Alkylative damage of mRNA leads to ribosome stalling and rescue by trans translation in bacteria

Abstract

Similar to DNA replication, translation of the genetic code by the ribosome is hypothesized to be exceptionally sensitive to small chemical changes to its template mRNA. Here we show that the addition of common alkylating agents to growing cultures of Escherichia coli leads to the accumulation of several adducts within RNA, including N(1)-methyladenosine (m¹A). As expected, the introduction of m¹A to model mRNAs was found to reduce the rate of peptide bond formation by three orders of magnitude in a well-defined in vitro system. These observations suggest that alkylative stress is likely to stall translation in vivo and necessitates the activation of ribosome-rescue pathways. Indeed, the addition of alkylation agents was found to robustly activate the transfer-messenger RNA system, even when transcription was inhibited. Our findings suggest that bacteria carefully monitor the chemical integrity of their mRNA and they evolved rescue pathways to cope with its effect on translation.

Keywords: E. coli; RNA damage; alkylation; biochemistry; chemical biology; quality control; ribosome; tmRNA; trans translation.

Figure 1.. Treatment of E. coli with MMS and MNNG results in significant accumulation of alkylative-damage adducts in RNA.

(A–E) Bar graphs showing the amount of the indicated modified nucleotides relative to their unmodified parent in untreated (white bars), MMS-treated (black bars), and MNNG-treated (gray bars) cells. The values plotted are the averages of three biological repeats and the error bars represent standard deviations around the mean. Significant differences in mean, as denoted by p<0.05, were determined by Welch’s t-test.

Figure 1—figure supplement 1.. LC-MS calibration curves for modified and unmodified nucleosides.

(A) The integrated peak area for absorbance at 260 nm for each indicated unmodified nucleotide plotted against its concentration. (B) The integrated peak area for cps intensity plotted against concentrations of modified nucleoside standards. For all plots in A and B, the data were fit to linear-regression lines forced to have zero x- and y-intercepts. Retention times and mass transitions shown in Supplementary file 1, which were determined empirically, were used to identify the peak for each nucleotide.

Figure 2.. N(1)-methyladenosine (m¹A) in mRNA significantly decreases the rate and endpoint of peptide bond formation in vitro.

(A) Chemical structure of m¹A. The N1-methyl group is highlighted in red, and the resonance structure of the molecule is represented. (B) Schematic representation of adenosine and m¹A initiation complexes encoding for the dipeptide Met-Glu. Both complexes contain the initiator fMet-tRNA^fMet in the P site; the A complex displays a GAA codon, while the m¹A complex displays a G^m1AA codon in the A site. (C) Representative time-courses of peptide bond-formation reactions between initiation complexes programmed with unmodified mRNA (blue) or an m¹A-modified one (red) m¹A, and Glu-tRNA^Glu ternary complex.

Figure 3.. Paromomycin does not rescue the effect of m¹A on peptide bond formation.

Representative time-courses of peptide bond-formation reactions between initiation complexes programmed with m¹A mRNA and Glu-tRNA^Glu ternary complexes in the absence (blue) and presence (red) of paromomycin.

Figure 4.. m¹A does not alter the reactivity of ribosomes with near-cognate and non-cognate aa-tRNA.

Phosphorimager scan of electrophoretic TLCs used to follow dipeptide-formation reactions between unmodified and m¹A-modified complexes with all canonical aa-tRNA ternary complexes.

Figure 5.. Alkylative stress activates trans translation in E. coli in a transcription-independent manner.

(A) Western blot analyses of total protein isolated from an E. coli strain expressing tmRNA-His₆. Cells were either untreated or treated with MMS, MNNG, or ciprofloxacin. Additionally, for each condition, cells were either mock pretreated or received a rifampicin pretreatment. Blots were probed with the indicated antibodies. (B) Bar graph showing the relative change in His signal (tmRNA activity) as a result of the addition of each of the indicated compounds. (C) Bar graph used to depict the fold-decrease of His₆ levels upon pre-treatment with rifampicin for each of the indicated treatments. In all cases, the initial His signal was normalized to that corresponding to RF2 levels before it was used to calculate the relative change. Three independent experiments were used to obtain the bar graphs, with the mean values plotted and the error bars representing the standard deviation around the mean.

Figure 5—figure supplement 1.. WT and ∆ssrA E. coli exhibit similar survival phenotypes after treatment with MMS.

(A) Spot assay of WT and ∆ssrA cells either after a mock treatment or treatment with 0.1% MMS for the indicated amount of time. (B) Quantification of colony forming units in (A) performed in triplicates. (C) Spot assay of WT and ∆ssrA cells either after no treatment or treatment with 0.5% MMS for the indicated amount of time. (D) Quantification of colony forming units in (C) performed in at least duplicates. In all cases, the data plotted is the average of three experiments and the error bars represent standard deviations from the mean.

Figure 5—figure supplement 2.. Deletion of ClpXP and Lon proteases results in further accumulation of tmRNA-induced His₆ tagging of peptides upon alkylative stress.

Western blot analysis collected from the indicated E. coli strains grown in the absence or presence of the denoted compounds. Blots were probed with the depicted antibodies.

Figure 5—figure supplement 3.. Different His antibodies display unique banding patterns on western blots.

Western blots of total protein isolated from E. coli expressing tmRNA-His₆ in the absence or presence of MNNG. For each of the two treatments, lysates were serially diluted by two- and fourfold before being resolved by SDS-PAGE next to their undiluted sample. Following the transfer, the blots were probed with one of three different anti-His antibodies (Santa Cruz Biotechnology, Abcam, or GenScript). α-RF2 was used as a loading control.

Figure 5—figure supplement 4.. Ciprofloxacin, but not mitomycin C, increases His₆ tagging by tmRNA.

Western blot analysis of total protein collected from E. coli expressing tmRNA-His₆. Cells were either untreated or treated with ciprofloxacin (Cipro) or mitomycin C (MMC). Additionally, cells either received (+) or did not receive (-) a pre-treatment with rifampicin before treatment with the indicated damaging agent. The blot was probed with the indicated antibodies.

Figure 5—figure supplement 5.. Optimal His₆ tagging and activation of Ada and RecA levels are achieved after 20 min of MMS treatment.

Western blot analysis of total protein collected from E. coli expressing tmRNA-His₆. Cells were either untreated or treated with MMS for the indicated lengths of time. The blot was probed with anti His, Ada, RecA, and RF2 antibodies.

Figure 5—figure supplement 6.. Significant transcriptional runoff is achieved after 10 s of rifampicin treatment.

Western blot analysis used to follow the effect of rifampicin pretreatment on ciprofloxacin (Cipro)- (A) and MMS-induced (B) activation of His tagging by tmRNA. The time on top of the blots indicate the amount of time cells were incubated in the presence of rifampicin before ciprofloxacin and MMS were added. The blots were probed with the depicted antibodies.

Figure 6.. Translation of MMS-treated mRNA results in tmRNA tagging in an S30 extract.

(A) Schematic of the mRNA used in the in vitro translation assays. (B) Fluorescence image of EtBr-stained gel was used to visualize the mock-treated and MMS-treated RNAs. (C) Top shows a phosphor-imager scan of a PVDF-transfer membrane of a bis-tricine gel that was used to separate products from the in-vitro-translation reactions containing the indicated mRNAs. Bottom is western-blotting analyses of the same membranes with the indicated antibodies. Shown is a representative of two assays.

Figure 6—figure supplement 1.. Deletion of ssrA gene results in stabilization of m¹A-modified mRNA.

(A) Top: fluorescence image of a denaturing agarose gel used to separate total RNA isolated from the indicated cells under the denoted conditions. Bottom: immunoblot analysis of the same samples using m⁶A antibody. (B) m⁶A-immunoblot analysis of the indicated RNA samples. Top blot shows analysis of samples that were transferred directly following electrophoresis. Bottom blot shows analysis of the same samples except that the agarose gel was soaked in an alkaline buffer (50 mM NaOH, 1.5 M NaCl) before transfer.

Figure 7.. Ribosome rescue by tmRNA is important for cellular recovery after treatment with alkylating agents.

(A–C) Growth curves, as measured by change in OD_600nm as a function of time, for the indicated cells following a treatment with the denoted compound. Average of three replicate growth assays is plotted.