PROJECT SUMMARY Synthesis of the correctly folded and functional proteins is crucial for maintaining the healthy state of the cell. Eukaryotic cells have evolved a variety of quality control (QC) mechanisms that recognize and eliminate abnormal polypeptides at different stages of their lifespan. One of such surveillance mechanisms, the co-translational protein QC, targets stalled ribosomes containing aberrant nascent polypeptides and promotes the release of the 60S subunits with a nascent chain attached. The aberrant polypeptides then undergo ubiquitination by the E3 ubiquitin ligase Ltn1 followed by proteasomal degradation facilitated by the ATPase Cdc48. Although the Ltn1-Cdc48-dependent segment of this QC pathway has been recently investigated, the identity of factors acting upstream in this pathway remains uncertain. We have recently discovered that the canonical release factors eRF1-eRF3 (eRFs) are responsible for generating most of the 60S-associated complexes containing nascent polypeptides that are eliminated through the Ltn1-Cdc48 pathway. Thus, eRFs accomplish two novel functions during co- translational protein QC: they initiate clearance of aberrant polypeptides and resolve translational stalls, thereby allowing translation to continue. We also found that in situations when protein QC malfunctions or is overwhelmed, accumulation of unresolved translational stalls triggers a specific endonucleolytic cleavage in the 25S rRNA of the large ribosomal subunit. Our central hypothesis is that eRF-mediated ribosome recycling and cleavage of stalled ribosomes constitute two key parts of the eukaryotic Translation REscue Mechanism (TREM), devoted to protecting the translational apparatus from terminal ribosomal stalling and preventing the escape of aberrant polypeptides. In this way, TREM likely plays a fundamental role in the defense against translational and proteotoxic stress. The broad objective of the current proposal is to obtain insight into the molecular basis of eukaryotic TREM using biochemical, genetic and molecular biology approaches in the yeast Saccharomyces cerevisiae. In this study, we will (1) define clients of the eRF-mediated translational rescue pathway; (2) elucidate the mechanistic function of eRF1 during translational rescue; and (3) evaluate the potential role of rRNA cleavage in the recovery from translational stalls. In addition to expanding our knowledge of the mechanisms of eRF1-eRF3 functioning during co- translational protein QC, this study will shed light on a crucial system that protects eukaryotic cells during proteotoxic stress. Because a number of pathological consequences are known to result from accumulation of harmful protein aggregates and defective translation when protein QC is dysfunctional or overloaded, this study may open previously unexplored avenues for therapeutic intervention.