UFMylation orchestrates spatiotemporal coordination of RQC at the ER

Abstract

Degradation of arrest peptides from endoplasmic reticulum (ER) translocon-bound 60S ribosomal subunits via the ribosome-associated quality control (ER-RQC) pathway requires covalent modification of RPL26/uL24 on 60S ribosomal subunits with UFM1. However, the underlying mechanism that coordinates the UFMylation and RQC pathways remains elusive. Structural analysis of ER-RQC intermediates revealed concomitant binding and direct interaction of the UFMylation and RQC machineries on the 60S. In the presence of an arrested peptidyl-transfer RNA, the RQC factor NEMF and the UFM1 E3 ligase (E3^UFM1) form a direct interaction via the UFL1 subunit of E3^UFM1, and UFL1 adopts a conformation distinct from that previously observed for posttermination 60S. While this concomitant binding occurs on translocon-bound 60S, LTN1 recruitment and arrest peptide degradation require UFMylation-dependent 60S dissociation from the translocon. These data reveal a mechanism by which the UFMylation cycle orchestrates ER-RQC.

Fig. 1.. Cryo-EM structure of the E3^UFM1-RQC-60S complex.

(A) Coomassie-stained SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel of a 3xFLAG-UFL1 pulldown from cells challenged with ANM. (B to D) Molecular model of the E3^UFM1-RQC-60S (RQC state 3) complex, shown as a side view on the intersubunit space (B), a top view (C), and a bottom view on the peptide tunnel exit (D). (E to G) Comparison of the E3^UFM1-RQC-60S complex with the posttermination E3^UFM1-60S complex (22) and RQC-60S complex (RQC state 4) (E) view of the tunnel exit region, (F) comparison of the E3^UFM1-RQC-60S structure (RQC state 3) with the RQC-60S class (RQC state 4, no E3^UFM1 bound) obtained from this study (see also Fig. 4D and figs. S1 and S2), and (G) view focusing on the overall position of the E3^UFM1. Molecular models in (E) to (G) are rendered as surfaces for the 60S and as ribbons for nonribosomal ligands. TE, tunnel exit; EBH, exit binding helix of DDRGK1; CTD, UFL1 C-terminal domain; WH, UFL1 winged helix domain; PT, posttermination.

Fig. 2.. Conformational change of the UFL1 C terminus and its interaction with NEMF.

(A and B) Molecular models of the E3^UFM1-RQC-60S complex (RQC state 3) (A) and E3^UFM1-60S complex (posttermination state) (22) (B). View focuses on the tRNA binding sites in the 60S intersubunit space, and thumbnails indicate the orientation. Note that in the E3^UFM1-RQC-60S complex, the UFL1 C terminus flips toward the L1 stalk and uL1 protein. (C) Schematic representation of NEMF and UFL1 domain organization and interactions between them. The vertical dashed line marks the site of the β-augmented residues. (D to F) Close-up views on the interactions between the UFL1 CTD and (D) uL1 (white), (E) 5S rRNA (gray), and (F) the NEMF C-terminal NFACT domain (violet; two views). CTD, UFL1 C-terminal domain; CIM, CTD-interacting motif of NEMF; WH, winged helix; CC, coiled-coil (NEMF and UFL1); M, middle domain of NEMF; NFACT, domain found in NEMF, FbpA, Caliban, and Tae2.

Fig. 3.. ER-AP but not cytosolic AP clearance depends on interaction between NEMF and UFL1.

(A) Schematic of the ER-targeted stalling reporters contained the following features: mRuby; V5 epitope tag; T2A (Thosea asigna virus 2A) peptide bond skipping sequences; signal sequence (SS) from bovine preprolactin; hemagglutinin epitope tag; blue fluorescent protein (BFP); N-glycosylation sequon; polylysine (K20) stalling sequence; superfolder green fluorescent protein (GFP). The protein species produced by each stalling reporter are indicated. (B) Model of the ER-targeted stalling reporter on a 60S ribosome at the ER. When the ER-AP is still attached to the ribosome by a P-tRNA, the N-glycosylation sequon is retained in the ribosome exit tunnel and is inaccessible to the glycosylation machinery in the ER lumen. If the ER-AP is released from the 60S and P-tRNA, then it will enter the ER lumen, and the sequon will be glycosylated. (C) Position of NEMF-UFL1 β-augmentation interface (red box). (D and E) Residues mutated in VLT UFL1, shown as molecular model (D) or schematic representation (E). The vertical dashed line marks the site of the β-augmented residues. (F) Degradation of ER but not cytosolic APs depends on UFL1-NEMF interaction. UFL1-dependent degradation of ER-AP but not cytosolic AP in WT or UFL1^KO cells stably rescued with WT, but not mutant Δ79 (deletion of residues L395-E473) and VLT (V393A/L395A/T397A) UFL1-FLAG. (G) Quantification of cytosolic and ER-AP reporter intensities from data in (F). For ER-APs, the sum of the −Gly and +Gly bands are quantified. Data show mean V5 normalized fold change ± SD relative to UFL1^KO cells rescued with WT UFL1-FLAG, P value from the indicated comparison derived from two-way analysis of variance (ANOVA) of n = 3 biological replicates. ns, not significant. *P > 0.05, ****P > 0.0001.

Fig. 4.. UFMylation promotes SEC61 displacement and LTN1 binding.

(A to D) Cryo-EM density maps of the four main classes found in the ANM-challenged UFL1 immunoprecipitation. (A) RQC state 1, a SEC61-bound ER-RQC intermediate with NEMF bound to a P-tRNA. (B) RQC state 2, same as (A) but with the E3^UFM1 bound to uL24-UFMylated 60S. (C) RQC state 3, E3^UFM1-60S-RQC complex as shown in Fig. 1. Compared to (B), no SEC61 is visible, instead the DDRGK1 EBH is positioned below the tunnel exit and LTN1 is bound. (D) RQC state 4, same as (C) but lacking the E3^UFM1. (E) LTN1 binding to ribosomes at the ER depends on UFMylation. Microsomes derived from WT or UFC1^KO HEK293 cells were treated with ANM and analyzed by immunoblot. Quantification is from n = 3 biological replicates, fold change ± SD relative to WT cells, P value from the indicated comparison derived from one-way ordinary ANOVA of n = 3 biological replicates. **P = 0.0052. (F) Loss of UFM1 reduces ER-AP ubiquitylation. WT or UFM1^KO HEK293 cells transfected with SS^VgVK20 from (17) were treated with dimethyl sulfoxide (DMSO) or BTZ for 4 hours before isolation of Ub conjugates using pan-ubiquitin (TUBE2) agarose. Quantification is from n = 3 biological replicates from TUBE affinity purification in the presence of BTZ. Data show mean FLAG smear in elution/total ubiquitin smear in elution (fig. S8), fold change ± SD relative to WT cells, P value from the indicated comparison derived from unpaired t test, ***P = 0.0004. (G) Loss of UFSP2 accumulates ER-APs. Human embryonic kidney (HEK) 293 WT or UFSP2^KO cells were transfected with the ER-targeted stalling reporter and analyzed by immunoblot. For ER-APs, both the −Gly and +Gly bands are quantified. Data show mean V5 normalized fold change ± SD relative to WT cells, P value from the indicated comparison derived from unpaired t test of n = 3 biological replicates, **P = 0.0021.

Fig. 5.. Model for E3^UFM1 and RQC cooperation in ER-RQC.

Stalled, translocon-docked ribosomes are split, yielding a translocon-engaged 60S subunit with a peptidyl-tRNA in the P-site, an ER-AP clogging the exit tunnel and the SEC61 translocon (RQC state 1). As in cytosolic RQC, binding of NEMF to the P-tRNA and empty A-site initiates TLT, forming a CAT tail (yellow circles) on the AP. Double headed arrows denote conformational flexibility of the NEMF NFACT-C domain. In RQC state 2, E3^UFM1 is bound with UFL1 CTD in the rotated conformation, forming a stabilizing interface with NEMF to pause TLT, and E3^UFM1 catalyzes the transfer of UFM1 to uL24. In RQC state 3, the EBH of DDRGK1 is stabilized at the tunnel exit, promoting dissociation of 60S from SEC61 and allowing LTN1 to position its RING domain near the tunnel exit to ubiquitylate the ER-AP. DeUFMylation allows E3^UFM1 to dissociate, producing RQC state 4 in which the NEMF NFACT-C domain regains its mobility and the P-tRNA-60S complex is no longer tethered to the ER membrane by E3^UFM1. DUF, DeUFMylation enzyme, UFSP2.