Transfer RNA Maturation Defects Lead to Inhibition of ribosomal RNA Processing via Synthesis of pppGpp

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

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The Essential Function of <em>B. subtilis</em> RNase III Is to Silence Foreign Toxin Genes

<div><p>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 <em>Bacillus subtilis</em>. 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 SPβ, through antisense RNA. Thus, while a growing number of organisms that use RNase III or its homologs as part of a viral defense mechanism, <em>B. subtilis</em> requires RNase III for viral accommodation to the point where the presence of the enzyme is essential for cell survival. We identify <em>txpA</em> and <em>yonT</em> as the two toxin-encoding mRNAs of Skin and SPβ that are sensitive to RNase III. We further explore the mechanism of RNase III–mediated decay of the <em>txpA</em> mRNA when paired to its antisense RNA RatA, both <em>in vivo</em> and <em>in vitro</em>.</p> </div

Cleavage of RatA, <i>txpA</i>, and the RatA/<i>txpA</i> hybrid by RNase III and RNase Y.

<p>(A) Cleavage of 5′ labeled <i>txpA</i> (0.5 pmol) alone or hybridized to a two-fold excess of unlabeled RatA by RNase III (3 ng) and RNase Y (2 µg) for the times indicated. A size standard is shown in lane M and an RNase T1 digestion of 5′ labeled <i>txpA</i> is shown in the lane labeled T1. (B) Cleavage of 5′ labeled RatA alone or hybridized to a two-fold excess of unlabeled <i>txpA</i> by RNase III and RNase Y for the times indicated. A size standard is shown in lane M and an RNase T1 digestion of 5′ labeled RatA is shown in the lane labeled T1.</p

The <i>rnc</i> gene can be deleted in strains lacking TxpA and YonT toxins.

<p>Histogram showing transformation efficiencies (number of Spc^R colonies/number Cm^R colonies) for strains lacking different toxin genes or mRNAs.</p

The <i>txpA</i> and RatA RNAs are stabilized in strains depleted for RNase III and RNase Y, respectively.

<p>(A) Chromosomal context of the <i>txpA</i>/RatA toxin/antitoxin cassette present in the Skin prophage. (B) and (C) Northern blots performed on RNAs isolated at times (min) after rifampicin addition (150 µg/ml) in strains depleted for RNase III (CCB288), RNase Y (CCB294) and RNase J1 (CCB034), probed for <i>txpA</i> and RatA, respectively. Northerns were re-probed for 5S rRNA (5S) for normalization. Half-lives are given below each panel. The band labeled D in panel C (RNase J1) is a degradation intermediate of RatA. Note that, in our hands, the <i>txpA</i> mRNA is about 45 nts longer than that proposed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003181#pgen.1003181-Silvaggi1" target="_blank">[24]</a> and consistent with the presence of a Rho-independent transcription terminator ∼270 nts from the mapped transcription start site. The overlap between RatA and <i>txpA</i> is predicted to be ∼120 nts.</p

Degradation of <i>txpA</i> (AUG→AAG) mRNA by RNase III is RatA-dependent.

<p>Northern blots performed on RNA isolated at times (min) after rifampicin addition in (A) strain CCB467 <i>txpA</i> (AUG→AAG) and (B) strain CCB468 <i>txpA</i> (AUG→AAG) P<i>ratA::ery</i>, depleted or not for RNase III. Northerns were re-probed for 5S rRNA (5S) for normalization. Half-lives are given below each panel.</p

Suppressors of the <i>rnc::spc</i> mutation lack the Skin prophage.

<p>(A) Agarose gel showing multiplex PCR analysis of <i>rnc::spc</i> suppressors in wild-type (WT) and ΔSPβ strains. A PCR product corresponding to the reconstituted <i>ypqP</i> and <i>sigK</i> genes is indicative of excision of the SPβ and Skin prophages, respectively. Spontaneous Spc^R colonies show a PCR product for the <i>rnc</i> gene, while successfully deleted <i>rnc</i> strains do not give a corresponding PCR product. A DNA marker (bp) is shown in the lane labeled M. (B) Sequence comparison of the <i>txpA</i> gene in wild-type (wt) and the sup5 mutant shown in panel A.</p