Bacillus subtilis RNase J1 endonuclease and 5′ exonuclease activities in the turnover of ΔermC mRNA

Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine of New York University, New York, New York 10029, USA.
RNA (Impact Factor: 4.94). 10/2009; 15(12):2331-9. DOI: 10.1261/rna.1749109
Source: PubMed


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. Previously, DeltaermC mRNA was used to show 5'-end dependence of mRNA turnover. Here we used DeltaermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. DeltaermC mRNA showed increased stability in a mutant strain that contained a reduced level of RNase J1. In agreement with the tracking concept, insertion of a strong stem-loop structure at +65 resulted in increased stability. Weakening this stem-loop structure resulted in reversion to wild-type stability. RNA fragments containing the 3' end were detected in a strain with reduced RNase J1 expression, but were undetectable in the wild type. The 5' ends of these fragments mapped to the upstream side of predicted stem-loop structures, consistent with an impediment to RNase J1 5' exonuclease processivity. A DeltaermC mRNA deletion analysis suggested that decay-initiating endonuclease cleavage could occur at several sites near the 3' end. However, even in the absence of these sites, stability was further increased in a strain with reduced RNase J1, suggesting alternate pathways for decay that could include exonucleolytic decay from the 5' end.

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Available from: Josh Sharp, Jun 25, 2014
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    • "Similar to the daa operon, pausing during ermC translation results in mRNA cleavage upstream of the paused ribosome and stabilization of the downstream sequence (Bechhofer and Zen, 1989; Drider et al., 2002). David Bechhofer and colleagues have discovered that this mRNA processing is mediated by RNase J1 (Yao et al., 2009), an essential enzyme with both endonuclease and 5′-to-3′ exonuclease activities (Mathy et al., 2007). Although tmRNA activity has not been examined during ErmC-mediated ribosome pausing, like the DaaP-arrested ribosome, the intact downstream message should preclude ribosome rescue. "
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    ABSTRACT: The bacterial tmRNA quality control system monitors protein synthesis and recycles stalled translation complexes in a process termed "ribosome rescue." During rescue, tmRNA acts first as a transfer RNA to bind stalled ribosomes, then as a messenger RNA to add the ssrA peptide tag to the C-terminus of the nascent polypeptide chain. The ssrA peptide targets tagged peptides for proteolysis, ensuring rapid degradation of potentially deleterious truncated polypeptides. Ribosome rescue also facilitates turnover of the damaged messages responsible for translational arrest. Thus, tmRNA increases the fidelity of gene expression by promoting the synthesis of full-length proteins. In addition to serving as a global quality control system, tmRNA also plays important roles in bacterial development, pathogenesis, and environmental stress responses. This review focuses on the mechanism of tmRNA-mediated ribosome rescue and the role of tmRNA in bacterial physiology.
    Preview · Article · Dec 2012
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    • "Consistent with the conclusion that RNase J1 is principally responsible for degrading monophosphorylated yhxA-glpP mRNA, the concentration of that decay intermediate increased threefold relative to its triphosphorylated precursor when RNase J1 was depleted (Figure 5B). Prior evidence that RNase J1 depletion prolongs the lifetime of ΔermC mRNA (Yao et al., 2009) suggests that the monophosphorylated form of that message is degraded by a similar mechanism. "
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    ABSTRACT: 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.
    Full-text · Article · Sep 2011 · Molecular cell
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    • "RNase J has been shown to play an extensive role in mRNA turnover, with B. subtilis being the best-characterized organism in this regard (Deikus et al., 2008; Mä der et al., 2008; Yao et al., 2009). RNase J is also involved in the maturation of 16S and 23S ribosomal RNA in both B. subtilis and S. meliloti, although in B. subtilis its function in 23S rRNA maturation is as a backup to the primary pathway catalyzed by Mini-RNase III (Madhugiri and Evguenieva-Hackenberg, 2009; Redko and Condon, 2010). "
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    ABSTRACT: RNase J is a key member of the β-CASP family of metallo-β-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 β-CASP domain to explain the enzyme's processive action.
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