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.

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.

The crystal structure of Trz1, the long form RNase Z from yeast.

tRNAs are synthesized as precursor RNAs that have to undergo processing steps to become functional. Yeast Trz1 is a key endoribonuclease involved in the 3΄ maturation of tRNAs in all domains of life. It is a member of the ß-lactamase family of RNases, characterized by an HxHxDH sequence motif involved in coordination of catalytic Zn-ions. The RNase Z family consists of two subfamilies: the short (250-400 residues) and the long forms (about double in size). Short form RNase Z enzymes act as homodimers: one subunit embraces tRNA with a protruding arm, while the other provides the catalytic site. The long form is thought to contain two fused ß-lactamase domains within a single polypeptide. Only structures of short form RNase Z enzymes are known. Here we present the 3.1 Å crystal structure of the long-form Trz1 from Saccharomyces cerevisiae. Trz1 is organized into two ß-lactamase domains connected by a long linker. The N-terminal domain has lost its catalytic residues, but retains the long flexible arm that is important for tRNA binding, while it is the other way around in the C-terminal domain. Trz1 likely evolved from a duplication and fusion of the gene encoding the monomeric short form RNase Z.

Trz1, the long form RNase Z from yeast, forms a stable heterohexamer with endonuclease Nuc1 and mutarotase.

Proteomic studies have established that Trz1, Nuc1 and mutarotase form a complex in yeast. Trz1 is a ß-lactamase-type RNase composed of two ß-lactamase-type domains connected by a long linker that is responsible for the endonucleolytic cleavage at the 3'-end of tRNAs during the maturation process (RNase Z activity); Nuc1 is a dimeric mitochondrial nuclease involved in apoptosis, while mutarotase (encoded by YMR099C) catalyzes the conversion between the α- and ß-configuration of glucose-6-phosphate. Using gel filtration, small angle X-ray scattering and electron microscopy, we demonstrated that Trz1, Nuc1 and mutarotase form a very stable heterohexamer, composed of two copies of each of the three subunits. A Nuc1 homodimer is at the center of the complex, creating a two-fold symmetry and interacting with both Trz1 and mutarotase. Enzymatic characterization of the ternary complex revealed that the activities of Trz1 and mutarotase are not affected by complex formation, but that the Nuc1 activity is completely inhibited by mutarotase and partially by Trz1. This suggests that mutarotase and Trz1 might be regulators of the Nuc1 apoptotic nuclease activity.

Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.

The initiation of mRNA degradation often requires deprotection of its 5' end. In eukaryotes, the 5'-methylguanosine (cap) structure is principally removed by the Nudix family decapping enzyme Dcp2, yielding a 5'-monophosphorylated RNA that is a substrate for 5' exoribonucleases. In bacteria, the 5'-triphosphate group of primary transcripts is also converted to a 5' monophosphate by a Nudix protein called RNA pyrophosphohydrolase (RppH), allowing access to both endo- and 5' exoribonucleases. Here we present the crystal structures of Bacillus subtilis RppH (BsRppH) bound to GTP and to a triphosphorylated dinucleotide RNA. In contrast to Bdellovibrio bacteriovorus RppH, which recognizes the first nucleotide of its RNA targets, the B. subtilis enzyme has a binding pocket that prefers guanosine residues in the second position of its substrates. The identification of sequence specificity for RppH in an internal position was a highly unexpected result. NMR chemical shift mapping in solution shows that at least three nucleotides are required for unambiguous binding of RNA. Biochemical assays of BsRppH on RNA substrates with single-base-mutation changes in the first four nucleotides confirm the importance of guanosine in position two for optimal enzyme activity. Our experiments highlight important structural and functional differences between BsRppH and the RNA deprotection enzymes of distantly related bacteria.

Real-time fluorescence detection of exoribonucleases.

The identification of RNases or RNase effectors is a continuous challenge, particularly given the current importance of RNAs in the control of genome expression. Here, we show that a fluorogenic RNA-DNA hybrid is a powerful tool for a real-time fluorescence detection and assay of exoribonucleases (RT-FeDEx). This RT-FeDEx assay provides a new strategy for the isolation, purification, and assay of known and unknown exoribonucleases.

Structure of the ubiquitous 3' processing enzyme RNase Z bound to transfer RNA.

The highly conserved ribonuclease RNase Z catalyzes the endonucleolytic removal of the 3' extension of the majority of tRNA precursors. Here we present the structure of the complex between Bacillus subtilis RNase Z and tRNA(Thr), the first structure of a ribonucleolytic processing enzyme bound to tRNA. Binding of tRNA to RNase Z causes conformational changes in both partners to promote reorganization of the catalytic site and tRNA cleavage.

Structural basis for substrate binding, cleavage and allostery in the tRNA maturase RNase Z.

Transfer RNAs (tRNAs) are synthesized as part of longer primary transcripts that require processing of both their 3' and 5' extremities in every living organism known. The 5' side is processed (matured) by the ubiquitously conserved endonucleolytic ribozyme, RNase P, whereas removal of the 3' tails can be either exonucleolytic or endonucleolytic. The endonucleolytic pathway is catalysed by an enzyme known as RNase Z, or 3' tRNase. RNase Z cleaves precursor tRNAs immediately after the discriminator base (the unpaired nucleotide 3' to the last base pair of the acceptor stem, used as an identity determinant by many aminoacyl-tRNA synthetases) in most cases, yielding a tRNA primed for addition of the CCA motif by nucleotidyl transferase. Here we report the crystal structure of Bacillus subtilis RNase Z at 2.1 A resolution, and propose a mechanism for tRNA recognition and cleavage. The structure explains the allosteric properties of the enzyme, and also sheds light on the mechanisms of inhibition by the CCA motif and long 5' extensions. Finally, it highlights the extraordinary adaptability of the metallo-hydrolase domain of the beta-lactamase family for the hydrolysis of covalent bonds.

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.

Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA.

Hfq protein is vital for the function of many non-coding small (s)RNAs in bacteria but the mechanism by which Hfq facilitates the function of sRNA is still debated. We developed a fluorescence resonance energy transfer assay to probe how Hfq modulates the interaction between a sRNA, DsrA, and its regulatory target mRNA, rpoS. The relevant RNA fragments were labelled so that changes in intra- and intermolecular RNA structures can be monitored in real time. Our data show that Hfq promotes the strand exchange reaction in which the internal structure of rpoS is replaced by pairing with DsrA such that the Shine-Dalgarno sequence of the mRNA becomes exposed. Hfq appears to carry out strand exchange by inducing rapid association of DsrA and a premelted rpoS and by aiding in the slow disruption of the rpoS secondary structure. Unexpectedly, Hfq also disrupts a preformed complex between rpoS and DsrA. While it may not be a frequent event in vivo, this melting activity may have implications in the reversal of sRNA-based regulation. Overall, our data suggests that Hfq not only promotes strand exchange by binding rapidly to both DsrA and rpoS but also possesses RNA chaperoning properties that facilitates dynamic RNA-RNA interactions.

Hfq variant with altered RNA binding functions.

The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS-lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq-RNA interaction but has probably no effect on the folding and the oligomerization of the protein.

Three-dimensional structures of fibrillar Sm proteins: Hfq and other Sm-like proteins.

Hfq is a nucleic acid-binding protein that functions as a global regulator of gene expression by virtue of its interactions with several small, non-coding RNA species. Originally identified as an Escherichia coli host factor required for RNA phage Qbeta replication, Hfq is now known to post-transcriptionally regulate bacterial gene expression by modulating both mRNA stability and translational activity. Recently shown to be a member of the diverse Sm protein family, Hfq adopts the OB-like fold typical of other Sm and Sm-like (Lsm) proteins, and also assembles into toroidal homo-oligomers that bind single-stranded RNA. Similarities between the structures, functions, and evolution of Sm/Lsm proteins and Hfq are continually being discovered, and we now report an additional, unexpected biophysical property that is shared by Hfq and other Sm proteins: E.coli Hfq polymerizes into well-ordered fibres whose morphologies closely resemble those found for Sm-like archaeal proteins (SmAPs). However, the hierarchical assembly of these fibres is dissimilar: whereas SmAPs polymerize into polar tubes (and striated bundles of such tubes) by head-to-tail stacking of individual homo-heptamers, helical Hfq fibres are formed by cylindrical slab-like layers that consist of 36 subunits arranged as a hexamer of Hfq homo-hexamers (i.e. protofilaments in a 6 x 6 arrangement). The different fibrillar ultrastructures formed by Hfq and SmAP are presented and examined herein, with the overall goal of elucidating another similarity amongst the diverse members of the Sm protein family.

Ribonuclease PH plays a major role in the exonucleolytic maturation of CCA-containing tRNA precursors in Bacillus subtilis.

In contrast to Escherichia coli, where all tRNAs have the CCA motif encoded by their genes, two classes of tRNA precursors exist in the Gram-positive bacterium Bacillus subtilis. Previous evidence had shown that ribonuclease Z (RNase Z) was responsible for the endonucleolytic maturation of the 3' end of those tRNAs lacking an encoded CCA motif, accounting for about one-third of its tRNAs. This suggested that a second pathway of tRNA maturation must exist for those precursors with an encoded CCA motif. In this paper, we examine the potential role of the four known exoribonucleases of B.subtilis, PNPase, RNase R, RNase PH and YhaM, in this alternative pathway. In the absence of RNase PH, precursors of CCA-containing tRNAs accumulate that are a few nucleotides longer than the mature tRNA species observed in wild-type strains or in the other single exonuclease mutants. Thus, RNase PH plays an important role in removing the last few nucleotides of the tRNA precursor in vivo. The presence of three or four exonuclease mutations in a single strain results in CCA-containing tRNA precursors of increasing size, suggesting that, as in E.coli, the exonucleolytic pathway consists of multiple redundant enzymes. Assays of purified RNase PH using in vitro-synthesized tRNA precursor substrates suggest that RNase PH is sensitive to the presence of a CCA motif. The division of labor between the endonucleolytic and exonucleolytic pathways observed in vivo can be explained by the inhibition of RNase Z by the CCA motif in CCA-containing tRNA precursors and by the inhibition of exonucleases by stable secondary structure in the 3' extensions of the majority of CCA-less tRNAs.

Ribonucleases J1 and J2: two novel endoribonucleases in B.subtilis with functional homology to E.coli RNase E.

Many prokaryotic organisms lack an equivalent of RNase E, which plays a key role in mRNA degradation in Escherichia coli. In this paper, we report the purification and identification by mass spectrometry in Bacillus subtilis of two paralogous endoribonucleases, here named RNases J1 and J2, which share functional homologies with RNase E but no sequence similarity. Both enzymes are able to cleave the B.subtilis thrS leader at a site that can also be cleaved by E.coli RNase E. We have previously shown that cleavage at this site increases the stability of the downstream messenger. Moreover, RNases J1/J2 are sensitive to the 5' phosphorylation state of the substrate in a site-specific manner. Orthologues of RNases J1/J2, which belong to the metallo-beta-lactamase family, are evolutionarily conserved in many prokaryotic organisms, representing a new family of endoribonucleases. RNases J1/J2 appear to be implicated in regulatory processing/maturation of specific mRNAs, such as the T-box family members thrS and thrZ, but may also contribute to global mRNA degradation.

The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation.

The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AU-rich single-stranded regions. In this paper, we study the ability of the Escherichia coli Hfq protein to bind to a polyadenylated fragment of rpsO mRNA. Hfq exhibits a high specificity for a 100-nucleotide RNA harboring 18 3'-terminal A-residues. Structural analysis of the adenylated RNA-Hfq complex and gel shift assays revealed the presence of two Hfq binding sites. Hfq binds primarily to the poly(A) tail, and to a lesser extent a U-rich sequence in a single-stranded region located between two hairpin structures. The oligo(A) tail and the interhelical region are sensitive to 3'-5' exoribonucleases and RNase E hydrolysis, respectively, in vivo. In vitro assays demonstrate that Hfq protects poly(A) tails from exonucleolytic degradation by both PNPase and RNase II. In addition, RNase E processing, which occurred close to the U-rich sequence, is impaired by the presence of Hfq. These data suggest that Hfq modulates the sensitivity of RNA to ribonucleases in the cell.

Fate of mRNA extremities generated by intrinsic termination: detailed analysis of reactions catalyzed by ribonuclease II and poly(A) polymerase.

In all living cells 3' ends of RNA are posttranscriptionally elongated or shortened by nucleotidyl transferases and ribonucleases. The detailed analysis of the rpsO mRNA of Escherichia coli presented here demonstrates that transcription terminates in vivo at two sites located seven and eight nucleotides downstream from the GC-rich hairpin of the intrinsic terminator and that primary transcripts can be shortened by RNase II. The shortest RNA identified in the cell result from nibbling of primary transcripts. Primary transcripts and nibbled molecules can also be adenylated by poly(A) polymerase I (PAP I). In addition, kinetics of decay performed in vitro demonstrate that RNase II rapidly degrades poly(A) tails longer than 7-8 As processively while it slowly nibbles shorter tails and non adenylated RNAs distributively. Comparison of the kinetics of nibbling of oligoadenylated rpsO mRNA in vivo and in vitro lead us to conclude that the rates of shortening and elongation of the oligo(A) tails detected in vivo are very slow: about 0.5-7 nucleotides per min. We finally speculate that the slowness of oligo(A) synthesis may explain why polyadenylation does not affect the stability of mRNAs whose degradation is controlled by RNase E.

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.

Genetic dissection of an inhibitor of the sporulation sigma factor sigma(G).

Sporulation in Bacillus subtilis is controlled by a cascade of four sigma factors that are held into inactive form until the proper stage of development. The Gin protein, encoded by csfB, is able to strongly inhibit the activity of one of these factors, sigma(G), in vivo. The csfB gene is present in a large number of endospore formers, but the various Gin orthologues show little conservation, in striking contrast to their sigma(G) counterparts. We have carried out a mutagenesis analysis of the Gin protein in order to understand its inhibitory properties. By measuring sigma(G) inhibition in the presence of Gin in vivo, assessing Gin ability to bind sigma(G) in a yeast two-hybrid assay, and quantifying Gin-sigma(G) interaction in B. subtilis, we have identified specific residues that play an essential role in binding sigma(G) or in preventing sigma(G) transcriptional activity. Two cysteine pairs, conserved in all Gin orthologues, are essential for Gin activity. Mutations in the first pair are partially complemented by mutations in the second pair, suggesting that Gin exists in oligomeric form, at least as a dimer. Dimerisation is consistent with our in vitro analysis of a purified Gin recombinant protein, which shows that Gin contains 0.5 zinc atom per monomer. Altogether, these results indicate that the conserved cysteines play a structural role, whereas another less conserved region of the protein is involved in interacting with sigma(G). Interestingly, some mutants have kept most of their ability to bind sigma(G) but are completely unable to inhibit sigma(G) transcriptional activity, raising the possibility that Gin might act by a mechanism more complex than just sequestration of sigma(G).