Role of ribosome assembly in Escherichia coli ribosomal RNA degradation

Abstract

DEAD-Box proteins (DBPs) constitute a prominent class of RNA remodeling factors that play a role in virtually all aspects of RNA metabolism. To better define their cellular functions, deletions in the genes encoding each of the Escherichia coli DBPs were combined with mutations in genes encoding different Ribonucleases (RNases). Significantly, double-deletion strains lacking Ribonuclease R (RNase R) and either the DeaD or SrmB DBP were found to display growth defects and an enhanced accumulation of ribosomal RNA (rRNA) fragments. As RNase R is known to play a key role in removing rRNA degradation products, these observations initially suggested that these two DBPs could be directly involved in the same process. However, additional investigations indicated that DeaD and SrmB-dependent rRNA breakdown is caused by delays in ribosome assembly that increase the exposure of nascent RNAs to endonucleolytic cleavage. Consistent with this notion, mutations in factors known to be important for ribosome assembly also resulted in enhanced rRNA breakdown. Additionally, significant levels of rRNA breakdown products could be visualized in growing cells even in the absence of assembly defects. These findings reveal a hitherto unappreciated mechanism of rRNA degradation under conditions of both normal and abnormal ribosome assembly.

Figure 1.

Growth defects in strains lacking RNase R and DBPs. (A) Growth of strains on LB-agar plates. Deletion alleles of each of the five Escherichia coli DBPs were transferred into WT or Δrnr strain backgrounds. The derivative strains were streaked on an LB-agar plate and grown overnight at 37°C. (B) Dilutions of saturated cultures necessary to yield single colonies were spotted on LB-agar plates and grown overnight at 37°C. (C) Doubling times were determined in LB medium at 37°C. Mean values and standard errors are derived from triplicate cultures for each strain.

Figure 2.

Accumulation of rRNA fragments in ΔdeaDΔrnr and ΔsrmBΔrnr strains. RNA was isolated from WT or mutant strains grown in LB medium at 37°C, as indicated, and analyzed by northern blotting using probes to 16S rRNA (A) or 23S rRNA (B). The full length rRNAs and rRNA degradation products are indicated.

Figure 3.

The rRNA degradation products in the ΔdeaDΔrnr and srmBΔrnr strains are the same as in the pnp^tsΔrnr strain. (A) RNA was isolated from WT, pnp^ts, Δrnr and pnp^tsΔrnr strains after a temperature shift to 42°C for 1 h following growth at 30°C to early-log phase. The RNAs were analyzed by northern blotting using a probe for 16S rRNA (lanes 7–10). Total RNA from ΔdeaDΔrnr, ΔsrmBΔrnr or derivative strains grown at 37°C were also analyzed in parallel (lanes 1–6). (B and C) Primer extension analysis. (B) RNA was isolated from WT, pnp^ts, Δrnr and pnp^tsΔrnr strains after a temperature shift to 42°C and analyzed by primer extension using an oligonucleotide complementary to 16S rRNA. The major reverse-transcription (RT) products are denoted by a bracket. A control lane (C) containing only the oligonucleotide is also included. The products that correspond to the first five reverse-transcribed nucleotides are indicated by red dots. (C) Primer extension was performed on RNA isolated from ΔdeaDΔrnr, ΔsrmBΔrnr or derivative strains grown at 37°C, as described in (B). (D) Suppression of rRNA fragment accumulation by PNPase over-expression. A control plasmid (pBR322) or a PNPase-expression plasmid (pKAK7) were transformed into ΔdeaDΔrnr (lanes 1 and 2) or ΔsrmBΔrnr strains (lanes 3 and 4). Total RNA was isolated from the resulting strains and analyzed by northern blotting using a 16S rRNA probe. The positions of the full-length RNAs and of the main rRNA degradation product are indicated.

Figure 4.

Two models for the accumulation of rRNA fragments in ΔdeaDΔrnr and ΔsrmBΔrnr strains. (A) A fraction of newly synthesized rRNAs (shown in red) is proposed to be cleaved by an endonuclease, depicted in gray. The resulting products are digested either by RNase R (green oval) or PNPase (yellow oval). PNPase-mediated digestion of structured rRNAs is facilitated via unwinding of base-pairing by SrmB or DeaD (blue ovals). (B) Newly synthesized rRNA is incorporated into a nascent ribosomal particle (show as a yellow oval). In the absence of delays, ribosomal subunit assembly rapidly proceeds to completion, resulting in the protection of the rRNAs from RNase action (top). When ribosome assembly is delayed, endonucleolytic cleavage occurs within exposed rRNA regions (bottom). The resulting fragments are primarily digested by RNase R (green oval) and therefore accumulate in its absence.

Figure 5.

Ribosome assembly kinetics. (A–C) Cultures of WT (A), ΔdeaD (B) or ΔsrmB (C) strains were pulse-labeled with ³²P inorganic phosphate for 1 min. Aliquots of the cultures were removed thereafter 5 or 15 min post-labeling and combined with unlabeled cells. Lysates derived from these cells were individually layered on 14–32% sucrose density gradients and ultracentrifuged. (Left) Ribosome profiles were determined at 254 nM. The positions of the peaks corresponding to the 30S and 50S ribosomal subunits, and to 70S ribosomes, are indicated. Sixteen ribosomal fractions were collected in each case. A₂₅₄, absorbance at 254 nM; (right) The ribosomal fractions were filtered through nitrocellulose membrane to capture labeled rRNAs present in ribosomal particles. The amount of radioactivity in each fraction was quantified by phosphorimaging of the dried membranes. Blue bars, relative amount of radiolabel in each fraction for cultures harvested 5 min after labeling; red bars, radiolabel incorporation for cultures harvested after 15 min of labeling; n = 3. The main fractions that correspond to the 70S, 50S and 30S particles are indicated. (D and E) The amount of radiolabel in 70S particles (fractions 2–5), relative to the total amount of radiolabel present in all fractions, was quantified for WT, ΔdeaD and ΔsrmB strains for cultures harvested 5 min (D) or 15 min (E) after labeling.

Figure 6.

Generation of rRNA fragments due to impaired ribosome assembly. (A) Ribosomal profiles of strains containing mutations in established RAFs. Cell lysates from WT, ΔrimP, ΔrbfA or ΔrimM strains were analyzed by ultracentrifugation, as described for Figure 5. (B and C) RNA was isolated from WT or Δrnr strains containing ΔrimP, ΔrbfA or ΔrimM mutations and analyzed by northern blotting as described in Figure 2 using probes for 16S rRNA (B) or 23S rRNA (C). (D and E) WT or Δrnr strains were grown in the absence or presence of the indicated amounts of antibiotics (Abs) and RNA derived from these strains was analyzed by northern blotting, as described in Figure 2 using probes for 16S rRNA (D) or 23S rRNA (E). (F) Doubling times were determined in LB medium at 37°C. Mean values and standard errors are based on triplicate cultures for each strain.

Figure 7.

Visualization of rRNA fragments in the absence of ribosome assembly defects. A ΔpnpΔrnr strain containing an IPTG-regulated PNPase or RNase R-expression plasmid was grown at 37°C in LB media supplemented with different concentrations of IPTG, and aliquots of these cultures were used to prepare total RNA or total protein. (A) RNA and protein analysis of the IPTG-regulated PNPase expression strain. Top, RNAs were analyzed by northern blotting, as described in Figure 2A; bottom, total cellular protein was analyzed by western blotting using anti-PNPase antibodies. (B) RNA analysis of the IPTG-regulated RNase R expression strain. (C) Comparison of 16S rRNA fragment levels and growth rates. Doubling times for the strains analyzed in (A) and (B) were measured and plotted against the relative fraction of rRNA degradation products observed. Blue, IPTG-regulated PNPase expression strain; red, IPTG-regulated RNase R expression strain. Numbers in brackets for each data point correspond to the concentration of IPTG, in micromolar, that was present in the growth medium; n = 3.

Figure 8.

Digestion of ribosomal particles by RNase E. (A) 30S or 70S ribosomal fractions were concentrated and treated with different amounts of purified RNase E for 1 h at 37°C. RNA was extracted from these reactions and analyzed by primer extension, as described in the Figure 3B legend. The products that correspond to the first five reverse-transcribed nucleotides are indicated by red dots. (B) Quantitation of RNase E cleavage efficiency. The amount of cleavage products in (A) was quantified by phosphorimaging, reduced by any signal observed without RNase E addition, and normalized with respect to the amount of product generated by using 1 μg of RNase E on 30S particles. Red bars, 70S particles; blue bars, 30S particles. Mean values and standard errors are based on four independent preparations of 30S and 70S particles. (C) Visualization of a RNase E cleavage site in the high-resolution Escherichia coli 70S ribosome structure (PDB ID: 4YBB, molecule A) shown via ribbon representation. The RNA and proteins components of the ribosomal large subunit are colored light gray and light cyan, respectively, whereas the corresponding residues on the small subunit are colored dark gray and light yellow. The residues abutting the RNase E cleavage site (16S rRNA nts 916–921) are shown as red spheres. The view shown is from the tRNA E site.