A ribosome profiling study of mRNA cleavage by the endonuclease RelE

Abstract

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.

Toxins that Trash Translation

“…Ribosomes are depleted on the 3’-fragment as they terminate normally and are not replaced (because the start codon has been lost); conversely, ribosomes are enriched on the 5’-fragment because initiation continues but ribosomes that reach the 3’-end are trapped (because the stop codon has been lost) (Figure 1C). We observed a similar polarity in ribosome density upon overexpression of another toxin, RelE (8). It seems certain that the machinery that rescues stalled ribosomes (tmRNA, ArfA, and ArfB) must play a critical role in the resumption of protein synthesis upon recovery from stress.…”

The RNA ‐binding protein Hfq is important for ribosome biogenesis and affects translation fidelity

“…From one aliquot, total RNA was extracted using TRI Reagent (Sigma-Aldrich), enriched by depleting small RNAs with GeneJET Purification Kit (Fermentas) and rRNA with MICROBExpress Bacterial mRNA Enrichment Kit (Ambion), and fragmented in alkaline solution (2 mM EDTA and 100 mM Na 2 CO 3 pH 9.2 for 40 min at 95°C) to fragments with size of 24-35 nts. Furthermore, to determine the reproducibility of our sequencing data sets, we used published data set serving as a truly independent biological replicate in which bacteria were grown under identical conditions (GEO accession number, GSE85540; Hwang & Buskirk, 2017). Cells were collected by filtration and flash-frozen without preincubation with antibiotics.…”

“…The same detection threshold was used for the corresponding ribosome profiling experiment. Furthermore, to determine the reproducibility of our sequencing data sets, we used published data set serving as a truly independent biological replicate in which bacteria were grown under identical conditions (GEO accession number, GSE85540; Hwang & Buskirk, 2017). The reproducibility is very high, R 2 = 0.865 and R 2 = 0.816 (Spearman correlation coefficient) for the RNA-Seq and ribosome profiling data sets, respectively.…”

Improving Bacterial Ribosome Profiling Data Quality

“…An approach for increased precision uses RelE in addition to MNase. RelE only cleaves the mRNA in the A-site of the ribosome if activated in a stress condition, and normally cleaves between the 2 nd and 3 rd base of a codon [31][32][33]. Therefore RelE is suitable for reading frame determination [31].…”

“…RelE only cleaves the mRNA in the A-site of the ribosome if activated in a stress condition, and normally cleaves between the 2 nd and 3 rd base of a codon [31][32][33]. Therefore RelE is suitable for reading frame determination [31]. Since RelE cleaves the footprint within the ribosomes, in addition to MNase cleaving the unprotected mRNA outside the ribosomes [34,35], the resulting fragments are even shorter, which can be harder to map accurately [31].…”