Ciarán Condon

5′-to-3′ Exoribonuclease Activity in Bacteria: Role of RNase J1 in rRNA Maturation and 5′ Stability of mRNA

Although the primary mechanism of eukaryotic messenger RNA decay is exoribonucleolytic degradation in the 5-to-3 orientation, it has been widely accepted that Bacteria can only degrade RNAs with the opposite polarity, i.e. 3 to 5. Here we show that maturation of the 5 side of Bacillus subtilis 16S ribosomal RNA occurs via a 5-to-3 exonucleolytic pathway, catalyzed by the widely distributed essential ribonuclease RNase J1. The presence of a 5-to-3 exoribonuclease activity in B. subtilis suggested an explanation for the phenomenon whereby mRNAs in this organism are stabilized for great distances downstream of roadblocks such as stalled ribosomes or stable secondary structures, whereas upstream sequences are never detected. We show that a 30S ribosomal subunit bound to a Shine Dalgarno-like element (Stab-SD) in the cryIIIA mRNA blocks exonucleolytic progression of RNase J1, accounting for the stabilizing effect of this element in vivo.

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 2.1u2009Å 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 β-lactamase family for the hydrolysis of covalent bonds.

Maturation of the 5' end of Bacillus subtilis 16s rRNA by the essential ribonuclease YkqC/RNase J1

Functional ribosomal RNAs are generated from longer precursor species in every organism known. Maturation of the 5′ side of 16S rRNA in Escherichia coli is catalysed in a two‐step process by the cooperative action of RNase E and RNase G. However, many bacteria lack RNase E and RNase G orthologues, raising the question as to how 16S rRNA processing occurs in these organisms. Here we show that the maturation of Bacillus subtilis 16S rRNA is also a two‐step process and that the enzyme responsible for the generation of the mature 5′ end is the widely distributed essential ribonuclease YkqC/RNase J1. Depletion of B.u2003subtilis of RNase J1 results in an accumulation of 16S rRNA precursors in vivo. The precursor species are found in polysomes suggesting that they can function in translation. Mutation of the predicted catalytic site of RNase J1 abolishes both 16S rRNA processing and cell viability. Finally, purified RNase J1 can correctly mature precursor 16S rRNA assembled in 70S ribosomes, showing that its role is direct.

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.

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.

Bacillus subtilis ribonucleases J1 and J2 form a complex with altered enzyme behaviour

Ribonucleases J1 and J2 are recently discovered enzymes with dual 5′‐to‐3′ exoribonucleolytic/endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild‐type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5′‐to‐3′ exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication.

Mini-III, an unusual member of the RNase III family of enzymes, catalyses 23S ribosomal RNA maturation in B. subtilis.

The late steps of both 16S and 5S ribosomal RNA maturation in the Gram‐positive bacterium Bacillus subtilis have been shown to be catalysed by ribonucleases that are not present in the Gram‐negative paradigm, Escherichia coli. Here we present evidence that final maturation of the 5′ and 3′ extremities of B.u2003subtilis 23S rRNA is also performed by an enzyme that is absent from the Proteobacteria. Mini‐III contains an RNase III‐like catalytic domain, but curiously lacks the double‐stranded RNA binding domain typical of RNase III itself, Dicer, Drosha and other well‐known members of this family of enzymes. Cells lacking Mini‐III accumulate precursors and alternatively matured forms of 23S rRNA. We show that Mini‐III functions much more efficiently on precursor 50S ribosomal subunits than naked pre‐23S rRNA in vitro, suggesting that maturation occurs primarily on assembled subunits in vivo. Lastly, we provide a model for how Mini‐III recognizes and cleaves double‐stranded RNA, despite lacking three of the four RNA binding motifs of RNase III.

The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor

Operons encoding stable toxins and their labile antidote are widespread in prokaryotes and play important roles in plasmid partitioning and cellular responses to stress. One such family of toxins MazF/ChpAK/PemK encodes an endoribonuclease that inactivates cellular mRNAs by cleaving them at specific, but frequently occurring sites. Here we show that the Bacillus subtilis ydcE gene encodes a member of this family of RNases, which we have called EndoA. Overexpression of EndoA is toxic for bacterial cell growth and this toxicity is reversed by coexpression of the gene immediately upstream, ydcD. Furthermore, YdcD inhibits EndoA activity directly in vitro. EndoA has similar cleavage specificity to MazF and PemK and yields cleavage products with 3′‐phosphate and 5′‐hydroxyl groups, typical of EDTA‐resistant degradative RNases. This is the first example of an antitoxin–toxin system in B. subtilis.

When all's zed and done: the structure and function of RNase Z in prokaryotes

RNase Z is a widely distributed and often essential endoribonuclease that is responsible for the maturation of the 3′-end of a large family of transfer RNAs (tRNAs). Although it has been the subject of study for more than 25 years, interest in this enzyme intensified dramatically with the identification of the encoding gene in 2002. This led to the discovery of RNase Z in bacteria, in which the final step in the generation of the mature 3′-end of tRNAs had previously been assumed to be catalysed by exoribonucleases. It also led inevitably to structural studies, and the recent resolution of the structure of RNase Z in complex with tRNA has provided a detailed understanding of the molecular mechanisms of RNase Z substrate recognition and cleavage. The identification of the RNase Z gene also allowed the search for alternative substrates for this enzyme to begin in earnest. In this Review, we outline the important recent developments that have contributed to our understanding of this enzyme, particularly in prokaryotes.

What is the role of RNase J in mRNA turnover

The discovery of the paralogous ribonucleases J1 and J2 has been a major advance in the study of RNA maturation and decay in Bacillus subtilis and related organisms. RNase J1 was the first bacterial enzyme shown to possess 5’-to-3’ exoribonuclease activity, reversing a dogma that suggested this type of activity was unique to eukaryotic mRNA decay. RNase J1 and J2 form a complex that also has endonuclease activity and these enzymes have been shown to play a key role in the turnover and maturation of many RNAs in B. subtilis. Here, I describe recent progress in our understanding of the role of these enzymes in RNA metabolism in this organism and argue that the 5’-to-3’ exoribonuclease activity may be the 10 more important of the complex’s two modes of action.