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Ribosome collisions induce mRNA cleavage and ribosome rescue in bacteria.

ABSTRACT: Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation^1,2. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes^3-6. Here, using a genetic screen in Escherichia coli, we discovered a new rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNAs³) to rescue upstream ribosomes. SmrB is recruited to ribosomes and is activated by collisions. Cryo-electron microscopy structures of collided disomes from E. coli and Bacillus subtilis show distinct and conserved arrangements of individual ribosomes and the composite SmrB-binding site. These findings reveal the underlying mechanisms by which ribosome collisions trigger ribosome rescue in bacteria.

SUBMITTER: Saito K

PROVIDER: S-EPMC8934283 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Ribosome collisions induce mRNA cleavage and ribosome rescue in bacteria.

Saito Kazuki K Kratzat Hanna H Campbell Annabelle A Buschauer Robert R Burroughs A Maxwell AM Berninghausen Otto O Aravind L L Green Rachel R Beckmann Roland R Buskirk Allen R AR

Nature 20220309 7901

Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation1,2. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes3-6. Here, using a genetic screen in Escherichia coli, we discovered a new rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmR ...[more]

PMID: 35264790

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Ribosome collisions in bacteria promote ribosome rescue by triggering mRNA cleavage by SmrB

Project description:Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes. In a genetic screen in E. coli, we discovered a novel rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNA) to rescue upstream ribosomes. SmrB is recruited by ribosome collisions. Cryo-EM structures of collided disomes from E. coli and B. subtilis reveal interactions between the 30S subunits and a possible SmrB binding site. These findings show that ribosome collisions trigger ribosome rescue in bacteria and reveal the mechanism by which this occurs.

2022-08-01 | GSE179691 | GEO

Ribosome collisions in bacteria promote ribosome rescue by triggering mRNA cleavage by SmrB

Project description:Ribosome collisions in bacteria promote ribosome rescue by triggering mRNA cleavage by SmrB

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Ribosome Rescue Pathways in Bacteria.

Project description:Ribosomes that become stalled on truncated or damaged mRNAs during protein synthesis must be rescued for the cell to survive. Bacteria have evolved a diverse array of rescue pathways to remove the stalled ribosomes from the aberrant mRNA and return them to the free pool of actively translating ribosomes. In addition, some of these pathways target the damaged mRNA and the incomplete nascent polypeptide chain for degradation. This review highlights the recent developments in our mechanistic understanding of bacterial ribosomal rescue systems, including drop-off, trans-translation mediated by transfer-messenger RNA and small protein B, ribosome rescue by the alternative rescue factors ArfA and ArfB, as well as Bacillus ribosome rescue factor A, an additional rescue system found in some Gram-positive bacteria, such as Bacillus subtilis. Finally, we discuss the recent findings of ribosome-associated quality control in particular bacterial lineages mediated by RqcH and RqcP. The importance of rescue pathways for bacterial survival suggests they may represent novel targets for the development of new antimicrobial agents against multi-drug resistant pathogenic bacteria.

| S-EPMC8012679 | biostudies-literature

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

Project description: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.

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Translation of poly(A) tails leads to precise mRNA cleavage and widespread ribosome rescue

Project description:Translation of poly(A) tails leads to mRNA cleavage but the mechanism and global pervasiveness of this “nonstop/no-go” decay process is not understood. Here we performed ribosome profiling of short 15-18 nt mRNA footprints to identify ribosomes stalled at 3’ ends of mRNA decay intermediates. We found mRNA cleavage extending hundreds of nucleotides upstream of ribosome stalling in poly(A) and predominantly in one reading frame. These observations suggest that cleavage is closely associated with the ribosome. Surprisingly, ribosomes appeared to stall when as few as 3 consecutive ORF-internal lysine codons were positioned in the A, P, and E sites though significant mRNA degradation was not observed. Endonucleolytic cleavage was widespread, however, at sites of premature polyadenylation and rescue of the ribosomes stalled at these sites was dependent on Dom34. These results suggest this process may be critical when changes in polyadenylation occur during development, tumorigenesis, or when translation termination/recycling is impaired.

2017-01-22 | GSE85944 | GEO

Kinetic modeling predicts a stimulatory role for ribosome collisions at elongation stall sites in bacteria.

Project description:Ribosome stalling on mRNAs can decrease protein expression. To decipher ribosome kinetics at stall sites, we induced ribosome stalling at specific codons by starving the bacterium Escherichia coli for the cognate amino acid. We measured protein synthesis rates from a reporter library of over 100 variants that encoded systematic perturbations of translation initiation rate, the number of stall sites, and the distance between stall sites. Our measurements are quantitatively inconsistent with two widely-used kinetic models for stalled ribosomes: ribosome traffic jams that block initiation, and abortive (premature) termination of stalled ribosomes. Rather, our measurements support a model in which collision with a trailing ribosome causes abortive termination of the stalled ribosome. In our computational analysis, ribosome collisions selectively stimulate abortive termination without fine-tuning of kinetic rate parameters at ribosome stall sites. We propose that ribosome collisions serve as a robust timer for translational quality control pathways to recognize stalled ribosomes.

| S-EPMC5446239 | biostudies-literature

Mechanism of ribosome rescue by alternative ribosome-rescue factor B.

Project description:Alternative ribosome-rescue factor B (ArfB) rescues ribosomes stalled on non-stop mRNAs by releasing the nascent polypeptide from the peptidyl-tRNA. By rapid kinetics we show that ArfB selects ribosomes stalled on short truncated mRNAs, rather than on longer mRNAs mimicking pausing on rare codon clusters. In combination with cryo-electron microscopy we dissect the multistep rescue pathway of ArfB, which first binds to ribosomes very rapidly regardless of the mRNA length. The selectivity for shorter mRNAs arises from the subsequent slow engagement step, as it requires longer mRNA to shift to enable ArfB binding. Engagement results in specific interactions of the ArfB C-terminal domain with the mRNA entry channel, which activates peptidyl-tRNA hydrolysis by the N-terminal domain. These data reveal how protein dynamics translate into specificity of substrate recognition and provide insights into the action of a putative rescue factor in mitochondria.

| S-EPMC7427801 | biostudies-literature

A comprehensive coverage insurance for cells: revealing links between ribosome collisions, stress responses and mRNA surveillance.

Project description:Cells of metazoans respond to internal and external stressors by activating stress response pathways that aim for re-establishing cellular homoeostasis or, if this cannot be achieved, triggering programmed cell death. Problems during translation, arising from defective mRNAs, tRNAs, ribosomes or protein misfolding, can activate stress response pathways as well as mRNA surveillance and ribosome quality control programs. Recently, ribosome collisions have emerged as a central signal for translational stress and shown to elicit different stress responses. Here, we review our current knowledge about the intricate mutual connections between ribosome collisions, stress response pathways and mRNA surveillance. A central factor connecting the sensing of collided ribosomes with degradation of the nascent polypeptides, dissociation of the stalled ribosomes and degradation of the mRNA by no-go or non-stop decay is the E3-ligase ZNF598. We tested whether ZNF598 also plays a role in nonsense-mediated mRNA decay (NMD) but found that it is dispensable for this translation termination-associated mRNA surveillance pathway, which in combination with other recent data argues against stable ribosome stalling at termination codons being the NMD-triggering signal.

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A ribosome profiling study of mRNA cleavage by the endonuclease RelE.

Project description:Implicated in persistence and stress response pathways in bacteria, RelE shuts down protein synthesis by cleaving mRNA within the ribosomal A site. Structural and biochemical studies have shown that RelE cuts with some sequence specificity, which we further characterize here, and that it shows no activity outside the context of the ribosome. We obtained a global view of the effect of RelE on translation by ribosome profiling, observing that ribosomes accumulate on the 5'-end of genes through dynamic cycles of mRNA cleavage, ribosome rescue and initiation. Moreover, the addition of purified RelE to cell lysates shows promise as a method for generating ribosome footprints. In bacteria, profiling studies have suffered from relatively low resolution and have yielded no information on reading frame due to problems inherent to MNase digestion, the method used to degrade unprotected regions of mRNA. In contrast, we find that RelE yields precise 3'-ends that for the first time reveal reading frame in bacteria. Given that RelE has been shown to function in all three domains of life, RelE has potential to improve reading frame and shed light on A-site occupancy in ribosome profiling experiments more broadly.

| S-EPMC5224514 | biostudies-literature

EDF1 coordinates cellular responses to ribosome collisions.

Project description:Translation of aberrant mRNAs induces ribosomal collisions, thereby triggering pathways for mRNA and nascent peptide degradation and ribosomal rescue. Here we use sucrose gradient fractionation combined with quantitative proteomics to systematically identify proteins associated with collided ribosomes. This approach identified Endothelial differentiation-related factor 1 (EDF1) as a novel protein recruited to collided ribosomes during translational distress. Cryo-electron microscopic analyses of EDF1 and its yeast homolog Mbf1 revealed a conserved 40S ribosomal subunit binding site at the mRNA entry channel near the collision interface. EDF1 recruits the translational repressors GIGYF2 and EIF4E2 to collided ribosomes to initiate a negative-feedback loop that prevents new ribosomes from translating defective mRNAs. Further, EDF1 regulates an immediate-early transcriptional response to ribosomal collisions. Our results uncover mechanisms through which EDF1 coordinates multiple responses of the ribosome-mediated quality control pathway and provide novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation.

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