Article

Initiation of decay of Bacillus subtilis rpsO mRNA by endoribonuclease RNase Y.

Department of Pharmacology and Systems Therapeutics, Box 1603, Mount Sinai School of Medicine of New York University, New York, NY 10029, USA.
Journal of bacteriology (Impact Factor: 3.94). 04/2010; 192(13):3279-86. DOI: 10.1128/JB.00230-10
Source: PubMed

ABSTRACT 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. Subsequent to endonuclease cleavage, the upstream products were degraded by polynucleotide phosphorylase (PNPase), and the downstream products were degraded by the 5' exonuclease activity of RNase J1. The rpsO mRNA half-life was unchanged in a strain that had decreased RNase J1 activity and no RNase J2 activity, but it was 2.3-fold higher in a strain with decreased activity of RNase Y, a recently discovered RNase of B. subtilis encoded by the ymdA gene. Accumulation of full-length rpsO mRNA and its decay intermediates was analyzed using a construct in which the rpsO transcription unit was under control of a bacitracin-inducible promoter. The results were consistent with RNase Y-mediated initiation of decay. This is the first report of a specific mRNA whose stability is determined by RNase Y.

0 Bookmarks
 · 
71 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: RNA processing and degradation are key processes in the control of transcript accumulation and thus in the control of gene expression. In Escherichia coli, the underlying mechanisms and components of RNA decay are well characterized. By contrast, Gram-positive bacteria do not possess several important players of E. coli RNA degradation, most notably the essential enzyme RNase E. Recent research on the model Gram-positive organism, Bacillus subtilis, has identified the essential RNases J1 and Y as crucial enzymes in RNA degradation. While RNase J1 is the first bacterial exoribonuclease with 5'-to-3' processivity, RNase Y is the founding member of a novel class of endoribonucleases. Both RNase J1 and RNase Y have a broad impact on the stability of B. subtilis mRNAs; a depletion of either enzyme affects more than 25% of all mRNAs. RNases J1 and Y as well as RNase J2, the polynucleotide phosphorylase PNPase, the RNA helicase CshA and the glycolytic enzymes enolase and phosphofructokinase have been proposed to form a complex, the RNA degradosome of B. subtilis. This review presents a model, based on recent published data, of RNA degradation in B. subtilis. Degradation is initiated by RNase Y-dependent endonucleolytic cleavage, followed by processive exoribonucleolysis of the generated fragments both in 3'-to-5' and in 5'-to-3' directions. The implications of these findings for pathogenic Gram-positive bacteria are also discussed.
    Molecular Microbiology 05/2012; 84(6):1005-17. · 5.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cells of Bacillus subtilis can either be motile or sessile, depending on the expression of mutually exclusive sets of genes that are required for flagellum or biofilm formation, respectively. Both activities are coordinated by the master regulator SinR. We have analyzed the role of the previously uncharacterized ymdB gene for bistable gene expression in B. subtilis. We observed a strong overexpression of the hag gene encoding flagellin and of other genes of the σ(D)-dependent motility regulon in the ymdB mutant, whereas the two major operons for biofilm formation, tapA-sipW-tasA and epsA-O, were not expressed. As a result, the ymdB mutant is unable to form biofilms. An analysis of the individual cells of a population revealed that the ymdB mutant no longer exhibited bistable behavior; instead, all cells are short and motile. The inability of the ymdB mutant to form biofilms is suppressed by the deletion of the sinR gene encoding the master regulator of biofilm formation, indicating that SinR-dependent repression of biofilm genes cannot be relieved in a ymdB mutant. Our studies demonstrate that lack of expression of SlrR, an antagonist of SinR, is responsible for the observed phenotypes. Overexpression of SlrR suppresses the effects of a ymdB mutation.
    Journal of bacteriology 08/2011; 193(21):5997-6007. · 3.94 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The genes encoding ribonucleases RNase J1 and Y have long been considered essential for B. subtilis cell viability, even before 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 to 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. Lastly, we combine 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.
    Journal of bacteriology 03/2013; · 3.94 Impact Factor

Full-text

View
0 Downloads
Available from