Translation dependent mRNA decay by Rae1 in Bacillus, Arabidopsis and Staphylococcus – BAS-Rae1

The BASRae1 project focuses on the Ribosome associated endoribonuclease Rae1 that we have recently shown cleaves mRNA in a translation and reading frame dependent manner in Bacillus subtilis. Rae1 is well-conserved in the Firmicutes, Cyanobacteria and in plants, where it localizes to the chloroplast. The crystal structure of Rae1 fits snugly into the A-site of the B. subtilis ribosome. We postulate that ribosome stalling allows Rae1 to enter the A-site to cleave mRNAs in a quality control mechanism. However, other sites of action are also possible, as long as the mRNA can be seen in frame by Rae1. Remarkably, a ?rae1 strain shows increased resistance to chloramphenicol compared to the wild-type strain, coherent with a link between Rae1 and ribosome stalling. Three confirmed targets of Rae1 appear to each use different stall/cleavage mechanisms; thus, the precise mode of Rae1 recruitment remains to be deciphered. The Rae1 homolog of Staphylococcus aureus can substitute for the enzyme in B. subtilis and potential Rae1 targets have been identified in S. aureus. In Arabidopsis, the levels of several mRNAs were also found to be governed by Rae1. Among them, the clpp1 mRNA contains a sequence motif that resembles the cleavage site of the best-characterised BsRae1 target S1025, suggesting some mechanistic conservation in plants. The objectives of the BASRae1 project are to determine (i) the key features of ribosome stall signals and sequence determinants around the cleavage site required for efficient mRNA cutting by Rae1, (ii) how Rae1 interacts with the ribosome (iii) whether physiological conditions that provoke translational stress accentuate the need for Rae1. This project will allow us to decipher the precise molecular mechanism of Rae1 recruitment to the ribosome, leading to ribosome rescue and the destruction of the non-functional mRNA in two Gram-positive models, and ask whether these properties are conserved from prokaryotes to eukaryotes.