Fig 1: Loss of BRAT1 impairs 3' end processing of capped UsnRNAs and snoRNAs.a Schematic representation of binding sites for primers used for RT-qPCR (arrows), canonical Integrator cleavage sites (scissors), and a downstream 3´box. b RT-qPCR analysis of unprocessed RNU1-1 transcripts in U2OS and BRAT1-/- (clone #8) cells transfected with control non-targeting siRNA (siNT) or siINTS11, as indicated. Data are represented as the mean ± SD (n = 3). Statistical significance was determined by a one-sided paired Student’s t-test (*p < 0.05, **p < 0.01). c RT-qPCR analysis of unprocessed RNU2-1 or RNU7-1 transcripts in wild-type (U2OS wt) and BRAT1-/- (clone #8) cells. Data are represented as the mean ± SD (n = 3). Statistical significance was determined by a one-sided paired Student’s t-test (*p < 0.05, **p < 0.01). d RNA-seq data for genes coding U1 snRNA transcripts in wild-type U2OS (wt #1 and wt #2) and BRAT1-/- (clone #6 and #8) cells. Read coverage is shown on the left and metaplot analysis of library-normalized averaged samples on the right. TSS transcription start site. See also Supplementary Fig. 3a. e Total RNA was isolated from U2OS and BRAT1-/- (clone #8) cells transfected with control non-targeting siRNA (siNT) or siINTS11, as indicated, resolved on urea-PAGE, Northern blotted and probed for levels of U1, U2, U7 snRNA, and 5S rRNA. Representative blots and quantification are shown. Data are represented as the mean ± SD (n = 4 for U1 and U2; n = 3 for U7). Statistical significance was determined by a one-sided paired Student’s t-test (*p < 0.05, **p < 0.01, ns not significant). f Total RNA was isolated from wild-type (U2OS wt) and BRAT1-/- (clone #8) cells, resolved on urea-PAGE, and silver-stained. The experiment was performed three times with similar results. Uncropped and unprocessed scans are provided in the Source Data file. g RNA-seq data for genes coding U3 snoRNA transcripts presented similarly as in d.
Fig 2: Altered Integrator function in BRAT1-mutated patient cells.a RNA-seq coverage plots for genes coding U1, U4, U5, or U6 snRNA in unaffected parents (Mother and Father) and BRAT1 patient-derived (Patient 1 and Patient 2) lymphoblastoid cell lines (LCLs) are shown, as indicated. b Metaplot analysis of mean reads coverage of individual U1, U4, U5, or U6 snRNA transcripts from unaffected parents (Mother and Father) and BRAT1 patient-derived (Patient 1 and Patient 2) LCLs. Data are presented as the smoothed mean coverage of the selected genes (n = 4) averaged across samples (n = 2) in the respective group with 95% confidence interval of the mean shown (ribbon). TSS transcription start site. c RT-qPCR analysis of total and unprocessed RNU1-1 transcripts in control, parent and BRAT1 patient-derived fibroblasts (top) and LCLs (bottom). Data are represented as the mean ± SD (n = 3). Statistical significance was determined by a one-sided paired Student’s t-test (*p < 0.05, ***p < 0.001).
Fig 3: BRAT1 deletion alters biogenesis of replication-dependent histone mRNAs and transcriptional regulation of protein-coding genes.a RNA-seq data for genes coding histones H2A, H2B, H3, or H4 in wild-type U2OS (wt #1 and wt #2) and BRAT1-/- (clone #6 and #8) cells, as indicated. Read coverage and metaplot analysis of averaged samples and histone coding region, 5’UTR, 3’UTR with a cleavage site (scissors) are shown. TSS transcription start site. See also Supplementary Fig. 4. b Schematic representation indicating pairs of primers (arrows) and RT-qPCR analysis of c-FOS mRNA and total RNA (pre-mRNA and mRNA) in wild-type (U2OS wt) and BRAT1-/- (clone #8) cells. The efficiency of splicing was determined by the ratio between the expression of pre-mRNA and mRNA. Data are represented as the mean ± SD (n = 6 for mRNA and total transcription; n = 3 for splicing). Statistical significance was determined by a one-sided paired Student’s t-test (**p < 0.01, ***p < 0.001, ns not significant). c A volcano plot of BRAT1-/- (clone #8 and #6) vs. wild-type U2OS (wt #1 and wt #2) transcription profiles showing differentially expressed genes (DEGs) and differentially expressed lncRNAs. Log2 ratio for upregulated genes =1.0 (FDR < 0.05) and for downregulated genes = -1.0 (FDR < 0.05), n = 27,006. d Venn diagrams demonstrating the overlap in identified differentially expressed genes and lncRNAs among cells depleted of BRAT1 (BRAT1-/-), INTS11, INTS9, INTS4, or INTS8 (shRNA transfected cells). Statistical significance of the overlap was determined by a hypergeometric test (p-values are indicated).
Fig 4: 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 5: The V62E mutation in BRAT1-mutated patient cells impairs BRAT1 interaction with INTS11 and INTS9.a Western blot of BRAT1, INTS11 and INTS9 levels in BRAT1 immunoprecipitates from control, parent, BRAT1 patient-derived fibroblasts and lymphoblastoid cell lines (LCLs), as indicated. Note that 2-fold more lysate from patient cells was employed for loading (input) and immunoprecipitation to ensure equivalent levels of INTS11. The black asterisk marks a nonspecific band. b BRAT1-/- (clone #8) cells transiently transfected with FLAG-tagged BRAT1WT and BRATV62E constructs were fixed 24 h post-transfection and stained with antibody against FLAG-tag (green) and DAPI (blue). c Levels of BRAT1, INTS11, INTS9 and FLAG-tagged BRAT1 in BRAT1 (left) and FLAG (right) immunoprecipitates from BRAT1-/- (clone #8) cells transiently transfected with FLAG-BRAT1WT or FLAG-BRATV62E, measured by Western blotting. a–c The experiment was performed three times with similar results. Uncropped and unprocessed scans are provided in the Source Data file.
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