Shutdown of multidrug transporter bmrCD mRNA expression mediated by the ribosome-associated endoribonuclease (Rae1) cleavage in a new cryptic ORF.

Rae1 is a well-conserved endoribonuclease among Gram-positive bacteria, cyanobacteria, and the chloroplasts of higher plants. We have previously shown that Rae1 cleaves the Bacillus subtilis yrzI operon mRNA in a translation-dependent manner within a short open reading frame (ORF) called S1025, encoding a 17-amino acid (aa) peptide of unknown function. Here, we map a new Rae1 cleavage site in the bmrBCD operon mRNA encoding a multidrug transporter, within an unannotated 26-aa cryptic ORF that we have named bmrX Expression of the bmrCD portion of the mRNA is ensured by an antibiotic-dependent ribosome attenuation mechanism within the upstream ORF bmrB Cleavage by Rae1 within bmrX suppresses bmrCD expression that escapes attenuation control in the absence of antibiotics. Similar to S1025, Rae1 cleavage within bmrX is both translation- and reading frame-dependent. Consistent with this, we show that translation-dependent cleavage by Rae1 promotes ribosome rescue by the tmRNA.

Structural studies of RNase M5 reveal two-metal-ion supported two-step dsRNA cleavage for 5S rRNA maturation.

All species transcribe ribosomal RNA in an immature form that requires several enzymes for processing into mature rRNA. The number and types of enzymes utilized for these processes vary greatly between different species. In low G + C Gram-positive bacteria including Bacillus subtilis and Geobacillus stearothermophilus, the endoribonuclease (RNase) M5 performs the final step in 5S rRNA maturation, by removing the 3'- and 5'-extensions from precursor (pre) 5S rRNA. This cleavage activity requires initial complex formation between the pre-rRNA and a ribosomal protein, uL18, making the full M5 substrate a ribonucleoprotein particle (RNP). M5 contains a catalytic N-terminal Toprim domain and an RNA-binding C-terminal domain, respectively, shown to assist in processing and binding of the RNP. Here, we present structural data that show how two Mg2+ ions are accommodated in the active site pocket of the catalytic Toprim domain and investigate the importance of these ions for catalysis. We further perform solution studies that support the previously proposed 3'-before-5' order of removal of the pre-5S rRNA extensions and map the corresponding M5 structural rearrangements during catalysis.

Walking from E. coli to B. subtilis, one ribonuclease at a time.

Most bacterial ribonucleases (RNases) known to date have been identified in either Escherichia coli or Bacillus subtilis. These two organisms lie on opposite poles of the phylogenetic spectrum, separated by 1-3 billion years of evolution. As a result, the RNA maturation and degradation machineries of these two organisms have little overlap, with each having a distinct set of RNases in addition to a core set of enzymes that is highly conserved across the bacterial spectrum. In this paper, we describe what the functions performed by major RNases in these two bacteria, and how the evolutionary space between them can be described by two opposing gradients of enzymes that fade out and fade in, respectively, as one walks across the phylogenetic tree from E. coli to B. subtilis.

La plupart des ribonucléases (RNases) bactériennes connues à ce jour ont été identifiées chez Escherichia coli ou Bacillus subtilis. Ces deux organismes se trouvent aux pôles opposés du spectre phylogénétique, séparés par 1­3 milliards d'années d'évolution. Par conséquent, les mécanismes de maturation et de dégradation de l'ARN de ces deux organismes se chevauchent peu, chacun possédant un ensemble distinct de RNases en plus d'un ensemble coeur d'enzymes hautement conservées dans tout le spectre bactérien. Dans cet article, nous décrivons les fonctions remplies par les principales RNases de ces deux bactéries, et comment l'espace évolutif qui les sépare peut être décrit par deux gradients opposés d'enzymes qui disparaissent et apparaissent, respectivement, lorsqu'on parcourt l'arbre phylogénétique de E. coli à B. subtilis.

Assay of Bacillus subtilis Ribonuclease Activity In Vitro.

Ribonucleases can cleave RNAs internally in endoribonucleolytic mode or remove one nucleotide at a time from either the 5' or 3' end through exoribonuclease action. To show direct implication of an RNase in a specific pathway of RNA maturation or decay requires the setting up of in vitro assays with purified enzymes and substrates. This chapter complements Chapter 24 on assays of ribonuclease action in vivo by providing detailed protocols for the assay of B. subtilis RNases with prepared substrates in vitro.

Analysis of Bacillus subtilis Ribonuclease Activity In Vivo.

Ribonucleases remodel RNAs to render them functional or to send them on their way toward degradation. In our laboratory, we study these pathways in detail using a plethora of different techniques. These can range from the isolation of RNAs in various RNase mutants to determine their implication in maturation or decay pathways by Northern blot, to proving their direct roles in RNA cleavage reactions using purified enzymes and transcribed substrates in vitro. In this chapter, we provide in-depth protocols for the techniques we use daily in the laboratory to assay RNase activity in vivo, with detailed notes on how to get these methods to work optimally. This chapter complements Chapter 25 on assays of ribonuclease action in vitro.

Structures of B. subtilis Maturation RNases Captured on 50S Ribosome with Pre-rRNAs.

The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.

tRNA Maturation Defects Lead to Inhibition of rRNA Processing via Synthesis of pppGpp.

rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.

The essential nature of YqfG, a YbeY homologue required for 3' maturation of Bacillus subtilis 16S ribosomal RNA is suppressed by deletion of RNase R.

Ribosomal RNAs are processed from primary transcripts containing 16S, 23S and 5S rRNAs in most bacteria. Maturation generally occurs in a two-step process, consisting of a first crude separation of the major species by RNase III during transcription, followed by precise trimming of 5' and 3' extensions on each species upon accurate completion of subunit assembly. The various endo- and exoribonucleases involved in the final processing reactions are strikingly different in Escherichia coli and Bacillus subtilis, the two best studied representatives of Gram-negative and Gram-positive bacteria, respectively. Here, we show that the one exception to this rule is the protein involved in the maturation of the 3' end of 16S rRNA. Cells depleted for the essential B. subtilis YqfG protein, a homologue of E. coli YbeY, specifically accumulate 16S rRNA precursors bearing 3' extensions. Remarkably, the essential nature of YqfG can be suppressed by deleting the ribosomal RNA degrading enzyme RNase R, i.e. a ΔyqfG Δrnr mutant is viable. Our data suggest that 70S ribosomes containing 30S subunits with 3' extensions of 16S rRNA are functional to a degree, but become substrates for degradation by RNase R and are eliminated.

Rae1/YacP, a new endoribonuclease involved in ribosome-dependent mRNA decay in Bacillus subtilis.

The PIN domain plays a central role in cellular RNA biology and is involved in processes as diverse as rRNA maturation, mRNA decay and telomerase function. Here, we solve the crystal structure of the Rae1 (YacP) protein of Bacillus subtilis, a founding member of the NYN (Nedd4-BP1/YacP nuclease) subfamily of PIN domain proteins, and identify potential substrates in vivo Unexpectedly, degradation of a characterised target mRNA was completely dependent on both its translation and reading frame. We provide evidence that Rae1 associates with the B. subtilis ribosome and cleaves between specific codons of this mRNA in vivo Critically, we also demonstrate translation-dependent Rae1 cleavage of this substrate in a purified translation assay in vitro Multiple lines of evidence converge to suggest that Rae1 is an A-site endoribonuclease. We present a docking model of Rae1 bound to the B. subtilis ribosomal A-site that is consistent with this hypothesis and show that Rae1 cleaves optimally immediately upstream of a lysine codon (AAA or AAG) in vivo.

Arabidopsis chloroplast mini-ribonuclease III participates in rRNA maturation and intron recycling.

RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 3' end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 3' extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation.

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.

Bacillus subtilis mutants with knockouts of the genes encoding ribonucleases RNase Y and RNase J1 are viable, with major defects in cell morphology, sporulation, and competence.

The genes encoding the ribonucleases RNase J1 and RNase Y have long been considered essential for Bacillus subtilis cell viability, even before there was concrete knowledge of their function as two of the most important enzymes for RNA turnover in this organism. Here we show that this characterization is incorrect and that ΔrnjA and Δrny mutants are both viable. As expected, both strains grow relatively slowly, with doubling times in the hour range in rich medium. Knockout mutants have major defects in their sporulation and competence development programs. Both mutants are hypersensitive to a wide range of antibiotics and have dramatic alterations to their cell morphologies, suggestive of cell envelope defects. Indeed, RNase Y mutants are significantly smaller in diameter than wild-type strains and have a very disordered peptidoglycan layer. Strains lacking RNase J1 form long filaments in tight spirals, reminiscent of mutants of the actin-like proteins (Mre) involved in cell shape determination. Finally, we combined the rnjA and rny mutations with mutations in other components of the degradation machinery and show that many of these strains are also viable. The implications for the two known RNA degradation pathways of B. subtilis are discussed.

Activation of tRNA maturation by downstream uracil residues in B. subtilis.

Ribonuclease (RNase) Z is involved in the maturation of the 3' ends of transfer RNAs (tRNAs) in all three kingdoms of life. To prevent futile cycles of CCA addition and removal, eukaryotic RNase Z discriminates against mature tRNAs bearing a CCA motif, with the first cytosine residue (C74) being the key antideterminant. Here, we show that, remarkably, the B. subtilis enzyme does not discriminate against cytosine in position 74, but rather is highly stimulated by uracil in this location. Consistent with this observation, the vast majority of B. subtilis tRNA precursor substrates of RNase Z naturally contain U74. Those tRNA precursors with a uracil further downstream are also substrates for RNase Z, but are matured in a two-step endo/exonuclease reaction. We solved the first crystal structure of B. subtilis RNase Z bound to a tRNA(Thr) precursor with U74 and show that the enzyme has a specific binding pocket for this nucleotide.

Three essential ribonucleases-RNase Y, J1, and III-control the abundance of a majority of Bacillus subtilis mRNAs.

Bacillus subtilis possesses three essential enzymes thought to be involved in mRNA decay to varying degrees, namely RNase Y, RNase J1, and RNase III. Using recently developed high-resolution tiling arrays, we examined the effect of depletion of each of these enzymes on RNA abundance over the whole genome. The data are consistent with a model in which the degradation of a significant number of transcripts is dependent on endonucleolytic cleavage by RNase Y, followed by degradation of the downstream fragment by the 5'-3' exoribonuclease RNase J1. However, many full-size transcripts also accumulate under conditions of RNase J1 insufficiency, compatible with a model whereby RNase J1 degrades transcripts either directly from the 5' end or very close to it. Although the abundance of a large number of transcripts was altered by depletion of RNase III, this appears to result primarily from indirect transcriptional effects. Lastly, RNase depletion led to the stabilization of many low-abundance potential regulatory RNAs, both in intergenic regions and in the antisense orientation to known transcripts.

The essential function of B. subtilis RNase III is to silence foreign toxin genes.

RNase III-related enzymes play key roles in cleaving double-stranded RNA in many biological systems. Among the best-known are RNase III itself, involved in ribosomal RNA maturation and mRNA turnover in bacteria, and Drosha and Dicer, which play critical roles in the production of micro (mi)-RNAs and small interfering (si)-RNAs in eukaryotes. Although RNase III has important cellular functions in bacteria, its gene is generally not essential, with the remarkable exception of that of Bacillus subtilis. Here we show that the essential role of RNase III in this organism is to protect it from the expression of toxin genes borne by two prophages, Skin and SPß, through antisense RNA. Thus, while a growing number of organisms that use RNase III or its homologs as part of a viral defense mechanism, B. subtilis requires RNase III for viral accommodation to the point where the presence of the enzyme is essential for cell survival. We identify txpA and yonT as the two toxin-encoding mRNAs of Skin and SPß that are sensitive to RNase III. We further explore the mechanism of RNase III-mediated decay of the txpA mRNA when paired to its antisense RNA RatA, both in vivo and in vitro.

Molecular basis for the recognition and cleavage of RNA by the bifunctional 5'-3' exo/endoribonuclease RNase J.

RNase J is a key member of the ß-CASP family of metallo-ß-lactamases involved in the maturation and turnover of RNAs in prokaryotes. The B. subtilis enzyme possesses both 5'-3' exoribonucleolytic and endonucleolytic activity, an unusual property for a ribonuclease. Here, we present the crystal structure of T. thermophilus RNase J bound to a 4 nucleotide RNA. The structure reveals an RNA-binding channel that illustrates how the enzyme functions in 5'-3' exoribonucleolytic mode and how it can function as an endonuclease. A second, negatively charged tunnel leads from the active site, and is ideally located to evacuate the cleaved nucleotide in 5'-3' exonucleolytic mode. We show that B. subtilis RNase J1, which shows processive behavior on long RNAs, behaves distributively for substrates less than 5 nucleotides in length. We propose a model involving the binding of the RNA to the surface of the ß-CASP domain to explain the enzyme's processive action.