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.

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 gene, is the major 3' exonuclease involved in mRNA turnover; in a deletion strain, numerous mRNA decay intermediates accumulate. Whether B.

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

The 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.

Polynucleotide phosphorylase and RNA helicase CshA cooperate in mRNA decay.

Polynucleotide phosphorylase (PNPase), a 3' exoribonuclease that degrades RNA in the 3'-to-5' direction, is the major mRNA decay activity in . PNPase is known to be inhibited by strong RNA secondary structure, and rapid mRNA turnover is thought to require an RNA helicase activity working in conjunction with PNPase. The most abundant RNA helicase in is 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 and , the model Gram-negative and Gram-positive organisms, from which most of our current knowledge has been derived.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Bacillus subtilis polynucleotide phosphorylase 3'-to-5' DNase activity is involved in DNA repair.

In the presence of Mn(2+), an activity in a preparation of purified Bacillus subtilis RecN degrades single-stranded (ss) DNA with a 3' --> 5' polarity. This activity is not associated with RecN itself, because RecN purified from cells lacking polynucleotide phosphorylase (PNPase) does not show the exonuclease activity. We show here that, in the presence of Mn(2+) and low-level inorganic phosphate (P(i)), PNPase degrades ssDNA.

Messenger RNA decay and maturation in Bacillus subtilis.

Our understanding of the ribonucleases that act to process and turn over RNA in Bacillus subtilis, a model Gram-positive organism, has increased greatly in recent years. This chapter discusses characteristics of B. subtilis ribonucleases that have been shown to participate in messenger RNA maturation and decay.

Assay of Bacillus subtilis ribonucleases in vitro.

Significant progress has been made recently regarding the identification of the ribonucleases involved in RNA maturation and degradation in Bacillus subtilis. More than half of these enzymes have no ortholog in Escherichia coli. To confirm that the in vivo effects of mutations in genes encoding RNases are direct, it is often necessary to purify the enzymes and assay their activity in vitro.