The RNA degradosome promotes tRNA quality control through clearance of hypomodified tRNA

Abstract

The factors and mechanisms that govern tRNA stability in bacteria are not well understood. Here, we investigated the influence of posttranscriptional modification of bacterial tRNAs (tRNA modification) on tRNA stability. We focused on ThiI-generated 4-thiouridine (s⁴U), a modification found in bacterial and archaeal tRNAs. Comprehensive quantification of Vibrio cholerae tRNAs revealed that the abundance of some tRNAs is decreased in a ΔthiI strain in a stationary phase-specific manner. Multiple mechanisms, including rapid degradation of a subset of hypomodified tRNAs, account for the reduced abundance of tRNAs in the absence of thiI Through transposon insertion sequencing, we identified additional tRNA modifications that promote tRNA stability and bacterial viability. Genetic analysis of suppressor mutants as well as biochemical analyses revealed that rapid degradation of hypomodified tRNA is mediated by the RNA degradosome. Elongation factor Tu seems to compete with the RNA degradosome, protecting aminoacyl tRNAs from decay. Together, our observations describe a previously unrecognized bacterial tRNA quality control system in which hypomodification sensitizes tRNAs to decay mediated by the RNA degradosome.

Keywords: RNA degradosome; Vibrio cholerae; stationary phase; tRNA modification; thiI.

Fig. 1.

Some tRNA species lacking s⁴U exhibit stationary phase-specific tRNA decay. A, Upper shows a polyacryamide gel of total RNA from WT and ΔthiI strains stained with SYBR Gold. A, Lower shows Northern blots probed for the indicated tRNA species. Log-phase cells were harvested at OD₆₀₀ = 0.5, and stationary-phase cells were harvested 24 h after inoculation. (B) Quantification of the indicated tRNAs from Northern blots in A and SI Appendix, Fig. S2. tRNA species that exhibited a greater than twofold change and a significant difference (P < 0.05 in t test) between WT and ΔthiI samples in stationary phase are colored in red. Bars show the mean values from three independent experiments, with error bars representing SD (calculated from SD of the WT and ΔthiI with error propagation). The list of tRNA sequences with names is shown in Dataset S3. (C) Amount of s⁴U detected (in 10 pmol) of each tRNA species based on HPLC analysis. Log-phase tRNAs were isolated from cultures with OD₆₀₀ = 0.4, and stationary-phase tRNAs were isolated from cultures 24 h after inoculation. Each bar represents the mean value of three independent measurements, with error bars representing SD. *s⁴U was not detected in tRNA-Ile1 from log-phase RNA; **s⁴U was not analyzed in stationary phase. (D) The relative levels of tRNA-Tyr, tRNA-Ser1, tRNA-Cys1, and tRNA-Ile1 over time based on Northern blotting. The signal at each time point is presented relative to its intensity in cultures at OD₆₀₀ = 0.5 (time 0) after normalization for RNA loading based on 5S rRNA. WT and ΔthiI curves are colored in blue and red, respectively. (E) Decay curves of tRNAs in log-phase cultures. Rifampicin was added at OD₆₀₀ = 0.3 followed by sampling for 1 h. Each point represents the mean value of three independent measurements, with error bars representing SD. For each strain background, RNA abundance at each time point is shown relative to abundance in that strain at t = 0. (F) Decay curves of tRNAs in early stationary-phase cultures as shown in E. Rifampicin was added 1 h after cultures reached OD₆₀₀ = 0.5 (∼OD₆₀₀ = 1.0) followed by sampling for 1 h.

Fig. 2.

TIS analyses reveal genetic interactions between thiI and other genes that mediate tRNA modifications. (A) Volcano plot of the results from Con-ARTIST analysis comparing WT and ΔthiI Tn libraries (Dataset S1). (B) Artemis plots showing normalized frequencies of Tn insertions in miaA, trmA, and truB. Insertion frequencies per locus are depicted as vertical lines for the WT (Upper) and ΔthiI (Lower) libraries. (C) Schematic secondary structure of tRNA with the chemical structures of the modified nucleosides that are synthesized by the genes identified in the TIS screen. The acceptor stem with 3′ terminus, D arm, anticodon arm, variable loop, T arm, and anticodon are colored in blue, red, light blue, green, yellow, and orange respectively. The names of the modifying enzymes are also shown. (D) Tertiary structure of tRNA-Tyr from T. thermophilus (49) (Protein Data Bank ID code 1H3E). The modified sites identified in the TIS screen are shown. The acceptor stem, D arm, anticodon arm, variable loop, and T arm are colored in blue, red, light blue, green, and yellow, respectively. The elbow and core regions of the tRNA are highlighted in light orange. (E) Growth of double-mutant strains on LB plates with (Right) or without (Left) 0.2% arabinose at 37 °C (Upper) and 25 °C (Lower) for 24 h.

Fig. 3.

Mutants lacking multiple tRNA modification genes exhibit tRNA decay mediated by the RNA degradosome. (A, Left) Colony morphology of the original Para-thiI/ΔtruB strain and suppressor strains (sup#1 and sup#2) on the same plate. A, Right shows the doubling time in LB medium at 37 °C of the indicated strains. (B) Predicted secondary structure of tRNA-Tyr with the mutation that is found in sup#1. Modifications are predicted based on those previously observed in E. coli. (C) Schematic depiction of the RNase E domain structure with the site of the RNase E Glu664STOP (sup#2) mutation annotated. (D) Relative abundance of tRNA-Tyr in log-phase (OD₆₀₀ = 0.5) cultures of the indicated strains grown in LB at 37 °C without arabinose. (E) Growth on LB plates with or without 0.2% arabinose of serially diluted double mutants containing either a multicopy vector expressing tRNA-Tyr or an empty vector. Plates were incubated at 37 °C for 24 h. (F) Decay curves of tRNA-Tyr in early log-phase (OD₆₀₀ = 0.3) cultures of the indicated strains grown in LB medium without arabinose. (G) Growth as in E of serially diluted strains producing either WT RNase E (rne) or RNase E lacking its CTD (rneΔCTD).

Fig. 4.

RNA degradosome-mediated tRNA decay in stationary phase in a ΔthiI mutant. Decay curves of tRNA-Tyr and tRNA-Ser1 in early stationary phase (1 h after OD₆₀₀ = 0.5) in the indicated strains.

Fig. 5.

RNA degradosomes mediate tRNA decay in vitro. (A) Coomasie-stained gel image of Flag-RNase E immunoprecipitated fraction. Indicated bands were identified through peptide mass spectrometry. (B) In vitro tRNA-Tyr decay assay. Time course of tRNA-Tyr resolved in 10% TBE-UREA gel stained with SYBR Gold at the indicated time. Upper and Lower show reactions with degradosomes from the WT and from the PNPase catalytic mutant, respectively. (C) Decay curves of tRNA-Tyr from WT and ΔthiI mutant strains based on data shown in B.

Fig. 6.

EF-Tu protects aminoacyl-tRNAs from RNA degradosome-mediated decay. (A) Aminoacylation levels of tRNA-Tyr in WT and ΔthiI strains. Total RNAs from early stationary phase (1 h after OD₆₀₀) were resolved on 6.5% acid gels and analyzed by Northern blotting. Means ± SD of aminoacylation level from three independent experiments are shown. Red and blue arrowheads indicate aminoacylated and deacylated tRNAs, respectively. (B and C) Decay of aminoacylated and deacylated tRNAs in early stationary-phase cultures after rifampicin treatment. Total RNAs were resolved on 6.5% acid gels and analyzed by Northern blotting with or without chloramphenicol (Cm) (B); decay curves of the lower band (deacylated tRNA), upper band (aminoacylated tRNA), and total tRNA-Tyr (calculated from neutral PAGE Northern blotting analysis) are shown in C. Red and blue arrowheads in B indicate aminoacylated and deacylated tRNAs, respectively. (D) Decay curves of tRNA-Tyr and tRNA-Ser1 from the ΔthiI strain containing a vector encoding arabinose-inducible EF-Tu (pEF-Tu) or an empty vector (pBAD) with or without 0.2% arabinose.