Effect of tRNA Maturase Depletion on Levels and Stabilities of Ribosome Assembly Cofactor and Other mRNAs in Bacillus subtilis

Abstract

The impact of translation on mRNA stability can be varied, ranging from a protective effect of ribosomes that shield mRNA from RNases to preferentially exposing sites of RNase cleavage. These effects can change depending on whether ribosomes are actively moving along the mRNA or stalled at particular sequences or structures or awaiting charged tRNAs. We recently observed that depleting Bacillus subtilis cells of their tRNA maturation enzymes RNase P and RNase Z led to altered mRNA levels of a number of assembly factors involved in the biogenesis of the 30S ribosomal subunit. Here, we extended this study to other assembly factor and non-assembly factor mRNAs in B. subtilis. We additionally identified multiple transcriptional and translational layers of regulation of the rimM operon mRNA that occur in response to the depletion of functional tRNAs. IMPORTANCE The passage of ribosomes across individual mRNAs during translation can have different effects on their degradation, ranging from a protective effect by shielding from ribonucleases to, in some cases, making the mRNA more vulnerable to RNase action. We recently showed that some mRNAs coding for proteins involved in ribosome assembly were highly sensitive to the availability of functional tRNA. Using strains depleted of the major tRNA processing enzymes RNase P and RNase Z, we expanded this observation to a wider set of mRNAs, including some unrelated to ribosome biogenesis. We characterized the impact of tRNA maturase depletion on the rimM operon mRNA and show that it is highly complex, with multiple levels of transcriptional and posttranscriptional effects coming into play.

Keywords: (p)ppGpp; RNA degradation; chloramphenicol; tRNA processing; translation.

FIG 1

Depletion of tRNA processing enzymes results in perturbed expression of some mRNAs encoding proteins involved in 30S subunit assembly. Northern blots showing (A) up-regulated mRNAs, (B) down-regulated mRNAs, (C) unaffected mRNAs, and (D) mRNAs unrelated to ribosome assembly, present in total mRNA isolated in the presence or absence of inducer as indicated. Note that the basal level of the bmrCD transcript, encoding a multidrug transporter, is higher in the Pspac-rnz and Pspac-rnpB strains because of the presence of erythromycin in the medium for stable maintenance of the construct. 16S rRNA levels (ethidium bromide stained) are shown as a loading control. Series of blots where a single loading control is shown were stripped and reprobed. The membrane used for panel B is the same as in panel C. The blots for era, yqeH, rimM, and cpgA were regenerated as in reference with independent RNA preparations, with permission granted by the publisher for reuse of previously published data. Numbers of repetitions are as follows: yqeH, 3; era, 3; rpsU, 4; ydaF, 2; yjcK, 2; rimM, 3; cpgA, 3; rbfA, 2; ylxS, 2; yfmL, 2; bmrCD, 2; yrzI, 2.

FIG 2

Upregulated mRNAs show increased stability upon tRNA maturase depletion and increased expression levels in the presence of chloramphenicol. Northern blots of total RNA isolated at different times after addition of rifampicin (Rif) to cells grown in the presence or absence of inducer for (A) rnpA or (B) rnpB expression. Transcript sizes are given in kilobases to the left of the blots, and half-lives are reported under each blot. Note that since yjcK gives no signal in the presence of inducer, we cannot rule out a transcriptional effect in this case. (C) Northern blots of total RNA isolated at different times after addition of 0.5× MIC and the MIC of Cm. 16S rRNA levels (ethidium bromide stained) are shown as a loading control. Series of blots where a single loading control is shown were stripped and reprobed. The membrane used to probe yqeH is the same as that probed with yjcK. Experiments were performed twice, with decay plots and their quantifications given in Fig. S1.

FIG 3

Downregulated mRNAs are subjected to a mixture of transcriptional and posttranscriptional effects upon tRNA maturase depletion and chloramphenicol addition. Northern blots of total RNA isolated at different times after addition of rifampicin (Rif) to cells grown in the presence or absence of inducer for (A) rnpA or (B) rnpB expression. Transcript sizes are given in kilobases to the left of the blot, and half-lives are reported under each blot. (C) Northern blots of total RNA isolated at different times after addition of 0.5× MIC and the MIC of Cm. 16S rRNA levels (ethidium bromide stained) are shown as a loading control. Series of blots where a single loading control is shown were stripped and reprobed. The membranes used in panel A (top, yqeH probe; bottom, era, ydaF, and yjcK probes) are the same as in Fig. 2A (top, rimM; bottom, cpgA and yfmL probes). Similarly, the membranes used in panel B (top and bottom) are the same as in Fig. 2B (top and bottom, respectively). The membrane used to probe rimH in panel C is the same as that probed with era and ydaF in Fig. 2C. Experiments were performed twice, with decay plots and their quantifications given in Fig. S2.

FIG 4

Three of the six transcripts emanating from the rimM operon are sensitive to tRNA maturase depletion. (A) Structure of the rimM operon. ORFs (not to scale) are shown as gray arrows and transcripts as colored wavy lines. Promoters (P₁ to P₃) are represented by black arrows and terminators (T₁ to T₃) as hairpins. A known RNase Y cleavage site is indicated. The asterisks indicate transcripts that may be processed by RNase Y but are not distinguishable from P₂ primary transcripts by Northern blotting. These are designated Y/P₂ in the text. The black boxes indicate the location of the probe used. (B) Northern blot analysis of total RNA from Pxyl-rnpA cells grown in the presence or absence of inducer, probed with oligonucleotides targeting different ORFs of the operon (indicated at the bottom). Colored dots correspond to the colors of the transcripts in panel A.

FIG 5

A determinant for downregulation of the rimM operon in response to tRNA maturase depletion is located within the ylqD ORF. (A) Northern blot of total RNA from Pspac-rnpB cells isolated in the presence or absence of IPTG showing the effect of RnpB depletion on expression of an ectopic ylqD-rimM short operon containing a WT or mutated (M) potential target sequence for trnD-Tyr pre-tRNA within the ylqD ORF. Colored dots identifying endogenous rimM transcripts follow the code used in Fig. 4. (B) Schematic of ectopic ylqD-rimM constructs placed under the control of the constitutive promoter (P) used for panel A. The enlargement shows the complementarity to the trnD-Tyr pre-tRNA and its disruption in the ylqD^M-rimM mutant construct. Coordinates are relative to the start codon of rimM. The black box indicates the location of the rimM probe. (C) Northern blot showing the effect of RNase P depletion (rnpA or rnpB) on expression of an ectopic rimM-only construct. (D) Schematic of ectopic rimM construct placed under the control of the constitutive promoter (P) used for panel C. Slower-migrating bands (marked with asterisks) are likely due to read-through of the terminator in the ectopic construct. 16S rRNA levels (ethidium bromide stained) are shown as a loading control.

FIG 6

Ethanol stress and stationary phase affect rnpA and rnpB expression without causing tRNA processing defects. (A) rnpB (black) and rnpA (red) transcript levels over 100 different growth conditions (from reference 10), adapted from the SubtiWiki website (http://subtiwiki.uni-goettingen.de/). The three conditions indicated in yellow (ethanol stress and stationary phase in complex and minimal medium) result in reduced rnpA RNA levels. For each condition, rnpA and rnpB RNA levels (log₂) are indicated in the yellow box. (B) Northern blot comparing rnpB (first panel; acrylamide gel because of small size, 401 nt), rnpA and rimM (second and third panels; agarose gel) RNA levels, after ethanol addition (EtOH) or during exponential (Exp) or stationary (Stat) phase in minimal (MM) or complex (2xTY) medium. Colored dots identifying rimM transcripts follow the code used in Fig. 4. Note that the 16S rRNA (loading control) is beginning to be degraded in MM in stationary phase. (C) Northern blot probed for trnJ-Lys (acrylamide gel) showing no pre-tRNA accumulation in the different conditions tested. See Figure S1 in reference for accumulation of trnJ-Lys precursors under conditions of RnpA and RnpB depletion.

FIG 7

Influence of (p)ppGpp on rimM expression. (A) Northern blot comparing the effect of RNase P depletion (RnpB or RnpA) on rimM expression in a WT and (p)ppGpp⁰ background. (B) Northern blot of rimM expression after induction of (p)ppGpp production using a xylose-inducible ywaC gene in a ΔyjbM ywaC relA [(p)ppGpp⁰] background. Colored dots identifying rimM transcripts follow the code used in Fig. 4. 16S rRNA levels (ethidium bromide stained) are shown as a loading control. Series of blots where a single loading control is shown were stripped and reprobed. Note that this Northern blot was generated by reprobing a membrane previously used in reference , with permission to reuse the ywaA control panel granted by the publisher.

FIG 8

Expression of the rimM operon is regulated by growth rate independently of (p)ppGpp. (A) Northern blot comparing the effect of RNase III deletion and RNase P or RNase III depletion on derepression of the CodY-regulated ywaA mRNA. (B) RNase III-depleted cells do not accumulate large amounts of (p)ppGpp compared to tRNA maturase-depleted ones. TLC analysis of ³²P-labeled nucleotides extracted from RNase III-depleted cells (rnc). Arginine hydroxamate (RHX; 250 mg/mL) was added to WT and (p)ppGpp⁰ strains as positive and negative controls. The top and bottom halves were exposed for different times. Note that this is a recrop of an image previously published in reference ; the first four (control) lanes are reproduced to show the migration position of (p)ppGpp, with permission from the publisher. (C) Northern blot comparing the effect of RNase III depletion or deletion on rimM expression. Colored dots identifying rimM transcripts follow the code used in Fig. 4. 16S rRNA levels (ethidium bromide stained) are shown as a loading control.