximal sites was an early step in turnover of mRNA.