Fig 2: INTS10 forms a module with INTS13–INTS14 in cells and binds to the MIDAS pocket of the INTS14 VWA domain.a Size exclusion chromatography of nuclear extract from HEK293T cells. All even fractions were analyzed by Western blotting. INTS1 and INTS7 served as marker proteins for the INT core, while INTS4–9–11 form the cleavage module. Elution points of molecular weight markers are indicated on the top. In addition, elution volumes of purified INTS13–INTS14 and INTS10–INTS13–INTS14 are indicated. b, c Coprecipitation of HA-INTS10 from HEK293T cells with V5-SBP-INTS13 b or V5-SBP-INTS14 c. Wild-type and binding patch double mutants were compared. Coprecipitation of endogenous INTS14 or INTS13 is detected as control for complex disruption. Inputs (αV5-blots 1%, αHA-blots 0.38% and 0.75%, αINTS14-blot 0.38%, αINTS13-blot 0.03%) and bound fractions (αV5-blots 3%, αHA-blots 20 and 15%, αINTS14/αINTS13-blots 20%) were analyzed by Western blotting. d Deletion constructs of V5-SBP-INTS14 were tested for copurification of HA-INTS10 and endogenous INTS13. Inputs (αV5-blot 1%, αHA-blot 1%, αINTS13-blot 0.1%) and bound fractions (αV5-blot 4%, αHA-blot 5%, αINTS13-blot 20%) were characterized by Western blotting. e Copurification of MBP-INTS10 with GST-INTS14-VWA from E. coli lysates. GST served as negative control. f Structure superposition (r.m.s.d. 3.6 Å over 160 residues) of the VWA domain of INTS14 (green) with the VWA domain of integrin-αL (ITGAL, gray, PDB-ID: 1T0P73) in complex with intercellular adhesion molecule 3 (ICAM3, purple). The black square highlights the position of the MIDAS pocket, residues lining the pocket are shown as sticks. g Focused view of the MIDAS pockets of both VWA domains highlights conservation of the site in INTS14. The Mg2+-ion in the ITGAL pocket that coordinates a Glu of the interaction partner ICAM3 is shown as a gray sphere. Ion coordination is indicated by black dotted lines. Coloring as in f. Residues mutated in INTS14 are underlined. h Coprecipitation of HA-INTS10 from HEK293T cells with V5-SBP-INTS14 wt or MIDAS pocket mutants (D8A, S10A, S12A [DA2SA] and L11E, R15A [LERA]). V5-SBP-MBP served as control. Inputs (αV5-blots 1%, αHA-blots 0.75%) and bound fractions (αV5-blots 3%, αHA-blots 15%) were analyzed by Western blotting.

Fig 3: INTS10–13–14 are required for termination after pausing.a Scheme of the RNAPII pause-termination reporter used in b. Reduced RNAPII pausing and termination at the HIV-1 TAR element results in elongation and increased RLuc levels. b Luciferase assay using the TAR pause-termination reporter. Cells depleted of individual INT subunits were transfected with the TAR reporter and FLuc transfection control plasmid. Statistical significance was determined for each knockdown condition relative to control. Bars show mean values of biological triplicates together with the individual replicates as dots. Error bars represent standard deviations. Statistical significance was determined by unpaired, two-tailed t-test: not significant (ns) p ≥ 0.05; significant *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Western blots in Fig. 6a. Source data are provided as a Source Data file. c Summary model of INT modules and their respective protein interaction networks. INTS10–INTS13–INTS14 brings cleavage module and target RNA into proximity.

Fig 4: INTS10–INTS13–INTS14 binds the INT cleavage module via a conserved motif in the INTS13 C-terminus.a Coomassie-stained gel and corresponding Western blots from copurification of the INT cleavage module (INTS4–INTS9–INT11) with 2S-INTS13–INTS14 or 2S-INTS10–INTS13–INTS14 using purified complexes. S-MBP served as negative control. b Coomassie stained gel and corresponding Western blots from copurification of INT cleavage module from insect cell extracts with GST-INTS13 C-term constructs from E.coli lysates. GST served as negative control. The asterisk marks endogenous GST from insect cells that also binds to GSH-beads. c Coprecipitation of endogenous INT subunits with V5-SBP-INTS13 wild type and mutants. V5-SBP-MBP is included as a control. Inputs (αV5-blot 1%, αINTS14 0.5%, and others 0.1%) and bound fractions (αV5-blot 5%, αINTS14 10%, and others 20%) were analyzed by Western blotting.

Fig 5: The INTS10–INTS13–INTS14 module has an accessory role in UsnRNA processing.a Representative Western blot of cells treated with siRNAs against INT subunits or non-targeting siRNA (corresponding to Fig. 7b). Actin served as loading control. b Scheme of qPCR-binding sites for amplification of mature (U1) and misprocessed (U1mis) U1 snRNA. c Relative U1 snRNA levels as determined by RT-qPCR. Clear bars represent U1 levels, while shaded bars represent levels of U1mis. All values were determined relative to U6 snRNA, which as an RNAPIII transcript is independent of INT, and were further normalized to the control knockdown condition. Statistical significance of U1 or U1mis levels was determined relative to their respective levels in the control depletion. d Scheme of U7 luciferase reporter construct used in e–g. Impaired U7 snRNA transcript cleavage by INT at the 3′-box leads to increased Renilla Luciferase (RLuc) levels. PAS polyadenylation signal. e, f Luciferase assay using the U7-reporter in cells depleted for individual INT subunits e or two INTS10–INTS13–INTS14 module subunits f. A plasmid expressing Firefly luciferase (FLuc) under control of a CMV promoter was used as transfection control. Statistical significance was determined for each knockdown relative to the control. g Rescue assays for INTS13 function in snRNA processing using the U7-reporter. Cells were treated with a control siRNA or INTS13 siRNA and transfected with HA-tagged INTS13 wt or mutants together with the reporter and FLuc transfection control. MBP served as negative control. All bar graphs depict mean values of biological triplicates shown together with individual values as dots, and error bars representing standard deviations. Statistical significance was determined by unpaired, two-tailed t-test: not significant (ns) p ≥ 0.05; significant *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Western blots for all experiments and immunofluorescence assays for INTS13 mutants are shown in Supplementary Fig. 8. Source data are provided as a Source Data file.