EF-P-resolved ribosome stalling and collision trigger Rae1-mediated mRNA decay
Ribosome stalling compromises proteome integrity and cellular fitness, and requires efficient mechanisms to eliminate problematic mRNAs and rescue blocked ribosomes. We previously identified Rae1 as an endoribonuclease that cleaves mRNAs at the tail end of stalled ribosomes to promote mRNA degradation in Bacillus subtilis. Here, we show that Rae1 has a broader role in the quality control of mRNAs that encounter translation problems. Global 5-end sequencing identified 31 previously unknown Rae1 cleavage sites, many of them associated with ribosome pausing at polyproline motifs in the absence of elongation factor P (EF-P). We also demonstrate that Rae1 cleaves mRNAs with ribosomes stalled on truncated transcripts lacking a stop-codon (non-stop mRNAs), provided that the transcript is sufficiently long to accommodate multiple collided ribosomes. Rae1 cleavage depends on a cytosine-poor context at the cut site, showing that both ribosome collisions and RNA sequence at the cleavage site govern the ability of Rae1 to promote the removal of stalled ribosomes from mRNA.