The coordinated action of UFMylation and the RQC pathways clears arrested polypeptides at the ER.

Clearance of arrested nascent polypeptides resulting from ribosomal stalling is essential for proteostasis. Stalled endoplasmic reticulum (ER)-bound ribosomes are marked by ubiquitin-fold modifier 1 (UFM1) on the large ribosomal subunit protein RPL26, but the precise role of this modification in ribosome-associated quality control (RQC) remains poorly understood. Here, we define the interplay between the UFMylation machinery and the RQC in clearing arrested polypeptides upon ribosome stalling at the ER. Proteomic analysis shows that RQC factors associate with UFMylated ribosomes. Functional assays demonstrate that ribosome rescue factors ZNF598 and ASC-1 recognize and split stalled ribosomes at the ER, a prerequisite for RPL26 UFMylation. The UFM1 E3 ligase complex then binds and UFMylates the post-split 60S-peptidyl-tRNA complex, facilitating access of RQC factors. Depletion of the NEMF/LTN1 complex leads to accumulation of UFMylated ribosomes, whereas impaired UFMylation weakens NEMF/LTN1 binding to ER-stalled ribosomes, supporting a physical link between these pathways. These findings demonstrate that RQC cooperates with the UFMylation machinery to overcome the topological constraints of clearing the arrested polypeptides at the ER.