PROJECT SUMMARY/ ABSTRACT Post-transcriptional regulation of gene expression is fundamental to normal cellular homeostasis. The current model in the field of biology is that mRNAs exist primarily as ‘full-length’ molecules that are ‘protected’ from decay by ribosomes during translation. The long-established understanding is that mRNA decay is initiated by deadenylation followed by decapping and subsequent exonucleolytic decay from both ends. Co-translational mRNA decay is known to occur mainly in defective mRNAs. Existing next-generation sequencing methods that profile mRNA decay target either the 5’ or 3’ ends, thus missing information about the other end that could illuminate new insights into mRNA decay. During my post-doctoral training, I developed several novel transcriptome-wide sequencing methods that concurrently select both ends of mRNAs. I discovered a novel mechanism of co-translational decay of canonical mRNAs that involves repeated endonucleolytic cleavage events, mediated by translating ribosomes that we named “ribothrypsis”. Ribothrypsis is conserved between humans and yeast. We identified the unexpected ribosome-phased mRNAs fragmentation and found that deadenylation is not a prerequisite for mRNA decay. Our discovery of ribothrypsis revealed that co-translational mRNA decay is more widespread than previously thought. The central hypothesis of this proposal is that ribothrypsis is an evolutionarily conserved mechanism for modulating gene expression that can be triggered by numerous factors to recruit an unknown endonuclease that we termed “ribothrypsin”. We propose here to capitalize on our past discoveries and leverage cutting-edge novel RNA sequencing methods to achieve a comprehensive understanding of the mechanistic underpinnings of ribothrypsis and its regulation. In this proposal, we will investigate (i) the impact of cellular conditions that induce ribosome stalling on RNA decay intermediates; (ii) the conservation of ribothrypsis in other eukaryotes; (iii) the identity of ribothrypsin; and (iv) the role of RNA modifications in triggering ribothrypsis. These goals are mirrored by our long-term objective to understand the mechanisms that underlie RNA decay dysregulation in human diseases. The molecular insights gained in this proposal could also broaden our understanding of ribosome biology and RNA modifications.