ILS disassembly assay in nematode extractSample preparation<\/p>\n
Total nematode extract was prepared from a C. elegans strain expressing endogenously 3\u00d7FLAG-tagged PRP19 (ref. 2<\/a>).<\/p>\n Disassembly assays in the presence and absence of ATP were performed in technical triplicates. A total of 500\u2009\u00b5l nematode extract was supplemented with either 5\u00d7 splicing buffer with ATP (50\u2009mM HEPES-KOH pH 7.9, 175\u2009mM KCl, 10\u2009mM ATP, 20\u2009mM MgCl2, 100\u2009mM creatine phosphate, 250\u2009\u00b5g\u2009ml\u22121 creatine kinase and 15% PEG 8000) or 5\u00d7 splicing buffer without ATP (50\u2009mM HEPES-KOH pH 7.9, 175\u2009mM KCl and 20\u2009mM MgCl2). The supplemented extracts were incubated at 20\u2009\u00b0C for 1\u2009h with gentle agitation. Of magnetic anti-FLAG bead slurry (Sigma) equilibrated in detergent wash buffer (20\u2009mM HEPES-KOH pH 7.9, 150\u2009mM KCl, 1\u2009mM TCEP, 2\u2009mM MgCl2, 0.05% Igepal 630 and 10% glycerol), 20\u2009\u00b5l was then added to each reaction. The samples were transferred to 4\u2009\u00b0C and incubated with the beads for 2\u2009h with gentle rotation. The beads were washed four times with 1\u2009ml detergent wash buffer, transferred to fresh tubes and then washed an additional four times with detergent-free wash buffer (20\u2009mM HEPES-KOH pH 7.9 and 150\u2009mM KCl). The washed beads were resuspended in detergent-free wash buffer, transferred to fresh tubes and submitted to the Vienna BioCenter Mass Spectrometry Facility for on-bead tryptic digestion and liquid chromatography\u2013tandem mass spectrometry (LC\u2013MS\/MS) analysis.<\/p>\n Assay analysis<\/p>\n The resulting raw MS data were processed for protein identification and label-free quantification. To identify the proteins that were depleted from the beads upon ATP treatment, protein abundances in the samples with and without ATP were compared. Proteins were considered significantly enriched based on statistical tests (moderated\u00a0t-test\u00a0in limma, P\u2009<\u20090.05) and an arbitrary log2 fold change cut-off of 1 (twofold enrichment over control on a linear scale). The data were visualized in GraphPad Prism 10.<\/p>\n Generation of human cell linesK562 cells stably overexpressing DHX35\u2013EGFP or YJU2B\u2013EGFP<\/p>\n N-terminally EGFP\u20133C-tagged DHX35 and YJU2B overexpression cell lines were generated using a lentivirus system. Lenti-X 293T cells (Takara) were polyethylenimine transfected with lentiviral carrier plasmids containing either pRRL-SFFV-EGFP-3C-DHX35 or pRRL-SFFV-YJU2B-3C-EGFP. These were transfected alongside viral helper plasmids pCMVR8.74 (Addgene #22036) and pCMV-VSV-G (Addgene #8454). After 2 days, supernatant containing the lentiviral particles was collected. Wild-type K562 cells (DSMZ) were infected at limiting dilutions and EGFP-expressing cells were isolated by fluorescence-activated cell sorting using a BD FACSAria III instrument (BD Biosciences). Integration and expression of the constructs were confirmed by in-gel EGFP fluorescence and immunoblotting for DHX35 (CAS HPA062700, Sigma; dilution 1:250) or YJU2B (CAS OAAB02156, Aviva SysBio; dilution 1:1,000). All cultures were routinely tested for mycoplasma contamination.<\/p>\n K562 cells inducibly overexpressing DHX35(K83A)\u2013EGFP or DBR1\u2013EGFP<\/p>\n To generate inducible cell lines, C-terminally 3C\u2013EGFP-tagged DBR1 and N-terminally EGFP\u20133C-tagged mutant DHX35(K83A) constructs were cloned into a PiggyBac transposon donor plasmid containing a doxycycline-inducible TRE Tight promoter. Wild-type K562 cells (DSMZ) were electroporated with equal amounts of transposon donor and PiggyBac transposase plasmids using a Neon NxT Electroporation System (NEON1S, Thermo). Cells were selected using blasticidin (R21001, Invitrogen). Expression of the transgene was induced using 0.1\u2009\u00b5g\u2009ml\u22121 doxycyclin (D9891, Sigma-Aldrich) for 18\u2009h and EGFP+ cells were isolated using fluorescence-activated cell sorting. Expression of the constructs was confirmed by in-gel EGFP fluorescence and immunoblotting for DBR1 (CAS PA5-30616, Invitrogen; dilution 1:500) or DHX35 (CAS HPA062700, Sigma; dilution 1:250). All cultures were routinely tested for mycoplasma contamination.<\/p>\n HEK293T cells with endogenous DBR1\u2013dTAG<\/p>\n HEK293T cells were endogenously tagged at the C terminus of DBR1 with GGGS3-FKBP12(F36V)-V5-IRES-lox5171-mScarlet3\/mNeonGreen-lox5171, where FKBP12 is the protein that brings DBR1 to the cellular ubiquitination machinery in the presence of the dTAGV-1 compound, and lox5171 is a Cre recombinase target site. An sgRNA targeting the DBR1 C terminus was designed using CHOPCHOP61<\/a> and cloned into a modified plasmid #217659 (Addgene), in which the mCherry coding sequence was replaced with a puromycin resistance cassette. The sgRNA plasmid was co-transfected with two donor plasmids. The donor plasmids contained the repair template with the desired tag followed by either mScarlet3 or mNeonGreen and flanked by the DBR1 homology arms. Five days after\u00a0transfection, cells co-expressing mScarlet3 and mNeonGreen, which indicated homozygous tagging, were sorted by flow cytometry and then allowed to recover for 4 days. The cells were then treated with homemade recombinant Cre recombinase for 2\u2009h at 37\u2009\u00b0C in serum-free medium to excise the coding sequences of the fluorescent protein markers from the DBR1 loci. After 3 days of recovery, mScarlet3\/mNeonGreen double negative cells were single-cell sorted into medium supplemented with extra 25% of FCS. The selected clones were expanded and genotyped by Sanger sequencing. The homozygous clones were selected, and protein depletion upon addition of the dTAGV-1 compound was validated by western blot using an antibody to DBR1 (CAS 66951-1-IG, Proteintech; dilution 1:10,000).<\/p>\n To generate HEK293T DBR1\u2013dTAG cell lines that also inducibly overexpress various DBR1 constructs (wild type, catalytic mutant (H85A) and deletion of peptide I residues 420\u2013434 (\u0394pep1)), the coding sequences of DBR1 were cloned into the PiggyBac transposon donor plasmid under a doxycycline-inducible TRE Tight promoter. The DBR1 constructs were tagged on the C terminus with a P2A self-cleaving peptide followed by mScarlet. The DBR1-P2A\u2013mScarlet expression cassettes were inserted into the HEK293T DBR1\u2013dTAG cells by electroporation, and the successful clones were selected by fluorescence-activated cell sorting, as described above for the DHX35(K83A)\u2013GFP and DBR1\u2013GFP K562 overexpression cell lines.\u00a0All cell lines were routinely tested for mycoplasma contamination.<\/p>\n HEK293T\u00a0cells inducibly overexpressing TurboID-tagged DBR1, C19L1, YJU2B, LENG1 or EGFP<\/p>\n To generate stable inducible cell lines for proximity labelling experiments, HEK293 Flp-In T-REx parental cells at approximately 80\u201390% confluency were co-transfected with a pcDNA5\/FRT\/TO plasmid, which carried the coding sequence of DBR1, C19L1, YJU2B, LENG1 or nuclear localized EGFP fused on the 3\u2032 end to the TurboID-V5 tag, and the pOG44 plasmid (Thermo Fisher Scientific) at a 1:5 mass ratio (donor:pOG44) using the JetOptimus transfection reagent (Polyplus-transfection). For a 24-well plate format, 350,000 cells were seeded per well onto the transfection complex. Twenty-four hours after\u00a0transfection, cells were expanded into a six-well plate. Forty-eight hours later, selection was initiated. The cells were seeded to achieve 25\u201330% confluency and allowed to attach for approximately 2\u2009h. The medium was then replaced with selection medium containing 100\u2009\u00b5g\u2009ml\u22121 hygromycin B and 15\u2009\u00b5g\u2009ml\u22121 blasticidin S HCl. The selection medium was refreshed every 3\u20134 days. The selection process was monitored using the parallel wild-type cell culture, and selection was continued until all wild-type cells had died (approximately 10\u201314 days), at which point the pooled colonies of resistant cells were expanded without antibiotics. Successful integration was confirmed by western blotting\u00a0using an\u00a0antibody to the V5 tag (CAS\u00a0R960-25,\u00a0Thermo Fisher Scientific, dilution 1:3,000). All cultures were routinely tested for mycoplasma contamination.<\/p>\n RPE-1 cells stably overexpressing LENG1\u2013EGFP<\/p>\n cDNA corresponding to LENG1 was amplified from cDNA by PCR and inserted into a PGK-EGFP-N1-IRES-PURO plasmid, thereby tagging LENG1 at its C terminus with EGFP. The annealed double-stranded DNA fragment of NLS (GGTGGTCCTAAGAAGAAGAGAAAGGTT) was similarly cloned into PGK-EGFP-C1-IRES-PURO as a control.<\/p>\n The plasmids expressing the tagged proteins were transfected into RPE-1 cells62<\/a> using polyethylenimine reagent (Brunschwig Chemie) following the manufacturer\u2019s instructions. Two days after transfections, antibiotic selection of cells was initiated by adding 1\u2009\u03bcg\u2009ml\u22121 puromycin to the growth media. The selection medium was replaced every second day until the cells reached confluency. The expression of tagged proteins was verified by western blot using an antibody to GFP\u00a0(CAS\u00a011814460001, Roche, dilution 1:1,000)\u00a0and fluorescence microscopy. All cultures were routinely tested for mycoplasma contamination.<\/p>\n Preparation of human nuclear extract<\/p>\n The nuclear extracts for cryo-EM, RNA sequencing and biochemical assays were prepared from the human K562 cells stably overexpressing EGFP-tagged DHX35 or YJU2B as previously described2<\/a>,63<\/a>. To prepare nuclear extract from the K562 cells overexpressing EGFP-tagged DHX35(K83A) or DBR1, expression of the tagged protein was first induced by addition of 0.1\u2009\u00b5g\u2009ml\u22121 doxycycline. The cells were cultured for 48\u2009h before collecting and preparing nuclear extract as previously described2<\/a>,63<\/a>.<\/p>\n Cryo-EM grid preparationPurification of DIS (DHX35-containing spliceosome)<\/p>\n Approximately 25\u2009ml of the nuclear extract of K562 cells stably overexpressing DHX35\u2013EGFP was applied to approximately 1.5\u2009ml of GFP-Trap Agarose beads (Chromotek) equilibrated in wash buffer (20\u2009mM HEPES-KOH pH 7.9, 100\u2009mM KCl, 0.5\u2009mM TCEP, 2\u2009mM MgCl2, 0.05% v\/v Igepal CA-630 and 8% v\/v glycerol). The beads were incubated with the nuclear extract for approximately 3.5\u2009h at 4\u2009\u00b0C with gentle rotation. The beads were then washed five times in 10\u2009ml wash buffer. The washed beads were incubated twice for approximately 1\u2009h at 4\u2009\u00b0C with gentle rotation in 0.5\u2009ml elution buffer (20\u2009mM HEPES-KOH pH 7.9, 100\u2009mM KCl, 0.5\u2009mM TCEP, 2\u2009mM MgCl2, 3% w\/v sucrose and 0.1\u2009\u00b5g\u2009ml\u22121 3C protease). The eluted samples were then applied to a GraFix64<\/a> 15\u201330% w\/v sucrose gradient containing 0.05% glutaraldehyde in the heaviest fraction in a gradient buffer (20\u2009mM HEPES-KOH pH 7.9, 100\u2009mM KCl, 0.5\u2009mM TCEP and 2\u2009mM MgCl2). The gradient samples were spun in an SW60 Ti rotor (Beckman Coulter) at 22,000\u2009rpm for 16\u2009h at 4\u2009\u00b0C. The gradients were fractionated manually, and each fraction was quenched with 45\u2009mM lysine. The sedimentation coefficients were estimated using the CowSuite software, and the peak spliceosome fractions were further confirmed by SDS\u2013PAGE. The peak spliceosome fractions were pooled and concentrated using Amicon Ultra 0.5-ml concentrators with 100-kDa molecular weight cut-off (Sigma). During concentrating, the sample was buffer exchanged to a cryo-EM buffer (20\u2009mM HEPES-KOH pH 7.9, 0.5\u2009mM TCEP, 100\u2009mM KCl and 2\u2009mM MgCl2) to remove sucrose and excess glutaraldehyde.<\/p>\n Purification of ILSAQR (YJU2B-containing spliceosome)<\/p>\n Approximately 25\u2009ml of the nuclear extract of K562 cells stably overexpressing YJU2B\u2013EGFP was incubated overnight with 2\u2009\u00b5g\u2009ml\u22121 benzonase before proceeding to anti-GFP affinity purification and gradient ultracentrifugation as described above for DHX35-containing spliceosome.<\/p>\n Grid preparation<\/p>\n The purified spliceosome samples were incubated with a 2-nm continuous carbon film (prepared in-house) for either overnight (DIS), approximately 7\u2009h (ILSAQR; dataset 1) or approximately 3\u2009h (ILSAQR; dataset 2) in a homemade humidity chamber at 4\u2009\u00b0C. The carbon film was picked up with a glow-discharged R3.5\/1 holey carbon grid (Quantifoil). Of ice-cold cryo-EM buffer, 3\u2009\u00b5l was applied to the grid before blotting at 4\u2009\u00b0C and 80% humidity and plunging into liquid ethane using a Leica EM GP2 instrument.<\/p>\n Cryo-EM data collectionILSAQR or YJU2B -containing spliceosome<\/p>\n Two datasets of ILSAQR were collected using a Titan Krios G4 equipped with a Falcon 4i direct electron detector (Vienna BioCenter). Dataset 1 contained 26,091 movies, whereas dataset 2 consisted of 27,327 movies. The energy filter was set to a width of 10\u2009eV. The C2 aperture was set to 50\u2009\u00b5m, whereas the objective aperture was retracted. In both datasets, the movies were collected at a total dose of 40\u2009e\u2212\u2009\u00c5\u22122 in EER format with a pixel size of 0.951\u2009\u00c5 per pixel. The target defocus of both datasets ranged from \u22121.0\u2009\u00b5m to \u22122.5\u2009\u00b5m.<\/p>\n DIS or DHX35-containing spliceosome<\/p>\n A dataset of 12,277 micrographs was collected using a Titan Krios G4 equipped with a Falcon 4i direct electron detector (Vienna BioCenter). The energy filter was set to a width of 10\u2009eV. The C2 aperture was set to 50\u2009\u00b5m, whereas the objective aperture was retracted. The movies were collected at a total dose of 50\u2009e\u2212\u2009\u00c5\u22122 in EER format with a pixel size of 0.951\u2009\u00c5 per pixel. The target defocus ranged from \u22121.25\u2009\u00b5m to \u22122.25\u2009\u00b5m.<\/p>\n Cryo-EM data analysisPre-processing and particle cleaning of human ILSAQR<\/p>\n Pre-processing (gain correction, motion correction and contrast transfer function estimation) of both datasets of the ILSAQR was performed in cryoSPARC live65<\/a>.<\/p>\n For dataset 1, 931,992 particles were picked on-the-fly using the Blob picker in cryoSPARC live with a minimum and maximum particle diameter set to 250\u2009\u00c5 and 450\u2009\u00c5, respectively. The picked particles were extracted with a box size of 128\u2009pixels at a binned pixel size of 4.01\u2009\u00c5 per pixel. A 2D classification was performed to select the particles representing 2D class averages with clear internal features. The selected particles were subsequently used to generate three 3D ab initio models. Three rounds of heterogeneous refinement were performed using the ab initio models as references. Only the particles from the best class were carried over to the next round of refinement, resulting in the final subset of 232,423 particles. Particles in dataset 1 were also picked in Warp66<\/a> using a model trained on manually picked particles with an expected particle diameter of 350\u2009\u00c5. The 1,172,730 Warp-picked particles were processed the same as above, resulting in a subset of 294,689 particles. Upon removal of duplicate particles, the combined Blob-picked and Warp-picked particles were re-extracted with a box size of 448\u2009pixels at a binned pixel size of 1.15\u2009\u00c5 per pixel and further processed by reference-based motion correction in cryoSPARC. In the end, 310,532 particles were obtained from dataset 1.<\/p>\n For dataset 2, particles were picked in Warp66<\/a> using a model trained on manually picked particles with an expected particle diameter of 350\u2009\u00c5. The picked 1,068,365 particles were extracted with a box size of 128\u2009pixels with a binned pixel size of 4.01\u2009\u00c5 per pixel. The three ab initio models from dataset 1 were used for three rounds of heterogeneous refinement with only the best class being carried over to the next round of refinement. The selected particles were then re-extracted with a box size of 448\u2009pixels at a binned pixel size of 1.15\u2009\u00c5 per pixel and further processed by reference-based motion correction in cryoSPARC. In the end, 197,936 particles were obtained from dataset 2.<\/p>\n Obtaining maps of human ILSAQR<\/p>\n The particles from both datasets were combined, resulting in a complete set of 508,031 particles. A non-uniform refinement generated a consensus map of the ILSAQR (map 1) at a global resolution of 2.40\u2009\u00c5. By performing a local refinement with a mask around the core of the spliceosome, we obtained map 2 at a resolution of 2.34\u2009\u00c5. To further improve the resolution of specific spliceosome regions, we performed local refinements with masks around the zinc-finger domain of YJU2B (map 3 at a nominal resolution of 2.32\u2009\u00c5), the helical bundle of the NTR subunits PRP19\u2013CDC5L\u2013SPF27 (map 9 at a nominal resolution of 3.83\u2009\u00c5), the helical repeats of the NTC subunits SYF1\u2013SYF2 (map 10 at a nominal resolution of 2.59\u2009\u00c5) and the U5 Sm ring (map 11 at a nominal resolution of 2.88\u2009\u00c5).<\/p>\n The GCFC domains and the coiled-coil interaction interface of TFIP11 and GCFC2 were poorly resolved in map 1, probably due to their flexibility and potentially their partial occupancy. To better resolve these termination factors in the ILSAQR, we performed two rounds of 3D classification with a mask around TFIP11\u2013GCFC2 on the combined 508,031 particles. First, we performed a hard 3D classification in cryoSPARC. The 139,130 particles from the best 7 out of 50 classes were selected for a 3D classification in RELION5 with Blush regularization67<\/a> with a T value of 40. The resulting best class containing 6,794 particles was locally refined in cryoSPARC with a mask around TFIP11\u2013GCFC2 to generate map 8 at a nominal resolution of 7.37\u2009\u00c5.<\/p>\n For certain spliceosomal regions, combining particles from two datasets did not result in significant improvements in density quality. Specifically, to produce ILSAQR maps 4\u20137, only the 310,532 particles from dataset 1 were used.<\/p>\n To improve the resolution of the termination factor C19L2, we performed a local refinement with a mask around C19L2, resulting in map 4 at a nominal resolution of 2.84\u2009\u00c5.<\/p>\n To better visualize AQR within ILSAQR, we performed a hard 3D classification on the particles from dataset 1 with a mask around AQR. Particles from 3D classes with the best-defined AQR density were used in a local refinement with a mask around AQR before proceeding to 3D variability analysis in cryoSPARC with the same mask. The 46,099 particles from the best cluster out of 10 were used for another local refinement with a mask around AQR, generating map 5 at a nominal resolution of 3.92\u2009\u00c5.<\/p>\n The region of ILSAQR containing the helical repeats of SYF1, the MPP domain of C19L1 and the interacting helix of YJU2B appeared to be flexible and, hence, poorly resolved in map 1. To better visualize this region, we performed 3D variability analysis of the 310,532 particles from dataset 1 after homogeneous refinement with a global mask. One out of 10 clusters with the best-defined region of interest containing 26,265 particles was selected and further locally refined with a mask around SYF1\u2013C19L1\u2013YJU2B. This produced map 6 at a nominal resolution of 6.32\u2009\u00c5.<\/p>\n To better resolve DHX15 that appeared to be flexibly tethered to the ILSAQR, we performed several 3D classification jobs varying the class similarity values with a mask around DHX15. The best classes from several classification jobs were combined, duplicate particles were removed and\u00a0locally refined with a broad mask around DHX15 and the surrounding regions, and further 3D classified with the same broad mask. The 9,659 particles from the two best classes were further locally refined with a mask around DHX15, resulting in map 7 at a nominal resolution of 8.62\u2009\u00c5.<\/p>\n Pre-processing and particle cleaning of human DIS<\/p>\n Pre-processing (gain correction, motion correction and contrast transfer function estimation) of the DIS dataset was performed in cryoSPARC live65<\/a>.<\/p>\n Particles were picked on-the-fly using the Blob picker in cryoSPARC live with a minimum and maximum particle diameter set to 250\u2009\u00c5 and 350\u2009\u00c5, respectively. The picked particles were extracted with a box size of 256\u2009pixels with a binned pixel size of 1.90\u2009\u00c5 per pixel. A 2D classification was performed to select the particles representing 2D class averages with the best internal features. The selected particles were subsequently used to generate three ab initio 3D models. Three rounds of heterogeneous refinement were performed using the ab initio models as references. Only the particles from the best class were carried over to the next round of refinement, resulting in the final subset of 140,678 particles. DIS particles were also picked in Warp66<\/a> using a model trained on manually picked particles with an expected particle diameter of 350\u2009\u00c5. The 514,099 Warp-picked particles were processed the same as above, resulting in a subset of 130,283 particles. Upon removal of duplicate particles, the combined Blob-picked and Warp-picked particles were re-extracted with a box size of 448\u2009px with a binned pixel size of 1.19\u2009\u00c5 per pixel, resulting in the final dataset of 153,470 particles.<\/p>\n Obtaining maps of human DIS<\/p>\n We used all 153,470 particles to perform a non-uniform refinement and obtained the overall consensus map of the DIS (map 1) at a global resolution of 3.14\u2009\u00c5. Although the core of the DIS in map 1 could be visualized at high resolution, many peripheral spliceosome regions were poorly defined and required further 3D classifications and local refinements to obtain high-quality cryo-EM densities.<\/p>\n To improve the density of the intron\u2013U6 snRNA duplex, we performed a 3D classification with all 153,470 particles with a mask around the RBM22\u2013intron\u2013U6 snRNA region. The best class from this classification was further refined using non-uniform refinement, resulting in map 2 at a nominal resolution of 4.67\u2009\u00c5.<\/p>\n To improve the density quality of the WD40 domain of PRP17, we performed a local refinement around this domain and obtained map 3 at a nominal resolution of 3.97\u2009\u00c5.<\/p>\n To improve the density quality of DHX35\u2013GPATCH1, we imported all 153,470 particles to RELION5 and performed a 3D classification with Blush regularization67<\/a> with a mask around DHX35\u2013GPATCH1 and a T value of 150. The best class from the 3D classification containing 63,636 particles was imported back into cryoSPARC. After homogeneous reconstruction, a local refinement with a mask around DHX35\u2013GPATCH1 was performed, generating map 4 at a nominal resolution of 2.90\u2009\u00c5.<\/p>\n We performed a 3D classification on all 153,470 particles to assess the homogeneity of the dataset. Although we did not observe multiple distinct spliceosome complexes in the dataset, we identified 45,958 particles that yielded a DIS map with the highest quality peripheral densities. We performed a non-uniform refinement on these particles to obtain a more complete DIS map at a nominal resolution of 3.62\u2009\u00c5 with better peripheral density quality than map 1. We used the same subset of particles to perform local refinements with masks around two NTC regions\u2014the PRP19\u2013CDC5L\u2013SPF27 helical bundle and the RBM22\u2013PPIL1\u2013PLRG1 region\u2014resulting in map 5 at a nominal resolution of 5.35\u2009\u00c5 and in map 9 at a nominal resolution of 4.04\u2009\u00c5, respectively.<\/p>\n To better visualize AQR within the DIS, we performed a hard 3D classification with a mask around AQR, producing a class with the best AQR density with 15,913 particles. After homogeneous reconstruction, a local refinement with a mask around AQR was performed, generating map 6 at a nominal resolution of 7.34\u2009\u00c5.<\/p>\n We consistently observed a low-resolution density near DHX35 that appeared to be shaped like a WD40 domain. Given our biochemical data and in silico protein interaction predictions (Fig. 1<\/a> and Extended Data Fig. 1<\/a>), we assigned this density as the GPATCH1 interactor WDR83. To improve this cryo-EM density, we performed multiple rounds of 3D classification. Specifically, we performed five 3D classification jobs with variable values of class similarity with a mask around the putative WDR83 density in cryoSPARC. The best classes from all jobs were selected. After removing duplicate particles, the selected particles were used in two rounds of 3D classification in RELION5 with a mask around WDR83 at T values of 80 for the first round and of 40 during the second round. The best class containing 1,826 particles was reconstructed using homogeneous reconstruction in cryoSPARC, generating map 7 at a nominal resolution of 9.65\u2009\u00c5. Given the limited resolution of map 7, we could not explicitly assign the orientation of WDR83 in the DIS.<\/p>\n In maps 1 and 2, the helical repeats of the NTR subunits SYF1\u2013SYF3 and the Sm ring of the U5 snRNP were not well resolved, probably due to the inherent flexibility of these spliceosomal regions. Therefore, we performed focus refinements with masks around SYF1\u2013SYF3 and the U5 Sm ring, resulting in map 8 at a nominal resolution of 5.19\u2009\u00c5 and map 10 at a nominal resolution of 3.35\u2009\u00c5, respectively.<\/p>\n Model building into cryo-EM mapsHuman ILSAQR<\/p>\n We generated the composite map of the human ILSAQR from ILSAQR maps 1\u201311 as previously described2<\/a>.<\/p>\n To prepare a model of the human ILSAQR, we started from our previously published model of the human ILS\u2033 (PDB 8RO2<\/a>)2<\/a>. First, we removed the following protein segments that were not observed in the cryo-EM density of the ILSAQR: SDE2, SYF1 residues 791\u2013824, CDC5L residues 57\u2013111 and 357\u2013382. To model the zinc-finger domain of YJU2B (residues 43\u2013118), we predicted its structure together with a Zn2+ ion using AlphaFold3 (ref. 36<\/a>) and rigid body fitted the predicted model of this domain. To model the helical region of YJU2B (residues 163\u2013233), we predicted the structures of the YJU2B\u2013SYF1\u2013SYF3\u2013C19L1 and YJU2B\u2013PRP8 complexes in AlphaFold3. Both models were rigid body fitted. We also added BRR2 residues 39\u201357 (as in PDB 8Q7X<\/a>)9<\/a>, PPIE RRM residues 1\u201380 (as in PDB 9FMD<\/a>)2<\/a> by rigid body fitting. We also modelled additional ESS2 regions (residues 65\u201387, 178\u2013189, 211\u2013234 and 240\u2013254) guided by AlphaFold3 predictions and ModelAngelo68<\/a>. The RNA in the active site of AQR was modelled as an unknown RNA sequence. The position of DHX15\u2013TFIP11 G-patch was adjusted by rigid body fitting into map 10. PAXBP1 was replaced with GCFC2 by first deleting the TFIP11\u2013PAXBP1 dimer from the model and then fitting an AlphaFold3 prediction of the TFIP11\u2013GCFC2 dimer into map 6. We truncated side chains for non-proline residues to their C\u03b2 atoms in map regions where side-chain density was lacking.<\/p>\n We modelled the RNA in the active site based on the structure of the human ILS assembled on the MINX pre-mRNA (PDB 6ID1<\/a>)3<\/a> but only assigned a sequence for a consensus 5\u2032 SS and a BP adenosine, whereas all other nucleotides were modelled as uridine and truncated to their ribose moiety. The portion of the intron bound to AQR and PPIE was modelled based on AlphaFold3 predictions.<\/p>\n All models were manually adjusted using ISOLDE69<\/a> in ChimeraX. The final model was real-space refined against the composite map in Phenix70<\/a>. We generated base-pairing and stacking restraints in Phenix to stabilize nucleic acid geometry. We further used the input model to generate reference model restraints. The non-bonded weight parameter was set to 1,000 and rotamers were fitted for side chains with poor density that were also outliers.<\/p>\n Human DIS<\/p>\n We generated the composite map of the human DIS from maps 1\u201310 as previously described2<\/a>.<\/p>\n To prepare a model of the DIS, we started from our previously published model of the human ILS\u2033 (PDB 8RO2<\/a>)2<\/a>. First, we deleted DHX15, TFIP11, PAXBP1, C19L1, C19L2, SDE2, ESS2 and U2 snRNA, which were not resolved in the cryo-EM density of the DIS. We also truncated U6 snRNA to residues 1\u201335 and the intron to 10 residues, six of which were base paired with U6 snRNA. Given that a mixture of introns is present in the endogenous DIS sample, no sequence was assigned to the intron.<\/p>\n To model DHX35 and GPATCH1, we predicted the structures of DHX35\u2013GPATCH1, GPATCH1\u2013PRP8 endonuclease domain and the GPATCH1\u2013PRP8 finger-palm domain using AlphaFold2 Multimer37<\/a>. The regions of GPATCH1 that were predicted with low confidence were truncated, and the predicted models were rigid body fitted into map 7. The segments of GPATCH1 towards its C terminus that were confidently predicted to interact with DHX35 but lacked experimental cryo-EM densities were also removed. The fitted models were adjusted using ISOLDE69<\/a> in ChimeraX. The segments of GPATCH1 that were not confidently predicted but were present in the cryo-EM density were manually built in ISOLDE.<\/p>\n The RNaseH domain of PRP8, which was relocated in the DIS compared with the ILS\u2033, was rigid body fitted into the DIS map. BRR2 residues 39\u201357 (as in PDB 8Q7X<\/a>)9<\/a> and the RRM domain of PPIE (residues 1\u201380; as in PDB 9FMD<\/a>)2<\/a> were added by rigid body fitting followed by manual adjustments in ISOLDE.<\/p>\n We also modelled the WD40 domains of PRP17 and of WDR83 bound to GPATCH1. The orientation of the PRP17 WD40 was assigned based on its position relative to the PRP17 N-terminal domain that anchors the protein on the surface of BUD31. We rigid body fitted the predicted AlphaFold2 Multimer model of WDR83\u2013GPATCH1 into map 9. Owing to the low local resolution of map 9, we could not unambiguously assign the orientation of WDR83\u2013GPATCH1. The resolution of map 9 was also not sufficient to unambiguously determine whether WDR83 was bound by GPATCH1. However, our biochemical data (Extended Data Fig. 6d<\/a>) and AlphaFold predictions (Fig. 1<\/a> and Extended Data Fig. 1<\/a>) strongly indicate that WDR83 is anchored by GPATCH1 in the DIS. We truncated side chains for non-proline residues to their C\u03b2 atoms in map regions where side-chain density was lacking.<\/p>\n The final model was real-space refined against the composite map in Phenix70<\/a> as described above for ILSAQR.<\/p>\n Revision of S. pombe B*D<\/p>\n To revise the structure of the defective fission yeast spliceosome B*D (PDB 9ESI<\/a>)23<\/a>, we first focused on identifying the protein chains that were originally modelled as \u2018unknown\u2019. We compared the published model to our models of the human ILSAQR and DIS. We noted that all but one of the unidentified chains were modelled in our new human spliceosome structures. We predicted the structures of the S. pombe proteins and their interactors in AlphaFold3 (ref. 36<\/a>) and rigid body fitted them into the published map (EMD-19942<\/a>)23<\/a>. This allowed us to add CDC5L (S. pombe name Cdc5) residues 456\u2013476, CCDC12 (Cwf18) residues 101\u2013142 and GPATCH1 (C20H4.06c) residues 260\u2013280.<\/p>\n We also identified an unassigned density on the surface of PPIL1 in the B*D spliceosome that was modelled as an N-terminal domain of SNW1 in our previously revised human P complex spliceosome (PDB 9FMD<\/a>)2<\/a>. We predicted the structure of S. pombe SNW1\u2013PPIL1 (S. pombe names Prp45\u2013Ppi1), rigid body fitted the model into the published map and further adjusted it manually using ISOLDE69<\/a>. This allowed us to add SNW1 (Prp45) residues 42\u201383 to the model of the S. pombe B*D spliceosome.<\/p>\n To identify the unknown density on the surface of CWC22 (S. pombe name Cwf22), we examined the previously published spliceosome structures for proteins modelled in the same location, as well as searched the human predicted interactome database20<\/a> for CWC22 interactors. The S. pombe orthologues of the\u00a0proteins that were identified as potential CWC22 interactors were then further tested for their predicted interaction with S. pombe CWC22 using AlphaFold3. This narrowed down the list of potential candidates to three proteins: CWC25 (S. pombe name Cwf25), SLU7 (Slu7) or LENG1 (SPCC5E4.10c). As described in the main text and in Extended Data Fig. 9a<\/a>, in the published map, we observed a segment of four residues with unambiguous side-chain densities: P-Y\/F-W followed by an amino acid with a side chain of at least two non-hydrogen atoms. Only the sequence of LENG1 matched the unambiguously resolved side-chain density (Extended Data Fig. 9a<\/a>). The S. pombe CWC22\u2013LENG1 prediction was rigid body fitted into the published map and then manually adjusted in ISOLDE, using a more confident prediction of the human proteins as a guide.<\/p>\n We also adjusted the RNA network of the B*D spliceosome. Specifically, we noticed implausible base-pairing geometry in the 5\u2032 SS\u2013U6 snRNA region, and adjusted the model based on an earlier S. pombe spliceosome structure (PDB 3JB9<\/a>)71<\/a>. Finally, we truncated intron nucleotides that were poorly resolved and outside of known consensus sequences to their ribose moiety.<\/p>\n We truncated side chains for non-proline residues to their C\u03b2 atoms in map regions where side-chain density was lacking. The final model was real-space refined against the published map (EMD-19942<\/a>)23<\/a> in Phenix70<\/a> as described above.<\/p>\n Endogenous DBR1 pulldown<\/p>\n Pulldowns with nuclear extracts from both induced and uninduced (negative control) K562 doxycycline-inducible DBR1\u2013EGFP cells were performed in technical triplicates. Of nuclear extract, 1\u2009ml was added to the GFP Trap beads slurry (Chromotek) equilibrated in wash buffer (20\u2009mM HEPES-KOH pH 7.9, 50\u2009mM, 0.5\u2009mM TCEP, 2\u2009mM MgCl2, 0.05% v\/v Igepal CA-630 and 8% v\/v glycerol). The beads were incubated for 3.5\u2009h at 4\u2009\u00b0C with gentle rotation. The supernatant was removed, and the beads were washed five times with 1\u2009ml wash buffer. After removing residual buffer, bead-bound proteins were eluted by addition of 150\u2009\u00b5l SDS loading dye. All three replicates of the pulldowns from extracts of both induced and control cells were run for 3\u2009min at 180\u2009V on a Novex Tris-glycine 4\u201312% 1.0\u2009mm WedgeWell gel (Thermo Fisher Scientific) and submitted to the Vienna BioCenter Mass Spectrometry Facility for in-gel tryptic digestion and LC\u2013MS\/MS analysis. Protein abundances in both DBR1\u2013GFP and control samples were compared, and the data were visualized in GraphPad Prism 10.<\/p>\n Endogenous LENG1 pulldown<\/p>\n Cell pellets were solubilized in EBC-1 (50\u2009mM Tris pH 7.5, 150\u2009mM NaCl, 0.5% NP-40, 2\u2009mM MgCl2, with protease and phosphatase inhibitor cocktails (Roche)) supplemented with 500\u2009U benzonase for 1\u2009h at 4\u2009\u00b0C on a rotator. The lysates were cleared from insoluble chromatin by centrifugation and were subjected to immunoprecipitation with GFP Trap beads (Chromotek) for 1.5\u2009h at 4\u2009\u00b0C under rotation. The beads were then washed five times with EBC-2 buffer (50\u2009mM Tris pH 7.5, 150\u2009mM NaCl, 0.5% NP-40 and 1\u2009mM EDTA) and boiled in Laemmli buffer for 10\u2009min at 95\u2009\u00b0C.<\/p>\n After the pulldown, the GFP beads were washed three times with 50\u2009mM ammonium bicarbonate, followed by overnight digestion using 0.5\u2009\u03bcg trypsin at 37\u2009\u00b0C under constant shaking. Digested peptides were separated from the beads using a 0.45-\u00b5m filter column (Merck) that was pre-washed with 50\u2009mM ammonium bicarbonate. Trypsin activity was quenched by acidifying the sample with trifluoroacetic acid to a final concentration of 1%. Peptides were desalted and concentrated using in-house-assembled triple-disc C18 stage-tip columns (66883-U; 3\u2009M) as previously described72<\/a>. In brief, the columns were first washed with methanol, then with buffer B (80% acetonitrile and 0.1% formic acid in water), followed by buffer A (0.1% formic acid in water). The samples were loaded using vacuum centrifugation. Finally, peptides were eluted with 32% acetonitrile and 0.1% formic acid in water, lyophilized and dissolved in 0.1% formic acid in water. All solvents were LC\u2013MS\/MS grade.<\/p>\n Mass spectrometry data were acquired at the proteomics facility of the Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER) using a Vanquish Neo liquid chromatographer (Thermo Scientific) coupled to an Orbitrap Astral mass spectrometer. Mass spectrometry RAW data were analysed using DIA-NN (v2.3.2 Academia)73<\/a>. Protein abundances in both LENG1\u2013GFP and control samples were compared, and the data were visualized in GraphPad Prism 10.<\/p>\n Proximity labellingLarge-scale proximity labelling and affinity purification<\/p>\n For proteomic analysis, each stable HEK293 Flp-In T-REx cell line that can inducibly overexpress a TurboID-tagged protein of interest (DBR1, C19L1, YJU2B or LENG1) and a control cell line (nuclear EGFP\u2013TurboID) were processed in biological triplicates. Three million cells per 10-cm dish were seeded. After 24\u2009h, TurboID expression was induced with 100\u2009ng\u2009ml\u22121 doxycycline for another 24\u2009h. This was followed by biotin labelling with 500\u2009\u00b5M d-biotin for 15\u2009min. After labelling, the reaction was stopped by placing dishes on ice. The medium was removed, and the cells were washed once with PBS. The cells were then collected by scraping them off in 5\u2009ml ice-cold PBS and transferred to a fresh tube. The cell pellet was washed for a total of four more times by centrifugation at 300g for 4\u2009min at 4\u2009\u00b0C before getting resuspended in 1\u2009ml ice-cold PBS. The final pellets were flash-frozen in liquid nitrogen and stored at \u221280\u2009\u00b0C.<\/p>\n The thawed cell pellets were lysed by addition of 120\u2009\u00b5l ice-cold RIPA buffer with Halt protease inhibitor cocktail (Thermo Fisher Scientific), followed by vigorous vortexing and incubation on ice for 20\u2009min. The lysates were clarified by centrifugation at 20,000g for 15\u2009min at 4\u2009\u00b0C. Protein concentration in the supernatant was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Of total protein, 300\u2009\u00b5g from each lysate was incubated for 3\u2009h at 4\u2009\u00b0C with 30\u2009\u00b5l of the MagReSyn Streptavidin MS magnetic bead slurry (ReSyn Biosciences) equilibrated in ice-cold RIPA buffer. The beads were washed three times with RIPA buffer supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific) with a 10-min rotation for each wash. This was followed by one wash in MS-grade PBS, during which the bead slurry was transferred to a fresh low-bind tube to minimize detergent carryover. Finally, five additional washes were performed with MS-grade PBS. After the final wash, all residual PBS was removed, and the dry bead pellets were flash-frozen in liquid nitrogen and stored at \u221280\u2009\u00b0C.<\/p>\n MS and data analysis<\/p>\n The final washed bead pellets were submitted either\u00a0to the Vienna BioCenter Mass Spectrometry Facility or to the Max Perutz Labs Mass Spectrometry Facility\u00a0on dry ice for on-bead tryptic digestion and analysis by LC\u2013MS\/MS. The resulting raw data were processed for protein identification and label-free quantification. To identify high-confidence proximity partners, protein abundances in each bait-TurboID sample were statistically compared against those from the control samples (nuclear-localized EGFP\u2013TurboID). Proteins were considered significantly enriched based on statistical tests (moderated\u00a0t-test\u00a0in limma, P\u2009<\u20090.05) and an arbitrary log2 fold-change cut-off of 0.75 (approximately 68% enrichment over control on a linear scale). Data visualization was performed in GraphPad Prism 10.<\/p>\n Purification of recombinant proteinsC19L1 MPP domain<\/p>\n The recombinant human C19L1 MPP domain (residues 1\u2013278), which carried an N-terminal His10-MBP tag followed by a 3C protease cleavage site, was expressed in Escherichia coli BL21 DE3 in LB media at 18\u2009\u00b0C overnight using a pOPINB+ plasmid.<\/p>\n The cell pellet was resuspended in lysis buffer (25\u2009mM HEPES-NaOH pH 7.9, 500\u2009mM NaCl and 5% glycerol) supplemented with cOmplete protease inhibitor tablets (Roche). The cells were lysed by sonication, and the lysate was cleared by centrifugation. The lysate was then applied to a 5-ml MBP Trap HP column (Cytiva) equilibrated in lysis buffer. The column was washed with lysis buffer before elution of the protein with a step gradient of 12\u2009mM maltose. The eluate was diluted fivefold with buffer A (25\u2009mM HEPES-NaOH and 5% glycerol) to reduce the salt concentration in the sample to 100\u2009mM NaCl. The diluted sample was applied to a 5-ml HiTrap Q anion exchange column (Cytiva) equilibrated in buffer M (25\u2009mM HEPES-NaOH, 100\u2009mM NaCl and 5% glycerol). The column was washed with buffer M before elution of the protein over a linear gradient of 100\u2013800\u2009mM NaCl. The peak fractions were concentrated using a Sartorius centrifugal concentrator with a 50-kDa molecular weight cut-off (Sigma) and subsequently loaded onto a HiLoad Superdex 75 16\/60 size-exclusion column (Cytiva) equilibrated in size-exclusion buffer (25\u2009mM HEPES-NaOH pH 7.9, 250\u2009mM NaCl, 1\u2009mM dithiothreitol and 5% glycerol). The peak size-exclusion fractions were pooled, concentrated and flash-frozen in liquid nitrogen.<\/p>\n DHX35<\/p>\n Human DHX35 was cloned into a pGB10 plasmid with an N-terminal His10\u22123\u00d7FLAG tag followed by a 3C protease cleavage site. The plasmid was introduced into E. coli DH10EMBacY cells to generate bacmids74<\/a>. The purified bacmid was transfected into Sf9 insect cells to produce a baculovirus that was then further amplified in Sf9 cell. The resultant virus was used to infect Hi5 insect cells at a volume ratio of 1:400. The cells were grown for approximately 72\u2009h before collecting by centrifugation.<\/p>\n The cell pellet was resuspended in lysis buffer (25\u2009mM HEPES pH 7.9, 500\u2009mM NaCl, 5% glycerol and 20\u2009mM imidazole) supplemented with cOmplete protease inhibitor tablets (Roche) and DNaseI (Sigma). The cells were broken open by sonication. The lysate was cleared by two rounds of centrifugation and subsequently applied to a 5-ml HisTrap column (Cytiva) equilibrated in lysis buffer. The column was washed with lysis buffer, and the protein was eluted over a linear gradient of 20\u2013350\u2009mM imidazole. The peak fractions containing DHX35 were pooled and diluted fivefold in buffer A (25\u2009mM HEPES-NaOH and 5% glycerol) to reduce the salt concentration in the sample to 100\u2009mM NaCl. The diluted sample was then loaded onto a 5-ml HiTrap Q anion exchange column (Cytiva) equilibrated in buffer M (25\u2009mM HEPES-NaOH, 100\u2009mM NaCl and 5% glycerol). The column was washed with buffer M before elution of the protein over a linear gradient of 0.1\u20131.4\u2009M NaCl. The peak fractions were concentrated using a Sartorius centrifugal concentrator with a 50-kDa molecular weight cut-off (Sigma) and subsequently loaded onto a HiLoad Superdex 200 16\/60 size-exclusion column (Cytiva) equilibrated in size-exclusion buffer (25\u2009mM HEPES-NaOH pH 7.9, 250\u2009mM NaCl, 1\u2009mM dithiothreitol and 5% glycerol). The peak size-exclusion fractions were pooled, concentrated and flash-frozen in liquid nitrogen.<\/p>\n GPATCH1 G-patch domain<\/p>\n The recombinant human wild-type and mutant (M163K V171R L236E) versions of the GPATCH1 G-patch domain (residues 137\u2013247), which carried an N-terminal MBP tag and a C-terminal His6 tag, were expressed in E. coli BL21 DE3 in LB media at 37\u2009\u00b0C for 3\u2009h using a pET plasmid.<\/p>\n Each cell pellet was resuspended in lysis buffer (25\u2009mM HEPES-NaOH pH 7.9, 500\u2009mM NaCl, 5% glycerol and 20\u2009mM imidazole) supplemented with cOmplete protease inhibitor tablets (Roche). The cells were lysed by sonication, and the lysate was cleared by centrifugation. The lysate was then applied to a 5-ml HisTrap column (Cytiva) equilibrated in lysis buffer. The column was washed with lysis buffer before elution of the protein over a linear gradient of 20\u2013350\u2009mM imidazole. The eluate was diluted fivefold with buffer A (25\u2009mM HEPES-NaOH and 5% glycerol) to reduce the salt concentration in the sample to 100\u2009mM NaCl. The diluted sample was applied to a 5-ml HiTrap Q anion exchange column (Cytiva) equilibrated in buffer M (25\u2009mM HEPES-NaOH, 100\u2009mM NaCl and 5% glycerol). The column was washed with buffer M before elution of the protein over a linear gradient of 100\u2013800\u2009mM NaCl. The peak fractions were concentrated using a Vivaspin centrifugal concentrator with a 30-kDa molecular weight cut-off (Sigma) and subsequently loaded onto a HiLoad Superdex 200 16\/60 size-exclusion column (Cytiva) equilibrated in size-exclusion buffer (25\u2009mM HEPES-NaOH pH 7.9, 200\u2009mM NaCl, 1\u2009mM dithiothreitol and 5% glycerol). The peak size-exclusion fractions were pooled, concentrated and flash-frozen in liquid nitrogen.<\/p>\n WDR83\u2013GPATCH1 complex<\/p>\n Human full-length WDR83 carrying an N-terminal His10-3\u00d7FLAG-3C tag and its binding region of human GPATCH1 (residues 560\u2013651) were cloned into a pGB10 plasmid. The plasmid was then used to generate a baculovirus and to\u00a0express the complex in Hi5 insect cells, as described above for DHX35.<\/p>\n The cell pellet was resuspended in lysis buffer (25\u2009mM HEPES-NaOH pH 7.9, 300\u2009mM NaCl, 1\u2009mM TCEP, 5% glycerol and 20\u2009mM imidazole) supplemented with cOmplete protease inhibitor tablets (Roche) and DNaseI (Sigma). The cells were broken open by sonication. The lysate was cleared by two rounds of centrifugation and subsequently applied to a 5-ml HisTrap column (Cytiva) equilibrated in lysis buffer. The column was washed with lysis buffer, and the protein was eluted over a linear gradient of 20\u2013350\u2009mM imidazole. The peak fractions containing WDR83\u2013GPATCH1 were pooled and diluted fivefold in buffer A (25\u2009mM HEPES-NaOH, 0.5\u2009mM TCEP and 5% glycerol) to reduce the salt concentration in the sample to 100\u2009mM NaCl. The diluted sample was then loaded onto a 5-ml HiTrap Q anion exchange column (Cytiva) equilibrated in buffer M (25\u2009mM HEPES-NaOH, 100\u2009mM NaCl, 0.5\u2009mM TCEP and 5% glycerol). The column was washed with buffer M before elution of the protein over a linear gradient of 0.1\u20131.4\u2009M NaCl. The peak fractions were concentrated using an Amicon centrifugal concentrator with a 30-kDa molecular weight cut-off (Sigma). Of the WDR83\u2013GPATCH1 complex at 79\u2009\u00b5M, 2\u2009ml was subsequently loaded via a 5-ml loop onto a HiLoad Superdex 200 16\/60 size-exclusion column (Cytiva) equilibrated in size-exclusion buffer (25\u2009mM HEPES-NaOH pH 7.9, 200\u2009mM NaCl, 0.5\u2009mM TCEP and 5% glycerol). SDS\u2013PAGE analyses of each chromatographic run showed that WDR83 and GPATCH1 co-purified throughout the procedure, indicating the formation of a stable protein complex. The peak size-exclusion fractions were pooled, concentrated and flash-frozen in liquid nitrogen.<\/p>\n DHX35(K83A)\u2013GPATCH1 1\u2013440 complex<\/p>\n Full-length human DHX35 carrying a K83A mutation and an N-terminal His10-3\u00d7FLAG tag, and the GPATCH1 construct encompassing the binding regions to both DHX35 and PRP8 (residues 1\u2013440) were cloned into a pGB10 plasmid. The plasmid was then used to generate a baculovirus and express the complex in Hi5 insect cells.<\/p>\n The 3\u00d7FLAG\u2013DHX35(K83A)\u2013GPATCH1(1\u2212440)\u2013TwStrep complex was purified as described for the WDR83\u2013GPATCH1 complex above, except for the size-exclusion step, in which the DHX35(K83A)\u2013GPATCH1(1\u2212440) complex was loaded onto a HiLoad Superdex 200 16\/60 size-exclusion column (Cytiva) equilibrated in size-exclusion buffer (25\u2009mM HEPES-NaOH pH 7.9, 250\u2009mM NaCl, 0.5\u2009mM TCEP and 5% glycerol).<\/p>\n Peptide synthesis<\/p>\n The peptides used in in vitro pulldown assays were synthesized at the Vienna BioCenter Peptide Synthesis facility as previously described2<\/a>. In brief, the peptides were synthesized with an N-terminal fluorescein and a C-terminal biotin tag on a Liberty Blue peptide synthesizer (CEM) using Fmoc chemistry. The lyophilized peptides were dissolved in 25\u2009mM HEPES-NaOH pH 7.9 to a concentration of 1\u2009mg\u2009ml\u22121. The following peptides were synthesized: DBR1 peptide I (420\u2013434) wild type: fluo-ALSSINPDEIMLDEE-biotin; DBR1 peptide I (420\u2013434) scrambled: fluo-EEIEMISLADDLSNP-biotin; YJU2B wild type (134\u2013178): fluo-EHEKKQKLETDAMFRLEHGEADRSTLKKALPTLSHIQEAQSAWKD-biotin; YJU2B(M146A L149K E150K) (134\u2013178): fluo-EHEKKQKLETDAAFRKKHGEADRSTLKKALPTLSHIQEAQSAWKD-biotin.<\/p>\n Pulldowns with purified proteinsMBP\u2013C19L1 MPP domain and DBR1 peptide I<\/p>\n Of MBP-tagged C19L1 MPP, 5\u2009\u00b5M was mixed with 50\u2009\u00b5M of either wild-type DBR1 peptide I or its scrambled version in the final reaction volume of 50\u2009\u00b5l. Reactions containing either MBP\u2013C19L1 MPP alone or the DBR1 peptides mixed with MBP as background binding controls were also prepared. The salt concentration in all reactions was adjusted to 100\u2009mM NaCl. Of each sample, 5\u2009\u00b5l was removed for SDS\u2013PAGE analysis as input. The binding reactions were incubated for 40\u2009min at 4\u2009\u00b0C while rotating. Subsequently, the samples were added to 30\u2009\u00b5l of amylose bead slurry (NEB) equilibrated in wash buffer (25\u2009mM HEPES-NaOH pH 7.9, 100\u2009mM NaCl, 1\u2009mM TCEP, 0.1% v\/v Igepal CA-630 and 5% v\/v glycerol). The beads were incubated for 2\u2009h rotating at 4\u2009\u00b0C. The supernatant was removed, and the beads were washed five times with 500\u2009\u00b5l wash buffer. Upon removal of residual buffer, 50\u2009\u00b5l elution buffer (25\u2009mM HEPES-NaOH pH 7.9, 50\u2009mM NaCl, 0.5\u2009mM TCEP, 5% v\/v glycerol and 20\u2009mM maltose) was added to each sample and incubated at 4\u2009\u00b0C for 30\u2009min while rotating. The eluates were collected, mixed with SDS loading dye and ran on a Novex Tris-Glycine 4\u201312% 1.0\u2009mm WedgeWell gel (Thermo Fisher Scientific) for 20\u2009min at 180\u2009V. The DBR1 peptides were visualized by fluorescence signal at 488\u2009nm on a ChemiDoc imager (Bio-Rad), whereas MBP\u2013C19L1 MPP and MBP were stained afterwards\u00a0with Coomassie blue (Vienna BioCenter Molecular Biology Service).<\/p>\n DHX35 and MBP\u2013GPATCH1 G-patch domain<\/p>\n Of DHX35, 4\u2009\u00b5M was mixed with 1\u2009\u00b5M of either wild-type or mutant (M163K V171R L236E) GPATCH1 G-patch domain (residues 137\u2013247) in the final reaction volume of 50\u2009\u00b5l. Reactions containing individual proteins were also prepared as background binding controls. The salt concentration in all reactions was adjusted to 50\u2009mM NaCl. All samples were incubated at 4\u2009\u00b0C for 35\u2009min while rotating. Of each sample, 5\u2009\u00b5l was removed for SDS\u2013PAGE analysis as input. Subsequently, each sample was added to\u00a040\u2009\u00b5l of amylose bead slurry (NEB) equilibrated in wash buffer (25\u2009mM HEPES-NaOH pH 7.9, 50\u2009mM NaCl, 0.5\u2009mM TCEP, 0.03% v\/v Igepal CA-630 and 5% v\/v glycerol). The proteins were incubated with the beads for 1.5\u2009h at 4\u2009\u00b0C while rotating. The beads were then washed five times with 0.5\u2009ml wash buffer. After removing residual wash buffer, 50\u2009\u00b5l of wash buffer supplemented with 40\u2009mM maltose was added to the beads and incubated for 1\u2009h at 4\u2009\u00b0C while rotating. The eluates were collected, mixed with SDS loading dye and ran on a Novex Tris-Glycine 4\u201312% 1.0\u2009mm WedgeWell gel (Thermo Fisher Scientific) for 30\u2009min at 180\u2009V. The gel was stained with Coomassie blue (Vienna BioCenter Molecular Biology Service).<\/p>\n WDR83\u2013GPATCH1 and biotin-tagged YJU2B peptide<\/p>\n Of either wild-type or mutant (M146A L149K E150K) YJU2B peptide (134\u2013178), which was predicted to interact with WDR83, 80\u2009\u00b5l was added at 25\u2009\u00b5M to 80\u2009\u00b5l StrepTactin Sepharose bead slurry (IBA) equilibrated in wash buffer (25\u2009mM HEPES-NaOH pH 7.9, 50\u2009mM NaCl, 1\u2009mM TCEP, 5\u2009mM MgCl2, 0.05% v\/v Igepal CA-630 and 10% v\/v glycerol). The beads were incubated with the peptides for 1\u2009h at 4\u2009\u00b0C while rotating and then washed four times with 0.4\u2009ml of wash buffer. The washed beads containing the immobilized peptides were split into two samples and\u00a0incubated either with or without 80\u2009\u00b5l of the WDR83\u2013GPATCH1(560\u2013651) complex at 29\u2009\u00b5M.\u00a0The\u00a0WDR83\u2013GPATCH1(560\u2013651) complex was also incubated with the beads lacking\u00a0immobilised peptides as a background\u00a0binding control.\u00a0The salt concentration in all samples was adjusted to 50\u2009mM NaCl and incubated at 4\u2009\u00b0C for 2\u2009h while rotating. The beads were then washed five times with 0.5\u2009ml wash buffer. After removal of residual wash buffer, 80\u2009\u00b5l of wash buffer supplemented with 50\u2009mM biotin was added to each reaction and incubated for 1\u2009h at 4\u2009\u00b0C while rotating. The eluates were collected, mixed with SDS loading dye and ran on a Novex Tris-Glycine 4\u201312% 1.0\u2009mm WedgeWell gel (Thermo Fisher Scientific) for 25\u2009min at 180\u2009V. The YJU2B peptides were visualized by fluorescence signal at 488\u2009nm on a ChemiDoc imager (Bio-Rad). The gel was then stained with Coomassie blue (Vienna BioCenter Molecular Biology Service) to visualize WDR83. The band corresponding to GPATCH1 in a Coomassie-stained gel could not be unambiguously identified, because its band overlaps with the bands of the YJU2B peptides.<\/p>\n DBR1 depletion rescue experiment<\/p>\n HEK293T homozygous DBR1\u2013dTAG-V5 cells containing the doxycycline-inducible mScarlet-tagged DBR1 constructs (wild type, catalytic mutant (H85A) and deletion of peptide I residues 420\u2013434 (\u0394pep1)) as well as a control line lacking DBR1 overexpression were mixed 1:1 with wild-type HEK293T cells that constitutively expressed GFP. Cells were split into two wells: one well was treated with the 500\u2009nM dTAGv-1 compound, whereas the other well was left untreated to serve as a control. Expression of the DBR1 constructs was induced using 1\u2009\u00b5g\u2009ml\u22121 doxycycline. Cells were split at a ratio of 1:3 every day to prevent them from reaching confluency. The percentage of GFP+ cells was measured using an iQue Screener Plus high-throughput cytometer (Sartorius AG) immediately after mixing and again after 8 days. The average percentage of GFP\u2212 cells per line was calculated from technical triplicates across three biological replicates and served as a proxy for cell growth phenotype.<\/p>\n Relative expression levels of the inducible DBR1 constructs were measured as the mScarlet signal in each well after 1 day, when the GFP signal, and hence, cell viability, did not differ across conditions. The average mScarlet signal was then normalized to that of the wild-type DBR1 construct.<\/p>\n In vitro ILSAQR-to-DIS remodelling assay<\/p>\n Of nuclear extract prepared from K562 cells stably overexpressing EGFP-tagged YJU2B, 120\u2009\u00b5l was incubated with the recombinant 3\u00d7FLAG\u2013DHX35(K83A)\u2013GPATCH1(1\u2212440)\u2013TwStrep complex at various final concentrations (0, 0.5\u2009\u00b5M or 4.5\u2009\u00b5M) either in the presence or absence of 5\u2009mM ATP. The reactions were incubated at 37\u2009\u00b0C for 40\u2009min before being applied to 45\u2009\u00b5l GFP Trap bead slurry (Chromotek) equilibrated in wash buffer (20\u2009mM HEPES-KOH pH 7.9, 100\u2009mM KCl, 0.5\u2009mM TCEP, 2\u2009mM MgCl2, 0.05% v\/v Igepal CA-630 and 8% v\/v glycerol) supplemented with 2\u2009mg\u2009ml\u22121 BSA. The binding reactions were incubated for 1.5\u2009h at 4\u2009\u00b0C. The beads were then washed three times with wash buffer and twice with harsh wash buffer (20\u2009mM HEPES-KOH pH 7.9, 200\u2009mM KCl, 0.5\u2009mM TCEP, 2\u2009mM MgCl2, 0.1% v\/v Igepal CA-630 and 8% v\/v glycerol). The proteins immobilized on the beads were eluted in 60\u2009\u00b5l detergent-free wash buffer (20 mM HEPES-KOH pH 7.9, 100 mM KCl, 0.5 mM TCEP, 2 mM MgCl2, 8% v\/v glycerol)\u00a0supplemented with 0.1\u2009\u00b5g\u2009ml\u22121 3C protease for 1\u2009h at 4\u2009\u00b0C.<\/p>\n The eluates from all samples were then analysed by western blot using standard protocols with the antibodies to spliceosome proteins YJU2B (CAS OAAB02156, Aviva Systems Biology; dilution 1:100), PRP8 (CAS AB190347, Abcam; dilution 1:1,000), BRR2 (CAS AB241589, Abcam; dilution 1:1,000), AQR (CAS A302-546A, Bethyl Laboratories; dilution 1:1,000), TFIP11 (CAS PA5-49275, Invitrogen; dilution 1:1,000), C19L1 (CAS 31700-1-AP, Proteintech; dilution 1:5,000) and C19L2 (CAS 24872-1-AP, Proteintech, dilution 1:200). 3\u00d7FLAG\u2013DHX35(K83A) and GPATCH1(1\u2212440)\u2013TwStrep were detected using antibodies to FLAG (CAS F3165, Sigma-Aldrich; dilution 1:500) and Strep (CAS 2-1509-001, iba; dilution 1:10,000) tags, respectively.<\/p>\n RNA sequencing assayExtraction of spliceosome-associated RNA<\/p>\n For RNA sequencing, endogenous human DIS was purified by anti-GFP affinity purification and gradient ultracentrifugation as described above for cryo-EM sample preparation, except that no glutaraldehyde was added to the gradient and that the pooled spliceosome-containing gradient fractions were not concentrated.<\/p>\n Endogenous human ILS\u2033 for the deletion rate experiment and endogenous human ILSAQR for the read truncation experiment were purified from K562 cells stably overexpressing TFIP11\u2013GFP or YJU2B\u2013GFP, respectively, using anti-GFP affinity purification and gradient ultracentrifugation as described above and previously2<\/a>, except that no glutaraldehyde was added to the gradient and the peak gradient fractions were not concentrated.<\/p>\n The pooled spliceosome fractions were incubated with 0.4\u2009mg\u2009ml\u22121 proteinase K in 2\u2009mM EDTA and 0.04% SDS for 1.5\u2009h at 37\u2009\u00b0C with gentle shaking. A 0.1 volume of 7.5\u2009M ammonium acetate was added to the sample and mixed thoroughly. One volume of phenol-chloroform solution (ITW reagents) was added to the sample and briefly vortexed. The sample was spun down at 20,000g for 5\u2009min. The top aqueous layer containing nucleic acids was transferred to a fresh tube, mixed with 2 volumes of isopropanol and 0.002 volumes of GlycoBlue co-precipitant (Thermo Fisher Scientific), and incubated overnight at \u221220\u2009\u00b0C. The sample was then centrifuged at 20,000g for 5\u2009min at 4\u2009\u00b0C. The supernatant was removed, and the remaining pellet was washed in ice-cold 70% ethanol. The washed pellet was briefly air dried and resuspended in 10\u2009\u00b5l nuclease-free water.<\/p>\n Preparation of short-read sequencing libraries (read truncation experiment)<\/p>\n For each sample, 3\u2009\u00b5l of RNA was polyadenylated by incubating in a reaction containing 2\u2009\u00b5l 5\u00d7SSIV reaction buffer (Invitrogen), 0.25\u2009\u00b5l E. coli polyA polymerase (NEB), 0.25\u2009\u00b5l RNasin (Promega), 0.25\u2009\u00b5l 0.1\u2009M dithiothreitol (Invitrogen) and 0.5\u2009\u00b5l 0.5\u2009mM ATP (Invitrogen) at 37\u2009\u00b0C for 15\u2009min. The samples were placed on ice, diluted with 9.25\u2009\u00b5l water, then heated at 95\u2009\u00b0C for 4\u2009min to fragment the RNA. To anneal reverse transcription primers, 1\u2009\u00b5l of 10\u2009mM dNTPs and 1\u2009\u00b5l of 10\u2009\u00b5M dT(22)V-P7 primer were added, and the samples were heated to 65\u2009\u00b0C for 5\u2009min before cooling to 42\u2009\u00b0C. To initiate reverse transcription, 2\u2009\u00b5l of 5\u00d7SSIV reaction buffer (Invitrogen), 0.5\u2009\u00b5l 0.1\u2009M dithiothreitol (Invitrogen) and 0.5\u2009\u00b5l SSIV enzyme (Invitrogen) were added to the samples, and the reactions were incubated at 42\u2009\u00b0C for 20\u2009min. The reactions were cooled to 37\u2009\u00b0C and incubated with 2\u2009\u00b5l exonuclease I (NEB) for 15\u2009min to remove excess reverse transcription primers. To further remove excess RT primers, the samples were size selected using SPRI beads with a final PEG8000 concentration of 7.5% and a final isopropanol concentration of 22.5%. The samples were eluted from the beads, then RNA was hydrolysed by the addition of 1\u2009\u00b5l 1\u2009M NaOH and incubated at 95\u2009\u00b0C for 10\u2009min. The pH of the samples was then neutralized by the addition of 1\u2009\u00b5l 1\u2009M HCl and 10\u2009\u00b5l 1\u2009M HEPES pH 7.9. The cDNA was then purified using 2 volumes of SPRI beads and 3 volumes of isopropanol. The cDNA was eluted in 5.3\u2009\u00b5l water, to which 1\u2009\u00b5l DMSO and 2\u2009\u00b5l 10\u2009\u00b5M L01clip2.0 adapter was added. The mixture was heated to 75\u2009\u00b0C for 2\u2009min and then placed on ice. Adapter ligation was initiated by the addition of 2\u2009\u00b5l 10\u00d7 T4 RNA ligase reaction buffer (NEB), 0.2\u2009\u00b5l 100\u2009mM ATP (NEB), 9\u2009\u00b5l 50% PEG 8000 (NEB) and 0.5\u2009\u00b5l T4 RNA ligase 1 high concentration (NEB), and the reactions were incubated overnight at room temperature. The reaction was size selected using 20\u2009\u00b5l SPRI beads and 13.33\u2009\u00b5l isopropanol (final concentration of 25%). The resulting cDNA was amplified in a 50\u2009\u00b5l quantitative PCR using Illumina-compatible i5\/i7 indexed primer pairs and NEBNext Ultra II Q5 (NEB) according to the manufacturer instructions until the libraries appeared to amplify linearly. The resulting libraries were purified using 1.5 volumes of SPRI beads, then sequenced using a NovaSeqX 10B XP 2\u2009\u00d7\u2009150bp flow cell (Illumina) at the VBCF Next Generation Sequencing Facility.<\/p>\n Analysis of short-read sequencing data (read truncation experiment)<\/p>\n Complete reproduction of all data analysis from the raw sequencing data to the final plots can be performed by executing the Snakemake pipeline (https:\/\/github.com\/rfara\/spliceosome_disassembly<\/a>). In brief, all read pairs were trimmed of adapter and polyA sequences using cutadapt (v4.9) and UMI sequences were extracted using UMI-tools (v1.1.5) pre-mapped against the full human rDNA repeat sequence and GENCODE (v44) basic snRNA sequences using bowtie2 (v2.5.4). Unmapped reads pairs were mapped using STAR (v2.7.11b) against the GRCh38 primary assembly and GENCODE (v44) basic annotation. Uniquely mapping reads were deduplicated using umitools (v1.1.5). Introns and exons were defined using MANE Select transcripts from GENCODE (v44). Branchpoint locations were obtained from publicly available CoLa-seq data75<\/a>, and the highest scoring branchpoint per intron was used for analysis. Read pairs for which the 3\u2032 end mapped to the 3\u2032 SS were considered to be derived from excised introns.<\/p>\n Preparation of long-read sequencing libraries (deletion rate experiment)<\/p>\n Of RNA, 5.5\u2009\u00b5l was poly-adenylated by addition of 7\u2009\u00b5l of the poly-adenylation reaction mix (4\u2009\u00b5l 5X SuperScript IV RT buffer (Invitrogen), 1\u2009\u00b5l 10\u2009mM ATP, 1\u2009\u00b5l 100\u2009mM dithiothreitol, 0.5\u2009\u00b5l RNasin (Promega) and 0.5\u2009\u00b5l E. coli polyA polymerase (NEB)) and incubation at 37\u2009\u00b0C for 20\u2009min. To begin primer annealing, 1\u2009\u00b5l of 25\u2009mM dNTPs and 1\u2009\u00b5l of 10\u2009\u00b5M barcoded reverse transcription primer were added to the reaction, and the reaction was heated to 65\u2009\u00b0C for 3\u2009min. The reaction was cooled to 42\u2009\u00b0C, and reverse transcription was initiated by the addition of 5.5\u2009\u00b5l of the pre-heated reverse transcription mix (4\u2009\u00b5l 5\u2009M betaine, 0.5\u2009\u00b5l 40\u2009\u00b5M template switching oligo, 0.5\u2009\u00b5l 200\u2009mM MgCl2 and 0.5\u2009\u00b5l SuperScript IV (Invitrogen)). The reaction was incubated at 42\u2009\u00b0C for 1\u2009h, then purified using homemade SPRI beads at a ratio of 1.8X. Library amplification quantitative PCR was performed using PCR mix (NEBNext Ultra II Q5 Master Mix, 1X EvaGreen (Biotium) and 500\u2009mM partial P5 and P7 primers) with a long extension time (98\u2009\u00b0C for 10\u2009s, 69\u2009\u00b0C for 20\u2009s and 72\u2009\u00b0C for 3\u2009min 30\u2009s). Samples were removed when linear amplification was observed and subsequently purified with 1.5X SPRI beads. The resulting libraries were quantified using the Qubit HS DNA kit, and the distribution of fragment sizes was quantified using the Fragment Analyzer. The libraries were sequenced using a Nanopore R10.4.1 Flow Cell.<\/p>\n Analysis of long-read sequencing data (deletion rate experiment)<\/p>\n The raw Nanopore data were base called using Dorado (v0.9). The libraries were demultiplexed and trimmed of adapter sequences and polyA sequences using cutadapt (v5.0). The reads were then mapped sequentially with minimap2 (v2.29)76<\/a> to rRNA sequences, snRNA sequences, spliced transcriptomic sequences and the genome, with the unmapped reads from mapping to each reference being passed to the next mapping. All reference sequences were derived from the hg38 human genome and the GENCODE (v44) annotation.<\/p>\n Branchpoint locations were annotated by CoLa-seq75<\/a>. Deletion positions within reads were identified, with the value of a deletion being divided by the length of the deletion, and metaprofiles of the deletions were calculated relative to these branchpoint positions by summing the deletions at every position and dividing by the number of input reads. For the proportion of spliced transcripts to intronic reads, the total number of transcriptomic mapping reads and intron mapping reads was used.<\/p>\n DIS release assaySample preparation<\/p>\n Of nuclear extract from K562 cells expressing either wild-type DHX35 or mutant DHX35(K83A), 3\u2009ml was applied to 500\u2009\u00b5l GFP Trap bead slurry (Chromotek) equilibrated in wash buffer (20\u2009mM HEPES-KOH pH 7.9, 100\u2009mM KCl, 0.5\u2009mM TCEP, 2\u2009mM MgCl2, 0.05% v\/v Igepal CA-630 and 8% v\/v glycerol). The beads were incubated for 3\u2009h at 4\u2009\u00b0C with gentle mixing. The beads were then washed five times with 3\u2009ml wash buffer, transferred to fresh tubes and washed five more times with 1.5\u2009ml detergent-free wash buffer (20\u2009mM HEPES-KOH pH 7.9, 100\u2009mM KCl, 0.5\u2009mM TCEP, 2\u2009mM MgCl2 and 8% glycerol). A small volume of both wild-type and mutant DHX35 bead samples was removed, incubated with 180\u2009\u00b5l detergent-free wash buffer supplemented with 0.1\u2009\u00b5g\u2009ml\u22121 3C protease overnight at 4\u2009\u00b0C to elute the bound DIS complexes that represented an assay input. The rest of the beads of the wild-type and mutant DHX35 samples were both distributed across six fresh tubes each, resulting in three technical replicates per sample. After removing residual wash buffer, 120\u2009\u00b5l detergent-free wash buffer was added to each tube and incubated for 15\u2009min at 37\u2009\u00b0C to allow temperature equilibration, which may cause nonspecific dissociation of various proteins from the beads. The supernatant was then removed and 120\u2009\u00b5l detergent-free wash buffer supplemented with 20\u2009mM MgCl2 and either 10\u2009mM ATP or 10\u2009mM ADP was added to three tubes each of both the wild-type and mutant DHX35 samples. The assays were incubated for 30\u2009min at 37\u2009\u00b0C shaking at 800\u2009rpm. The samples were then spun down, and the supernatants containing the proteins released from the beads were collected, run for 3\u2009min at 180\u2009V on a Novex Tris-glycine 4\u201312% 1.0\u2009mm WedgeWell gel (Thermo Fisher Scientific) and submitted to the Vienna BioCenter Mass Spectrometry Facility for in-gel tryptic digestion and LC\u2013MS\/MS analysis.<\/p>\n Release assay analysis<\/p>\n The resulting raw MS data were processed for protein identification and label-free quantification. We found that one replicate of the DHX35(K83A) ADP sample had not been injected correctly for LC\u2013MS\/MS, leaving two instead of three technical replicates for this condition.\u00a0To identify the proteins that are released from the beads upon ATP treatment, protein abundances in each ATP sample were compared with protein abundances in the corresponding ADP samples. The samples of wild-type DHX35 and DHX35(K83A) were compared separately to account for potential differences in the starting protein amounts on the beads. Proteins were considered significantly enriched based on statistical tests (moderated\u00a0t-test\u00a0in limma, P\u2009<\u20090.05) and an arbitrary log2 fold-change cut-off of 1 (twofold enrichment over control on a linear scale). Data were visualized in GraphPad Prism 10.<\/p>\n Release assays were also analysed by western blot using standard protocols to validate that DHX35 that could not be detected by MS was not non-specifically released from the beads. Input samples as well as the released protein samples form a single replicate of wild-type DHX35 and DHX35(K83A) each were probed with the antibodies to the spliceosomal proteins DHX35 (CAS HPA062700, Sigma; dilution 1:250), WDR83 (CAS PA-60143, Invitrogen; diluted 1:500), BRR2 (CAS AB241589<\/a>, Abcam; dilution 1:1,000), PRP19 (CAS AB228694<\/a>, Abcam; dilution 1:1,000) and CWC15 (CAS 25293-1-AP, Proteintech; dilution 1:1,500). The primary antibodies were incubated with the milk-blocked membranes overnight at 4\u2009\u00b0C. A horseradish peroxidase-coupled goat anti-rabbit secondary antibody (CAS 31466, Invitrogen; dilution 1:10,000) was applied to each membrane before developing with Pierce ECL reagent (Thermo Fisher Scientific).<\/p>\n Reporting summary<\/p>\n