Distinct tmRNA sequence elements facilitate RNase R engagement on rescued ribosomes for selective nonstop mRNA decay

Abstract

trans-Translation, orchestrated by SmpB and tmRNA, is the principal eubacterial pathway for resolving stalled translation complexes. RNase R, the leading nonstop mRNA surveillance factor, is recruited to stalled ribosomes in a trans-translation dependent process. To elucidate the contributions of SmpB and tmRNA to RNase R recruitment, we evaluated Escherichia coli-Francisella tularensis chimeric variants of tmRNA and SmpB. This evaluation showed that while the hybrid tmRNA supported nascent polypeptide tagging and ribosome rescue, it suffered defects in facilitating RNase R recruitment to stalled ribosomes. To gain further insights, we used established tmRNA and SmpB variants that impact distinct stages of the trans-translation process. Analysis of select tmRNA variants revealed that the sequence composition and positioning of the ultimate and penultimate codons of the tmRNA ORF play a crucial role in recruiting RNase R to rescued ribosomes. Evaluation of defined SmpB C-terminal tail variants highlighted the importance of establishing the tmRNA reading frame, and provided valuable clues into the timing of RNase R recruitment to rescued ribosomes. Taken together, these studies demonstrate that productive RNase R-ribosomes engagement requires active trans-translation, and suggest that RNase R captures the emerging nonstop mRNA at an early stage after establishment of the tmRNA ORF as the surrogate mRNA template.

Figure 1.

tmRNA ORF alterations lead to accumulation of nonstop mRNA. (A) The top panel depicts a representative northern blot showing the steady state abundance of the λ-cI-NS nonstop mRNA in the presence of the hybrid variants of SmpB and tmRNA. The accumulation of nonstop mRNA in the presence of single and double tmRNA ORF hybrids (lanes 3 and 4) is ∼4-fold higher than that of Escherichia coli derived SmpB–tmRNA. P-values were calculated by performing Student's t-test analysis with SmpB–tmRNA and SmpB^FT–tmRNA (ns, not statistically significant), SmpB–tmRNA and SmpB–tmRNA^FT (*P = 0.035), or SmpB–tmRNA and SmpB^FT–tmRNA^FT (*P = 0.049). The accompanying graph represents the fold change in the steady state level of λ-cI-NS mRNA in the presence of indicated SmpB and tmRNA variants with respect to SmpB–tmRNA. The data are from three independent experiments (mean ± SEM, standard error of means). (B) A representative northern blot showing similar steady state levels of the control λ-cI-S stop mRNA in the presence of indicated SmpB and tmRNA hybrids.

Figure 2.

tmRNA ORF alterations do not affect the tagging activity or the steady state levels of RNase R. (A) A representative western blot showing the expression levels of the λ-cI stop (lanes 2–5) and nonstop (lanes 7–10) reporter protein in the presence of indicated SmpB and tmRNA hybrids. The λ-cI reporter protein is tagged in the presence of SmpB–tmRNA hybrids whereas the corresponding stop reporter is untagged. (B) A representative western blot showing similar steady state levels of Escherichia coli RNase R (RNase R^EC) in the presence of indicated SmpB–tmRNA hybrids.

Figure 3.

Ribosomes rescued by hybrid tmRNA exhibit defects in RNase R recruitment. (A) Total ribosomes were obtained from cells co-expressing the indicated SmpB and tmRNA hybrid variants with the λ-cI-S or λ-cI-NS reporter mRNAs. Ribosomes translating the reporter mRNAs were isolated from the total ribosomes pool using Ni²⁺-NTA affinity chromatography. Equal numbers of captured ribosomes were resolved by electrophoresis on 10% SDS-polyacrylamide gels. Western blot analysis was performed to detect the presence of Escherichia coli RNase R (RNase R^EC). The intensity of the band corresponding to RNase R^EC was quantified using ImageJ. The top panel shows a representative western blot, using an RNase R^EC specific antibody, and the bottom panel shows a section of the corresponding Coomassie stained gel displaying equal protein loading. (B) The graph represents fold RNase R^EC enrichment on ribosomes translating the λ-cI-NS reporter compared to ribosomes translating the control λ-cI-S reporter (mean ± SEM). P-values were calculated by performing Student's t-test analysis on the association level of RNase R^EC with ribosomes translating λ-cI-S and λ-cI-NS in the presence of SmpB^FT–tmRNA (**P = 0.0022, n = 5), SmpB–tmRNA^FT (ns, n = 4), or SmpB^FT–tmRNA^FT (*P = 0.01, n = 5). (C) A representative western blot showing SmpB^EC is enriched on ribosomes translating the λ-cI-NS reporter. Ribosomes translating the stop and nonstop reporter mRNAs were isolated from the total ribosomes pool using Ni²⁺-NTA affinity chromatography. Equal numbers of captured ribosomes were resolved by electrophoresis on 10% SDS-polyacrylamide gels. Western blot analysis was performed to detect the presence of E. coli SmpB.

Figure 4.

tmRNA ORF alterations lead to nonstop mRNA accumulation in the presence of Francisella tularensis RNase R. (A) A representative northern blot showing similar steady state abundance of λ-cI-NS nonstop mRNA in the presence of Escherichia coli- and F. tularensis-derived RNase R (RNase R^EC and RNase R^FT), respectively. (B) The top panel depicts a representative northern blot showing the steady state level of λ-cI-NS nonstop mRNA in the presence of the indicated SmpB and tmRNA hybrid variants and RNase R^FT. The level of nonstop mRNA accumulation in the presence of single and double tmRNA ORF hybrids (lanes 3 and 4) is ∼5-fold higher than that of E. coli derived SmpB–tmRNA. P-values were calculated by performing Student's t-test analysis with SmpB–tmRNA and SmpB^FT–tmRNA (P = ns), SmpB–tmRNA and SmpB–tmRNA^FT (*P = 0.007), or SmpB–tmRNA and SmpB^FT–tmRNA^FT (*P = 0.0135). The accompanying graph shows the fold change in the steady state level of λ-cI-NS mRNA in the presence of indicated SmpB and tmRNA variants with respect to WT E. coli SmpB–tmRNA. The data are from four independent experiments (mean ± SEM).

Figure 5.

Escherichia coli RNase R exhibits defects in enrichment on ribosomes rescued by tmRNA^DD. (A) Total ribosomes were obtained from cells expressing the tmRNA^ED or the tmRNA^DD variants with the λ-cI-S or λ-cI-NS reporter mRNAs. Ribosomes translating the reporter mRNAs were isolated from the total ribosomes pool using Ni²⁺-NTA affinity chromatography. Equal numbers of ribosomes were resolved by electrophoresis on 10% SDS-polyacrylamide gels. Western blot analysis was performed to detect the presence of wild type E. coli RNase R. The intensity of the band corresponding to RNase R was quantified using ImageJ. The top panel shows a representative Western blot, using an RNase R specific antibody, and the bottom panel shows a section of the Coomassie stained gel displaying equal protein loading. (B) The graph represents fold RNase R enrichment on ribosomes translating the λ-cI-NS reporter compared to ribosomes translating the control λ-cI-S reporter (mean ± SEM). P-values were calculated by performing Student's t-test analysis on the association level of RNase R with ribosomes translating λ-cI-S and λ-cI-NS mRNAs in the presence of tmRNA^ED (**P = 0.004, n = 3) or tmRNA^DD (**P = 0.0042, n = 4).

Figure 6.

RNase R initiates nonstop mRNA decay after establishment of the tmRNA reading frame. (A) A representative northern blot showing the steady state abundance of λ-cI-NS nonstop mRNA in the presence of SmpB–tmRNA, SmpB–tmRNA^DD, SmpB^DE–tmRNA and SmpB^G132E–tmRNA. The accompanying graph shows the relative steady state abundance of the λ-cI-NS nonstop mRNA in the presence of indicated variants with reference to SmpB–tmRNA. RNase R exhibits severe defect in degrading nonstop mRNA when ribosomes are rescued by SmpB^DE–tmRNA or SmpB^G132E–tmRNA. RNase R exhibits a moderate defect in degrading nonstop mRNA in the presence of SmpB–tmRNA^DD, wherein the reading frame is accurately established. P-values were calculated by performing Student's t-test analysis on the relative steady state abundance of the λ-cI-NS nonstop mRNA in the presence of SmpB–tmRNA and SmpB–tmRNA^DD (**P = 0.0039), SmpB–tmRNA and SmpB^DE–tmRNA (**P = 0.0017) or SmpB–tmRNA and SmpB^G132E–tmRNA (**P = 0.002). The data are from three independent experiments (mean ± SEM). (B) A representative western blot showing similar expression levels of RNase R in the presence of indicated SmpB and tmRNA variants.

Figure 7.

Escherichia coli RNase R exhibits defects in enrichment on ribosomes rescued by SmpB C-terminal tail variants. (A) Total ribosomes were obtained from cells co-expressing SmpB^DE or SmpB^G132E variants with the λ-cI-S or λ-cI-NS reporter mRNAs. Ribosomes translating the reporter mRNAs were isolated from the total ribosomes pool using Ni²⁺-NTA affinity chromatography. Equal numbers of ribosomes were resolved by electrophoresis on 10% SDS-polyacrylamide gels. Western blot analysis was performed to detect the presence of RNase R. The intensity of the band corresponding to RNase R was quantified using ImageJ. The top panel shows a representative western blot, using an RNase R specific antibody, and the bottom panel shows a section of the corresponding Coomassie stained gel displaying equal protein loading. (B) The graph represents fold RNase R enrichment on ribosomes translating the λ-cI-NS reporter compared to ribosomes translating the control λ-cI-S reporter. The data are from three independent experiments (mean ± SEM). P-values were calculated by performing Student's t-test analysis on the association level of RNase R with ribosomes translating λ-cI-S and λ-cI-NS in the presence of SmpB^DE (P = ns) and SmpB^G132E (*P = 0.022).