Moonlighting in Bacillus subtilis: The Small Proteins SR1P and SR7P Regulate the Moonlighting Activity of Glyceraldehyde 3-Phosphate Dehydrogenase A (GapA) and Enolase in RNA Degradation

Moonlighting proteins are proteins with more than one function. During the past 25 years, they have been found to be rather widespread in bacteria. In Bacillus subtilis, moonlighting has been disclosed to occur via DNA, protein or RNA binding or protein phosphorylation. In addition, two metabolic enzymes, enolase and phosphofructokinase, were localized in the degradosome-like network (DLN) where they were thought to be scaffolding components. The DLN comprises the major endoribonuclease RNase Y, 3′-5′ exoribonuclease PnpA, endo/5′-3′ exoribonucleases J1/J2 and helicase CshA. We have ascertained that the metabolic enzyme GapA is an additional component of the DLN. In addition, we identified two small proteins that bind scaffolding components of the degradosome: SR1P encoded by the dual-function sRNA SR1 binds GapA, promotes the GapA-RNase J1 interaction and increases the RNase J1 activity. SR7P encoded by the dual-function antisense RNA SR7 binds to enolase thereby enhancing the enzymatic activity of enolase bound RNase Y. We discuss the role of small proteins in modulating the activity of two moonlighting proteins.

Quasi-essentiality of RNase Y in Bacillus subtilis is caused by its critical role in the control of mRNA homeostasis

ABSTRACTRNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase – α, β, β′. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the β or β’ subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed massive decreases in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.

SR7 - a dual function antisense RNA from Bacillus subtilis

SummaryHere, we describe SR7, a dual-function antisense RNA from the Bacillus subtilis chromosome. This RNA was earlier published as the SigB-dependent regulatory RNA S1136 and reported to reduce the amount of the small ribosomal subunit under ethanol stress. We found that the 5’ portion of SR7 encodes a small protein composed of 39 amino acids which we designated SR7P. It is translated from a 185 nt SigB-dependent mRNA under five different stress conditions and a longer SigB-independent RNA constitutively. Two- to three-fold higher amounts of SR7P were detected in B. subtilis cells exposed to salt, ethanol or heat stress. Co-elution experiments with SR7PC-FLAG and Far-Western blotting demonstrated that SR7P interacts with the glycolytic enzyme enolase. Enolase is a scaffolding component of the B. subtilis degradosome where it interacts with RNase Y and phosphofructokinase PfkA. We found that SR7P increases the amount of RNase Y bound to enolase without affecting PfkA. RNA does not bridge the SR7P-enolase-RNase Y interaction. In vitro-degradation assays with the known RNase Y substrates yitJ and rpsO mRNA revealed enhanced enzymatic activity of enolase-bound RNase Y in the presence of SR7P. Northern blots showed a major effect of enolase and a minor effect of SR7P on the half-life of rpsO mRNA indicating a fine-tuning role of SR7P in RNA degradation. Moreover, SR7P impacts survival of B. subtilis under stress conditions. We suggest that the SR7P-dependent modification of the degradosome affects targets in different physiological pathways.

Dynamic Membrane Localization of RNase Y in Bacillus subtilis

ABSTRACT Metabolic turnover of mRNA is fundamental to the control of gene expression in all organisms, notably in fast-adapting prokaryotes. In many bacteria, RNase Y initiates global mRNA decay via an endonucleolytic cleavage, as shown in the Gram-positive model organism Bacillus subtilis. This enzyme is tethered to the inner cell membrane, a pseudocompartmentalization coherent with its task of initiating mRNA cleavage/maturation of mRNAs that are translated at the cell periphery. Here, we used total internal reflection fluorescence microscopy (TIRFm) and single-particle tracking (SPT) to visualize RNase Y and analyze its distribution and dynamics in living cells. We find that RNase Y diffuses rapidly at the membrane in the form of dynamic short-lived foci. Unlike RNase E, the major decay-initiating RNase in Escherichia coli, the formation of foci is not dependent on the presence of RNA substrates. On the contrary, RNase Y foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. The Y-complex of three proteins (YaaT, YlbF, and YmcA) has previously been shown to play an important role for RNase Y activity in vivo. We demonstrate that Y-complex mutations have an effect similar to but much stronger than that of depletion of RNA in increasing the number and size of RNase Y foci at the membrane. Our data suggest that the Y-complex shifts the assembly status of RNase Y toward fewer and smaller complexes, thereby increasing cleavage efficiency of complex substrates like polycistronic mRNAs. IMPORTANCE All living organisms must degrade mRNA to adapt gene expression to changing environments. In bacteria, initiation of mRNA decay generally occurs through an endonucleolytic cleavage. In the Gram-positive model organism Bacillus subtilis and probably many other bacteria, the key enzyme for this task is RNase Y, which is anchored at the inner cell membrane. While this pseudocompartmentalization appears coherent with translation occurring primarily at the cell periphery, our knowledge on the distribution and dynamics of RNase Y in living cells is very scarce. Here, we show that RNase Y moves rapidly along the membrane in the form of dynamic short-lived foci. These foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. This contrasts with RNase E, the major decay-initiating RNase in E. coli, where it was shown that formation of foci is dependent on the presence of RNA substrates. We also show that a protein complex (Y-complex) known to influence the specificity of RNase Y activity in vivo is capable of shifting the assembly status of RNase Y toward fewer and smaller complexes. This highlights fundamental differences between RNase E- and RNase Y-based degradation machineries.

Maturation of polycistronic mRNAs by the endoribonuclease RNase Y and its associated Y-complex in Bacillus subtilis

Endonucleolytic cleavage within polycistronic mRNAs can lead to differential stability, and thus discordant abundance, among cotranscribed genes. RNase Y, the major endonuclease for mRNA decay in Bacillus subtilis, was originally identified for its cleavage activity toward the cggR-gapA operon, an event that differentiates the synthesis of a glycolytic enzyme from its transcriptional regulator. A three-protein Y-complex (YlbF, YmcA, and YaaT) was recently identified as also being required for this cleavage in vivo, raising the possibility that it is an accessory factor acting to regulate RNase Y. However, whether the Y-complex is broadly required for RNase Y activity is unknown. Here, we used end-enrichment RNA sequencing (Rend-seq) to globally identify operon mRNAs that undergo maturation posttranscriptionally by RNase Y and the Y-complex. We found that the Y-complex is required for the majority of RNase Y-mediated mRNA maturation events and also affects riboswitch abundance in B. subtilis. In contrast, noncoding RNA maturation by RNase Y often does not require the Y-complex. Furthermore, deletion of RNase Y has more pleiotropic effects on the transcriptome and cell growth than deletions of the Y-complex. We propose that the Y-complex is a specificity factor for RNase Y, with evidence that its role is conserved in Staphylococcus aureus.

Role of RNase Y in Clostridium perfringens mRNA Decay and Processing

ABSTRACT RNase Y is a major endoribonuclease that plays a crucial role in mRNA degradation and processing. We study the role of RNase Y in the Gram-positive anaerobic pathogen Clostridium perfringens, which until now has not been well understood. Our study implies an important role for RNase Y-mediated RNA degradation and processing in virulence gene expression and the physiological development of the organism. We began by constructing an RNase Y conditional knockdown strain in order to observe the importance of RNase Y on growth and virulence. Our resulting transcriptome analysis shows that RNase Y affects the expression of many genes, including toxin-producing genes. We provide data to show that RNase Y depletion repressed several toxin genes in C. perfringens and involved the virR-virS two-component system. We also observe evidence that RNase Y is indispensable for processing and stabilizing the transcripts of colA (encoding a major toxin collagenase) and pilA2 (encoding a major pilin component of the type IV pili). Posttranscriptional regulation of colA is known to be mediated by cleavage in the 5′ untranslated region (5′UTR), and we observe that RNase Y depletion diminishes colA 5′UTR processing. We show that RNase Y is also involved in the posttranscriptional stabilization of pilA2 mRNA, which is thought to be important for host cell adherence and biofilm formation. IMPORTANCE RNases have important roles in RNA degradation and turnover in all organisms. C. perfringens is a Gram-positive anaerobic spore-forming bacterial pathogen that produces numerous extracellular enzymes and toxins, and it is linked to digestive disorders and disease. A highly conserved endoribonuclease, RNase Y, affects the expression of hundreds of genes, including toxin genes, and studying these effects is useful for understanding C. perfringens specifically and RNases generally. Moreover, RNase Y is involved in processing specific transcripts, and we observed that this processing in C. perfringens results in the stabilization of mRNAs encoding a toxin and bacterial extracellular apparatus pili. Our study shows that RNase activity is associated with gene expression, helping to determine the growth, proliferation, and virulence of C. perfringens.