Fig 2: BRAT1 interacts with the Integrator catalytic cleavage heterodimer.a A volcano plot of proteins enriched in BRAT1 immunoprecipitates from U2OS cells. The –Log10 (Student’s t-test FDR) is plotted against the difference of mean intensities between wild-type and BRAT1-/- samples. Red dots indicate significantly enriched proteins (intensity difference >1; FDR < 0.05). b, c Levels of BRAT1, INTS11 and INTS9 in BRAT1 (b) and INTS11 (c) immunoprecipitates from wild-type (U2OS wt) and BRAT1-/- (clone #8) cells, measured by Western blotting. The black asterisk marks a nonspecific band. d Levels of BRAT1, INTS11 and INTS9 in BRAT1 immunoprecipitates from BRAT1-/- (clone #8), U2OS wt, and U2OS cells transiently transfected with siINTS9 or siINTS11 as indicated. The black asterisk marks a nonspecific band. b–d The experiment was performed three times with similar results. e Immunoblot of indicated proteins and quantification in U2OS wt and BRAT1-/- cells (clones #8, #6, and #16). Represented as the mean ± SD (n = 3 for INTS1, INTS3, INTS4 and INTS9; n = 4 for INTS11). Statistical significance was determined by a one-sided paired Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ns not significant). Samples derived from the same experiment and blots were processed in parallel. Uncropped and unprocessed scans are provided in the Source Data file.

Fig 3: Termination of snRNA Transcription Is Partially Impaired by Integrator Depletion(A) Schematic of the SMASh tag system. Under normal conditions, the degron-encoded NS3 protease (green) removes the tag and the protein is stable. Addition of ASN (red) prevents this promoting proteosomal degradation.(B) Western blot demonstrating ASN-dependent depletion of INTS11 after 48 h. Unmodified cells are shown as a control and the lower tubulin blot shows loading.(C) qRT-PCR analysis of unprocessed (UC, uncleaved) RNU4-1 and RNU5A-1 transcripts in INTS11-SMASh cells treated or not with ASN. Levels in treated cells are shown relative to untreated cells after normalizing to spliced actin levels. The schematic shows snRNA (black rectangle), canonical Integrator cleavage site (scissors), downstream 3' box (hatched box), and primers used (arrows). n = 4, error bars are standard error of the mean (SEM).(D) qRT-PCR of RNU5 and RNU1 variants in HCT116 or INTS11-SMASh cells treated or not with ASN. Primers were used to detect read-through downstream of the annotated 3' ends of each gene, with the distance indicated under each set of bars. Values are expressed as a fold change versus untreated HCT116 cells after normalizing to spliced actin levels. n = 3, error bars are SEM.(E) Metagene plot of snRNA genes from nuclear RNA-seq performed on INTS11-SMASh cells treated or not with ASN. Signal is reads per kilobase of transcript, per million mapped reads (RPKM). TSS, transcription start site; TES, annotated snRNA 3' end.(F) Metagene plot of read-through at all expressed snRNA genes in chromatin-associated RNA-seq from HCT116 cells treated with control or INTS1-specific siRNAs. Signal is RPKM.

Fig 4: 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 (aV5-blots 1%, aHA-blots 0.38% and 0.75%, aINTS14-blot 0.38%, aINTS13-blot 0.03%) and bound fractions (aV5-blots 3%, aHA-blots 20 and 15%, aINTS14/aINTS13-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 (aV5-blot 1%, aHA-blot 1%, aINTS13-blot 0.1%) and bound fractions (aV5-blot 4%, aHA-blot 5%, aINTS13-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-aL (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 (aV5-blots 1%, aHA-blots 0.75%) and bound fractions (aV5-blots 3%, aHA-blots 15%) were analyzed by Western blotting.

Fig 5: snRNA Precursors Are Targeted for 3'?5' Degradation by the Exosome(A) qRT-PCR analysis of INTS11-SMASh cells transfected with control or EXOSC3-siRNAs before treatment or not with ASN. Unprocessed RNU4-1 and RNU5A-1 are quantitated relative to control siRNA transfected cells untreated with ASN (siC -ASN) after normalizing to spliced actin. n = 3, error bars are SEM.(B) qRT-PCR analysis of DIS3-AID cells transfected with control or INTS1 siRNAs before treatment or not with auxin (2 h). Unprocessed RNU4-1 and RNU5A-1 are shown relative to control siRNA transfected cells not treated with auxin (siC -IAA) after normalizing to spliced actin. n = 4, error bars are SEM.(C) qRT-PCR analysis of DIS3-AID cells transfected with control or INTS1 siRNAs before treatment or not with auxin (2 h). RNU1-1 and RNU4-2 extended read-through is quantitated relative to siCont-IAA samples after normalizing to spliced actin levels. n = 3, error bars are SEM.(D) qRT-PCR analysis of snRNA precursor stability in DIS3-AID cells transfected with control or INTS1 siRNAs before treatment or not with auxin. Quantitation shows the proportion of precursor remaining after 15 min of actD treatment versus the respective untreated condition (t0). n = 4, error bars are SEM.(E) Meta-analysis of DIS3 occupancy on snRNA genes as determined by PAR-CLIP performed in HEK293 cells (Szczepinska et al., 2015) and of INTS11 occupancy on snRNAs derived from published eCLIP in HeLa cells (Barra et al., 2020). Data from two different INTS11 antibodies are displayed. The y axes shows RPKM (left for DIS3, right for INTS11).