Antibodies and recombinant proteins
Details of the antibodies and recombinant proteins used in this study are provided in Supplementary Table 1.
Mammalian cell culture and cell line generation
Flp-In T-REx HEK293 cells (Invitrogen, R78007) were cultured in high-glucose DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), 50 mg ml−1 penicillin–streptomycin and 2 mM l-glutamine. Cells were maintained at 37 °C under 5% CO2 in an incubator in a humidified environment and routinely checked for mycoplasma. CDK5RAP3-KO cells were generated using CRISPR–Cas9. CRISPR sense and anti-sense guides were cloned into pX335 (DU64982) and pBABED puro U6 (DU64977) plasmids, respectively. In a separate strategy, single guide RNAs were cloned into the px459 vector (Addgene, 48139). In brief, around 2 million cells were seeded into a 10 cm dish in antibiotic-free Dulbecco’s modified Eagle medium (DMEM) and transfected with 1 μg plasmid DNA using Lipofectamine 2000 (Invitrogen, 1168019) according to the manufacturer’s instructions. Then, 24 h after transfection, cells were selected in 2 μg ml−1 puromycin for 24 h followed by a 24 h recovery period in preconditioned medium. Cells were submitted for single-cell sorting, expanded and knockouts were confirmed by sequencing and immunoblot analysis.
Cytosolic and membrane fractionation
For chemical induction of ribosome stalling, cells were treated with 200 nM anisomycin for 4 h before collection. The parent cell line (Flp-In T-REx HEK293 cells) and KOs were washed once in ice-cold PBS, collected in ice-cold PBS and pelleted by centrifugation at 800g. Cell pellets (around 2 × 106 cells) were resuspended in 125 μl of 0.02% (w/v) digitonin, 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl2, and 1× cOmplete protease inhibitor cocktail EDTA-free (Roche). Cells were incubated on ice for 10 min and centrifuged at 17,000g for 10 min at 4 °C. The supernatant was transferred to a fresh tube (cytoplasmic extract). The remaining pellet was washed with 1× PBS and centrifuged at 7,000g for 5 min at 4 °C. The pellet was resuspended in 125 μl 1% Triton X-100, 50 mM HEPES pH 7.5, 150 mM NaCl and 1× EDTA-free protease inhibitor cocktail tablet. This was further incubated on ice for 10 min and centrifuged at 17,000g for 10 min at 4 °C. The supernatant was transferred to a new Eppendorf tube (membrane extract). Equal volumes of the cytosolic and membrane fractions were resolved by SDS–PAGE and analysed using immunoblotting.
Expression and purification of recombinant proteins
UBA5, UFC1, UFM1, UFL1–UFBP1 and CDK5RAP3 were expressed and purified as described previously3. GST–3C–UFBP1(178–204) was applied onto Glutathione Sepharose 4B beads (Cytiva) followed by 3C-protease cleavage on the beads and purified on the HiLoad 26/600 Superdex 75 pg column, pre-equilibrated with 25 mM HEPES pH 7.5 and 200 mM NaCl. E3mUU constructs were applied onto HisTrap columns, pre-equilibrated with 50 mM Tris pH 7.5, 200 mM NaCl, 20 mM imidazole (pH 8.0) and eluted with the same buffer containing 300 mM imidazole. Constructs with a cleavable His-tag were incubated with 1:50 TEV protease and dialysed against 25 mM Tris pH 7.5, 200 mM NaCl at 4 °C overnight and further purified on the HiLoad 26/600 Superdex 75 pg column, pre-equilibrated with 25 mM HEPES pH 7.5, 200 mM NaCl and 1 mM DTT. For crystallization of the E3mUU(ΔUFIM)–UFC1 complex, 6×His-TEV-UFL1(1–179) was co-expressed with a UFC1-UFBP1 204-C fusion construct. Here, the His-tag was not cleaved.
Discharge assays
Single-turnover lysine discharge assays were performed to analyse the activity of UFC1 and UFL1–UFBP1 as described previously3. In brief, UFC1 was charged by incubating 0.5 μM UBA5, 10 μM UFC1 and 10 μM UFM1 in reaction buffer containing 50 mM HEPES pH 7.5, 50 mM NaCl, 0.5 mM DTT, 10 mM ATP and 10 mM MgCl2 for 20 min. The reaction was quenched by addition of 50 mM EDTA (pH 8.0) to the reaction mix followed by incubation for 10 min at room temperature. Discharge was performed in the presence of 50 mM lysine (pH 8.0). The reaction was stopped at the indicated timepoints and analysed under non-reducing conditions on a 4–12% SDS–PAGE gel followed by Coomassie staining.
Preparation of 80S ribosomes and polysomes
HEK293 cells (around 80% confluency) grown in five 15 cm dishes were washed briefly with ice-cold PBS and collected in a 15 ml falcon tube. Cells were lysed in buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg ml−1 cycloheximide, 1% Triton X-100, 1× cOmplete protease inhibitor cocktail, EDTA-free (Roche) and RNasin for 10 min on ice followed by centrifugation at 13,000g for 10 min. The clarified supernatant was collected and layered onto a 10–50% sucrose gradient containing 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg ml−1 cycloheximide and 1% Triton X-100, followed by centrifugation at 36,000 rpm for 3 h using the SW41 Ti rotor. The fractions containing 80S ribosomes and polysomes were collected and layered onto a 50% sucrose cushion and centrifuged at 40,000 rpm for 12 h in a Type 70 Ti rotor. Ribosome pellets were then resuspended in buffer containing 20 mM HEPES pH 7.6, 100 mM KCl, 5 mM Mg(OAc)2, 10 mM NH4Cl and 1 mM DTT and stored at −80 °C until further use.
Purification of stable 60S ribosomes from HEK293 cells
60S ribosomes were purified as described previously39,40 with minor changes. HEK293 cells were grown to around 80% confluency in fifteen 15 cm dishes with medium containing high-glucose DMEM supplemented with 10% (v/v) FBS, 50 mg ml−1 penicillin–streptomycin and 2 mM l-glutamine. To collect cells, the medium was first removed by aspiration, washed with ice-cold PBS followed by removal of PBS by aspiration. Cells were scrapped in residual PBS and transferred to a 15 ml falcon. Cells were pelleted by centrifugation at 1,000g for 3 min and the supernatant was discarded. Next, the cell pellets were resuspended in lysis buffer (containing 15 mM Tris pH 7.6, 1,500 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 2 mM DTT, RNAsin (60 U), 1× cOmplete mini protease inhibitor cocktail (Roche)) and mixed gently followed by incubation on ice for 10 min. The cell lysates were then centrifuged at 17,000g for 10 min and the supernatant was collected. The collected supernatant was layered directly onto a high-salt sucrose cushion containing 20 mM Tris pH 7.5, 500 mM KCl, 30% (v/v) sucrose, 10 mM MgCl2, 0.1 mM EDTA pH 8.0 and 2 mM DTT. Total ribosomes were sedimented by centrifugation at 63,000g (24,800 rpm) for 18 h using a Type 70 Ti rotor (Beckman Coulter). The sedimented ribosomes were then resuspended in buffer containing 20 mM Tris pH 7.5, 500 mM KCl, 7.5% (v/v) sucrose, 2 mM MgCl2, 75 mM NH4Cl, 2 mM puromycin and 2 mM DTT. The resolubilized pellet containing ribosomes was incubated at 4 °C for 1 h and then at 37 °C for 1.5 h. To isolate 40S and 60S ribosomal subunits, the solution was layered directly onto a linear 10–30% sucrose gradient containing 20 mM Tris pH 7.5, 500 mM KCl, 6 mM MgCl2 and 2 mM DTT. The 60S and 40S were separated by centrifugation at 49,123g (16,800 rpm) for 9 h 42 min at 4 °C using a SW41 Ti rotor (Beckman Coulter). Gradients were fractionated into 0.5 ml fractions using the BioComp fractionating system. The fractions containing 60S ribosomal subunits were collected and exchanged into buffer containing 20 mM HEPES pH 7.2, 100 mM KCl, 5 mM MgCl2, 2 mM DTT and stored at −80 °C.
In vitro UREL–ribosome association assays
Approximately, 0.2 µM of preformed UREL was added to a mixture of 0.2 µM of 60S ribosomes (1×) and 0.5 µM of 80S ribosomes (2.5×) and incubated for 15 min at 23 °C. After incubation, the mix was layered onto a 10–50% sucrose gradient containing 20 mM HEPES pH 7.5, 50 mM KCl, 5 mM MgCl2 and centrifuged at 36,000 rpm for 6 h at 4 °C. The samples were then manually fractionated into 22 fractions (100 µl each) and analysed for co-migration by immunoblotting using the indicated antibodies.
Cryo-EM sample preparation
Reconstitution of stable ribosome–E3 complexes
Approximately 10 µM of UREL complexes was incubated with 1 µM of purified 60S ribosomes in the presence of excess UFC1–UFM1 conjugate (5 µM) in buffer containing 20 mM HEPES pH 7.2, 50 mM KCl, 5 mM MgCl2 and 0.25 mM TCEP for 2 h at 4 °C. After incubation, the samples were mixed with 0.05% glutaraldehyde for 30 s at 23 °C followed by quenching with 100 mM Tris pH 8.0 (final concentration). The cross-linked sample was then layered onto a 10–30% sucrose gradient containing 20 mM HEPES pH 7.5, 50 mM KCl, 5 mM MgCl2 and 0.25 mM TCEP, and centrifuged using the TLS55 rotor at 24,000 rpm for 6 h at 4 °C. The sucrose gradient of 2.2 ml volume was then manually fractionated into 100 µl fractions and analysed for co-migration of UREL components, 60S ribosomes and UFC1–UFM1 by immunoblotting. The fractions containing UREL–60S ribosome–UFC1–UFM1 were then pooled and concentrated to 7.7 mg ml−1 and buffer-exchanged to remove excess sucrose.
Reconstitution of UFMylated 60S ribosome–UREL complexes
First, an in vitro UFMylation reaction was performed by incubating 0.1 µM UBA5, 5 µM UFC1, 10 µM UFM1, 3 µM UFL1–UFBP1, 5 µM CDK5RAP3 and 1 µM 60S ribosomes in the presence of 5 mM MgCl2 and 5 mM ATP. After the reaction, 10 µM UFC1–UFM1 was added to the reaction and further incubated at 4 °C for 2 h. The reaction products were then separated on a sucrose gradient and the fractions containing 60S–UREL–UFC1–UFM1 were collected as described in the previous section.
Cryo-EM data collection and image processing
UREL–60S EM grid preparation
Cryo-grids were prepared with 0.05% glutaraldehyde-cross-linked 60S–UREL–UFC1–UFM1 complex at 7.7 mg ml−1 in 25 mM HEPES pH 7.5, 50 mM KCl, 5 mM MgCl2, 2 mM DTT. Quantifoil R3.5/1 copper 200 mesh holey grids were glow discharged using the PELCO easiGlow glow discharge unit at 15 mA for 30 s. Cryo-grids were prepared using the Thermo Fisher Scientific Vitrobot MK IV with a chamber temperature of 4 °C and 100% humidity. A total of 3 μl of protein was applied to the grid and immediately blotted for 6 s with blot force 1, followed by rapid plunge-freezing into liquid ethane.
UREL–60S cryo-EM data collection
Single-particle cryo-EM data were collected on the Thermo Fisher Scientific Titan Krios G2 transmission electron microscope with a Thermo Fisher Scientific Falcon 4i direct electron detector and SelectrisX energy filter. Data were collected with an accelerating voltage of 300 kV and nominal magnification of ×165,000, which corresponds to a pixel size of 0.74 Å (full data acquisition settings are shown in Extended Data Table 1). A total of 59,394 cryo-EM videos was acquired.
UREL–60S image processing
Cryo-EM videos were imported, beam-induced motion corrected (MOTIONCOR2) and the CTF parameters were estimated (CTFFIND4.1) using RELION (v.3.1)41,42,43. Approximately 2.2 million particles were picked from motion-corrected micrographs using crYOLO (v.1.6.1)44 untrained particle picking (2019 general model) with a particle box size of 400 pixels and a picking confidence threshold of 0.2. Picked particles were extracted in RELION with a particle box size of 588 pixels, rescaled to 128 pixels (rescaled pixel size, ~3.4 Å). Extracted particles were imported into cryoSPARC (v.3.2)45 for processing. Seven rounds of reference-free 2D class averages were generated with the initial classification uncertainty factor set between 2 and 7, the number of online-EM iterations set to 40 and batchsize per class set to 200, and all ribosome-like particles were taken forward. The selected 1.6 million particles were used to generate an initial 3D model with C1 symmetry. The initial 3D model was further refined using the non-uniform refinement algorithm with the dynamic masking start resolution set to a value below the resolution of the data (that is, 1 Å) to generate a refined 3D model and a mask that encompasses the entire box size. The mask and model were input for 3D variability analysis asking for three classes. Particles from the class containing ligase-bound 60S ribosomes were taken forward for another round of 3D refinement, this time with dynamic masking start resolution set to default (12 Å) and the dynamic mask threshold set to 0.1. This was then followed by several more rounds of 3D variability analysis, asking for two classes to separate ligase-bound 60S ribosomes from unbound 60S ribosomes, resulting in 356,394 ligase-bound ribosome particles. Particles were then downsampled to 128 pixels and a cryoDRGN (v.3.2.0)46 model was trained with 8 latent dimensions and 50 training iterations. CryoDRGN particle filtering removed 57,386 junk particles, resulting in a final particle stack of 299,008 particles. The homogenous particle population containing ligase-bound ribosomes were re-extracted in Relion at the full box size. A 3D model was generated with C1 symmetry, followed by non-uniform refinement with per particle defocus optimization, Ewald sphere correction and CTF refinement in cryoSPARC (v.4.2.1) to generate the ligase-bound 60S ribosome map.
To further refine the density for the ligase complex, two masks were created from the final 3D refinement volume using UCSF ChimeraX (v.1.2.5)47: one that encompasses the ligase complex plus RPL10a and another that encompasses the ribosome. The ribosome mask was used for particle signal subtraction. Signal-subtracted particles were then used for local refinement of the ligase complex plus RPL10a using the ligase mask to generate a ligase-only map. A cryoSPARC (v.4.2.1) 3DFlex19 training model was generated for the ligase with 6 latent dimensions and a rigidity prior of 2. The resulting 3DFlex model was used for 3DFlex reconstruction with 40 max BFGS iterations to generate the final ligase map.
UREL–60S model building
The ligase-bound 60S map was sharpened using Phenix (v.1.2.1)48 autosharpen map job and the ligase-only map was sharpened using the DeepEMhancer49 tight target sharpening protocol. Atomic models were built using Coot (v.0.9.8.1)50. For the ligase-bound 60S ribosome map, PDB 7QWR (ref. 51) was used as a starting model for the 60S ribosome by rigid-body fitting the model into the density map, followed by rebuilding in Coot. No ligase components were built into the ligase-bound ribosome map except for the UFL1 PTC loop. For the ligase complex, AlphaFold2 models of the individual proteins were separated into smaller segments and then rigid-body fitted into the density map, followed by manual rebuilding in Coot. The UFL1 CTD (residues 515–786), CDK5RAP3 UUBD (residues 15–116) and UFM1 displayed poor side-chain density and the side chains of these regions were therefore set to an occupancy of 0. Atomic models were refined using Phenix real space refinement and validated using MolProbity. All 3D density maps were visually inspected in UCSF ChimeraX (v.1.2.5)47.
UFMylated ribosome data collection and image processing
Cryo-EM grids were prepared as described above with 1.5 mg ml−1 sample. Single-particle cryo-EM data were collected on the Thermo Fisher Scientific Titan Krios G2 transmission electron microscope with a Thermo Fisher Scientific Falcon 4 direct electron detector. Data were collected with an accelerating voltage of 300 kV and a nominal magnification of ×96,000, corresponding to a pixel size of 0.82 Å (full data-acquisition settings shown in Extended Data Table 1). A total of 3,028 cryo-EM videos was acquired. The data were processed as previously, with the final map being generated from the particles after several rounds of 3D variability analysis.
XL-MS sample preparation and analysis
Approximately 1.2 µM UFL1–UFBP1, 2 µM CDK5RAP3, 0.2 µM ribosomes and 10 µM of UFC1–UFM1 were incubated with 1 mM DSBU (disuccinimidyl dibutyric urea) in buffer containing 50 mM HEPES pH 7.5, 50 mM KCl, 6 mM MgCl2, 0.5 mM TCEP for 30 min at 23 °C. The reaction was quenched by addition of 50 mM Tris pH 8.0. Cross-linked samples were processed for MS analysis using S-Trap micro spin columns (Protifi) according to the manufacturer’s protocol. In brief, cross-linked samples were reduced by adding 20 mM DTT (10 min, 50 °C), and then alkylated with 40 mM iodoacetamide (30 min, 20 °C). The samples were acidified by the addition of phosphoric acid to a final concentration of 5%, and subsequently diluted with 90% methanol in 100 mM triethylammonium bicarbonate (TEAB) pH 7.1 (1:7 (v/v) sample: buffer). A total of 1 µg trypsin (Promega) was added, and the samples were then bound to a S-Trap micro spin column (Protifi). Subsequently, the column was washed three times with 90% methanol in 100 mM TEAB. An additional 0.6 µg of trypsin was applied to the column, and digestion was then performed by incubating the S-trap column at 47 °C for 90 min. Peptides were recovered by washing the column sequentially with 50 mM TEAB (40 µl), 0.2% (v/v) formic acid (40 µl) and 50% acetonitrile/0.2% (v/v) formic acid (40 µl). The eluate was then evaporated to dryness in a vacuum centrifuge and the peptides were resuspended in 5% (v/v) acetonitrile/0.1% (v/v) formic acid (20 μl) before MS analysis. Peptides (5 µl) were injected onto the Vanquish Neo LC (Thermo Fisher Scientific) system and the peptides were trapped on the PepMap Neo C18 trap cartridge (Thermo Fisher Scientific, 5 µm particle size, 300 µm × 0.5 cm) before separation using the Easy-spray reverse-phase column (Thermo Fisher Scientific, 2 µm particle size, 75 µm × 500 mm). Peptides were separated by gradient elution of 2–40% (v/v) solvent B (0.1% (v/v) formic acid in acetonitrile) in solvent A (0.1% (v/v) formic acid in water) over 80 min at 250 nl min−1. The eluate was infused into an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) operating in positive-ion mode. Orbitrap calibration was performed using FlexMix solution (Thermo Fisher Scientific). Data acquisition was performed in data-dependent analysis mode and fragmentation was performed using higher-energy collisional dissociation. Each high-resolution full scan (m/z 380–1,400, R = 60,000) was followed by high-resolution product ion scans (R = 30,000), with a stepped normalized collision energies of 21%, 26% and 31%. A cycle time of 3 s was used. Only charge states 3–8+ were selected for fragmentation. Dynamic exclusion of 60 s was used. Cross-link identification was performed using Proteome discoverer (v.3.0) and the in-built XlinkX module (Thermo Fisher Scientific) using the following settings: crosslinker: DSBU, mass deviation tolerances of 10 ppm in MS and 0.02 Da for Sequest HT and 20 ppm for XlinkX tandem MS (MS/MS). Carbamidomethylation of Cys residues was set as a static modification, and dynamic modifications were set as Met oxidation and DSBU dead-end modifications (DSBU-amidated, DSBU Tris and DSBU hydrolysed) (maximum of three modifications per peptide). Only results with scores corresponding to a false-discovery rate of <1% were taken forward. Finally, a minimum XlinkX score of 45 was used to filter cross-linked peptides52,53.
Ribosome UFMylation assays
Ribosome UFMylation assays were performed as described previously3. Purified 60S ribosomes (approximately 0.05 µM) were mixed with 0.5 μM UBA5, 1 μM UFC1, 1 μM UFM1 and 0.1 µM UFL1–UFBP1 in a reaction buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl2 and 5 mM ATP and incubated at 37 °C for 10 min or the indicated time duration. The reaction was stopped by the addition of SDS loading buffer and run on a 4–12% SDS–PAGE gel under reducing conditions followed by immunoblotting using the indicated antibodies. In reactions containing CDK5RAP3, approximately 0.15 µM of CDK5RAP3 was added to the reaction along with 0.1 µM of UFL1–UFBP1.
Polysome profiling using HEK293 cell lysates
Polysomes were isolated from HEK293 cells as described previously with slight modification. In brief, HEK293 cells were seeded one night before the experiment. On the day of the experiment, cells were treated with either 0.1% DMSO or 200 nM anisomycin for around 20 min before collection. Cells were washed with ice-cold PBS, scraped off and pelleted by centrifugation at 800g for 5 min. The pellet was then resuspended in lysis buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg ml−1 cycloheximide, 0.02% Digitonin, cOmplete protease inhibitor cocktail, EDTA-free (Roche) and RNasin. Digitonin-treated cells were incubated for 5 min on ice and centrifuged at 17,000g for 10 min at 4 °C. The supernatant containing the cytoplasmic extract was discarded and the remaining pellet was washed with 20 mM Tris pH 7.5, 150 mM NaCl and 5 mM MgCl2 and centrifuged at around 7,000g for 5 min. Supernatant was discarded and the pellet was resuspended in lysis buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg ml−1 cycloheximide, 0.5% Triton X-100, cOmplete protease inhibitor cocktail, EDTA-free (Roche) and RNasin. The resuspended pellets were incubated on ice for 10 min and centrifuged at 17,000g for 10 min at 4 °C. The supernatant containing the membrane fraction extract was transferred to a new Eppendorf tube and the amount of RNA was quantified for each sample using the NanoDrop system. RNA-normalized samples were then layered onto a 10–50% sucrose gradient containing 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg ml−1 cycloheximide and RNAsin, and then centrifuged at 36,000 rpm for 3 h. Polysomes were then separated by fractionation using the Biocomp fractionating system and analysed using western blotting.
Comparison of 60S and 80S UFMylation in vitro
In vitro UFMylation reactions were performed by incubating 0.1 µM 60S ribosome, 0.2 µM or 0.3 µM enriched 80S (two or threefold excess over 60S) with 0.5 µM UBA5, 1 µM UFC1, 1 µM UFM1, 0.3 µM UFL1–UFBP1 and 0.3 µM CDK5RAP3 in the presence of 5 mM MgCl2 and 5 mM ATP at 37 °C for 15 min. After incubation, the reaction mix was layered over a 10–50% sucrose gradient containing 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT and centrifuged at 36,000 rpm for 3 h at 4 °C using a SW41 Ti rotor. The gradients were fractionated using the BioComp fractionation system. The sucrose gradient fractions were then run on a 4–12% SDS–PAGE gel and analysed for UFMylation of RPL26 by immunoblotting.
Preparation of membrane-associated 60S ribosomes
Parental cells (WT HEK293) or CDK5RAP3-KO cells (around 80% confluency) grown in ten 15 cm dishes were washed briefly with ice-cold PBS and collected in a 15 ml falcon tube. Cells were pelleted down by centrifugation at 500g for 5 min. Cell pellets were resuspended in buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg ml−1 cycloheximide, 0.02% (w/v) digitonin, 1× cOmplete protease inhibitor cocktail, EDTA-free (Roche) and RNasin for 10 min on ice followed by centrifugation at 17,000g for 10 min. The clarified supernatant is the cytosolic fraction and was discarded. The remaining membrane pellet was resuspended in lysis buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg ml−1 cycloheximide, 1% (w/v) decyl maltose neopentyl glycol (DMNG), 1× cOmplete protease inhibitor cocktail, EDTA-free (Roche) and RNasin for 15 min on ice, and then centrifuged at 17,000g for 10 min. The clarified supernatant was collected and layered onto a 10–30% sucrose gradient containing 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg ml−1 cycloheximide and 0.01% DMNG, and then centrifuged at 36,000 rpm for 3 h using the SW41 Ti rotor. Fractions containing 60S ribosomes were collected and exchanged into buffer containing 20 mM HEPES pH 7.2, 100 mM KCl, 5 mM MgCl2 and 2 mM DTT and stored at −80 °C until use.
In vitro 60S ribosome–SEC61 dissociation assays
The in vitro 60S–SEC61 dissociation reaction was performed by incubating 0.05 µM membrane solubilized 60S ribosomes (60S–SEC61 solubilized and enriched from CDK5RAP3-KO cells) with 0.5 µM UBA5, 1 µM UFC1, 1 µM UFM1, 0.1 µM UFL1–UFBP1, 0.1 µM CDK5RAP3 in the presence of 5 mM MgCl2 and 5 mM ATP at 37 °C for 25 min. At the end of the reaction, the reaction mix was layered over a 10–50% sucrose gradient containing 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2 and 1 mM DTT, and centrifuged at 36,000 rpm for 3 h using the SW41 Ti rotor. Sucrose gradients were fractionated using the BioComp fractionation system. The sucrose gradient fractions were separated on a 4–12% SDS–PAGE gel and analysed for co-migration of SEC61β with 60S ribosomes by immunoblotting.
LC–MS/MS sample preparation, data acquisition and analysis
First, an in vitro ribosome UFMylation reaction was performed to generate UFMylated ribosomes in the presence of either UFL1–UFBP1 or UREL. Then, the reaction products were run on a 4–12% SDS–PAGE gel to separate the mono- and di-UFMylated ribosomes. Next, the bands corresponding to mono- and di-UFMylated ribosomes were excised and in-gel digestion was performed according to a previously described protocol54. Digested peptides were analysed by liquid chromatography coupled with MS/MS (LC–MS/MS) on the Exploris 240 (Thermo Fisher Scientific) system coupled to the Evosep One (Evosep). The samples were loaded onto the Evotips according to the manufacturer’s recommendations and analysed using the 30 SPD method. Peptides were then analysed in on the Exploris 240 system using data-dependant acquisition with an MS1 resolution of 60,000, an AGC target of 300% and a maximum injection time of 25 ms. Peptides were then fragmented using TOP 2 s method, MS2 resolution of 15,000, NCE of 30%, AGC of 100% and maximum injection time of 100 ms. Peptide identification was performed in MaxQuant (v.2.0.2.0) against UniProt SwissProt Human containing isoforms (released 5 May 2021) with match between runs enabled. Carbamidomethylation (C) was set as a fixed modification and oxidation (M), acetyl (protein N-term) and the addition of the dipeptide valine–glycine (K) were set as variable modifications. The other parameters were left as the default.
Preparation of isopeptide-linked UFC1–UFM1 conjugate
First, 30 µM UBA5, 30 µM UFC1(C116K) and 60 µM UFM1 were incubated in 25 mM HEPES pH 7.5, 200 mM NaCl, 10 mM MgCl2 and 10 mM ATP. The pH of the reaction mixture was adjusted to 9.8 with 0.5 M CAPS, pH 11.5, and incubated for 18 h at 23 °C. UFC1–UFM1 was subsequently separated from UBA5 and unreacted UFC1 and UFM1 using the HiLoad 26/600 Superdex 75 pg column, pre-equilibrated with 25 mM HEPES pH 7.5, 200 mM NaCl, 1 mM DTT.
Crystallization and structure determination
UFC1–UFM1 conjugate
UFC1–UFM1 crystals were obtained using the sitting-drop vapour diffusion technique whereby UFC1–UFM1 (22.8 mg ml−1) was 1:1 mixed with 30% (v/v) PEG 400, 0.1 M Tris pH 8.5, 0.2 M Na citrate and incubated at 19 °C. Single crystals appeared within 2–3 days. Crystals were flash-frozen in crystallization buffer containing 30% (v/v) ethylene glycol. Datasets were collected at Diamond Light Source (DLS), beamline I04, and processed with Xia2 (ref. 55) and DIALS56. The crystal structure was solved by molecular replacement (PHASER)57 using the crystal structures of UFC1 (PDB: 3EVX)58 and UFM1 (PDB: 5IA7)59 as the starting model. Refinement and model building was performed using REFMAC60 and Coot50 (CCP4i2 suite), respectively. The statistics for data collection and refinement are listed in Extended Data Table 2.
UFL1–UFBP1–UFC1 complex
UFL1–UFBP1–UFC1 crystals were obtained using the sitting-drop vapour diffusion technique whereby UFL1–UFC1–UFBP1 (20.2 mg ml−1) was 1:1 mixed with 1.03 M Li2SO4, 0.1 M HEPES pH 7.2 and incubated at 19 °C. Single crystals appeared within 1–2 days. Crystals were flash-frozen in crystallization buffer containing 30% (v/v) ethylene glycol. Datasets were collected at the European Synchrotron Radiation Facility (ESRF), beamline ID23-EH2, and processed with the autoPROC suite61 (including XDS62, Pointless63 Aimless64, CCP4 (ref. 65) and STARANISO66). The crystal structure was solved by molecular replacement (PHASER)57 using the AlphaFold11 predicted models for UFL1 and UFBP1 and the crystal structure of UFC1 (PDB: 3EVX)58 as starting models. Refinement and model building was performed using REFMAC60 and Coot50 (CCP4i2 suite), respectively. The statistics for data collection and refinement are listed in Extended Data Table 2.
SEC
Analytical SEC runs were performed using the Superdex 200 Increase 3.2/300 column, pre-equilibrated with 25 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM TCEP. A total of 50 µl protein of the different components was mixed and incubated on ice for 30 min before loading onto the column.
ITC
ITC experiments were performed using a MicroCal PEAQ-ITC (Malvern). Proteins were first dialysed into ITC buffer containing 25 mM HEPES pH 7.5, 200 mM NaCl, 0.44 mM TCEP. Each experiment consisted of 13 injections for a duration of 6 s each followed by a 150 s spacing between injections except the experiment for UFBP1 UFIM–UFM1, which consisted of 19 injections instead. All of the experiments were performed at 25 °C.
Figures
Adobe Illustrator, BioRender and ChimeraX47 were used to make figures.
Materials availability
All cDNA constructs in this study were generated by H.M.M., J.J.P. and the cloning team at the MRC PPU Reagents and Services team. All of the plasmids have been deposited at the MRC PPU Reagents and Services and are available at https://mrcppureagents.dundee.ac.uk/.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
