Abstract Ribosome-associated quality control (RQC) is the process by which cells deal with prolonged translational stalls caused by genetic errors, insufficient charged tRNA, or faulty mRNA which results in ribosome collisions and potentially cytotoxic, incomplete, translation products. Ribosomes are split into a free 40S and 60S subunit with an incomplete nascent chain (arrest product or AP) as a peptidyl tRNA adduct which obstructs the exit tunnel and P-site of the 60S. In the cytosol, an intricate process has been defined for how cells remove the AP, recycle the 60S subunit, and degrade the AP. However, corresponding research into how this process is managed for proteins produced at the endoplasmic reticulum (ER) membrane is lacking. The key difference between cytosolic and ER-RQC is that at the ER membrane the AP is integrated into the SEC61 translocon and thus separated from the cytosolic RQC and degradative machinery. How cells manage this topological restraint is the focus of this proposal. The Kopito lab and others have shown that ER localized ribosome collisions result in the conjugation of the small ubiquitin-like protein UFM1 to ribosomal protein L26 (RPL26) on the 60S subunit (UFMylation). UFM1 knockout cells (UFM1KO) prevent proteasomal degradation of ER localized, but not cytosolic, APs suggesting a link between UFMylation and AP degradation in ER-RQC. I hypothesize that UFMylation of RPL26 severs as mark to recruit proteins to stalled ER ribosomes necessary for resolving the 60S-AP- translcoon complex so that the AP may be degraded. In Aim 1 and Aim 2 of this proposal I will identify the readers of UFMylation using two complimentary approaches: proximity labeling via fusion of a biotin ligase to UFM1 and affinity purification of UFM1 binding proteins. These experiments are designed such that I will identify proteins that interact with conjugated UFM1 and not free UFM1. Preliminary results show that ER localized, translocon associated proteins, are enriched in proximity labeling experiments which supports the model that UFMylation occurs at the ER membrane near the translocon. Preliminary affinity purification experiments reveal that known cytosolic RQC proteins are enriched on UFMylated ribosomes supporting the role of UFMylation in ER-RQC. In Aim 3 of this proposal, I will define the mechanism by which UFMylation leads to AP degradation. I will first evaluate the role of UFMylation readers identified in Aims 1 and 2 in AP stability via emetine chase and metabolic pulse/chase experiments. I will also test if UFMylation facilitates release of the AP from the ribosome or translocon. The UFMylation pathway is conserved amongst most eukaryotic species and is essential for hematopoietic and neurological development in metazoan cells. Mutations in this pathway have been linked to a wide variety of human diseases. Despite its importance, we do not fully understand what this pathway is doing. In this proposal, I will uncover the readers of...