Deves, V. | bioRxiv Science

bioRxiv2025

The endoribonuclease Rae1 from Bacillus subtilis cleaves mRNA upstream of stalled ribosomes

The ribosome-associated endoribonuclease 1 (Rae1) cleaves mRNAs in a translation-dependent manner. Here, we identify a new Rae1 target, the fliY mRNA, which is cleaved by Rae1 in the absence of the elongation factor P (EF-P). The Rae1 site was mapped 12 nucleotides upstream of the second proline codon of an SPP stalling motif in fliY. Remarkably, Rae1 cleavages also occur 12 nucleotides upstream of the stop codon within two validated Rae1 mRNA targets, bmrX and spyA (S1025). Shifting the stop codon relative to the Rae1 cutting site abolished Rae1 sensitivity of bmrX and spyA mRNAs. We show that ribosome pausing occurs at the spyA stop codon, confirming its crucial role, and positioning the Rae1 cleavage at the tail end of the stalled ribosome, rather than in the A-site as previously proposed. These findings reveal a compelling novel mechanism by which Rae1 mediates mRNA cleavage in coordination with immobile ribosomes.

molecular biology↗

bioRxiv2023

Identification of a new substrate for the ribosome associated endoribonuclease Rae1 reveals a link to the B. subtilis response and sensitivity to chloramphenicol

Rae1 is a well-conserved endoribonuclease among Gram-positive bacteria, cyanobacteria and the chloroplasts of higher plants. We have previously shown that Rae1 cleaves the Bacillus subtilis yrzI operon mRNA in a translation-dependent manner, within a short open reading frame (ORF) called S1025, encoding a 17-amino acid (aa) peptide of unknown function. Here, we map a new Rae1 cleavage site in the bmrBCD operon mRNA encoding a multidrug transporter, within a previously unannotated 26-aa short ORF that we have named bmrX. Similar to S1025, Rae1 cleavage within bmrX is both translation- and reading frame-dependent. Both mRNAs were previously shown to be induced by the presence of the protein synthesis inhibitor chloramphenicol (Cm). Strikingly, a rae1 deletion strain shows greater resistance to Cm than the wild-type strain, while its over-expression leads to increased Cm sensitivity, suggesting a link to translation quality control. Consistent with this, we show that cleavage by Rae1 promotes ribosome rescue by the tmRNA. Globally, our data point to a role of Rae1 in mRNA surveillance by eliminating mRNAs that encounter problems with translation.

microbiology↗