Journal: bioRxiv

Article Title: The Integrator complex regulates microRNA abundance through RISC loading

doi: 10.1101/2021.09.21.461113

Figure Lengend Snippet: a , Immunoblot detection of successful knock-down of INTS1, INTS3, INTS6, INTS7, and INTS11 before and after shRNA induction with Doxycyclin (Dox) at 1 µg/ml. GAPDH was used as loading control. b , Immunoblot of shControl before and after induction using the same INTS antibodies. c-f , Volcano plot comparing statistical significance and miRNA log 2 fold change between control and knock-down cells. Significantly regulated miRNAs are depicted in red. c , uninduced shControl compared to induced shControl. d , shINTS1 compared to induced shControl. e , shINTS3. f , shINTS7. g , Heat map of normalized miRNA expression from shControl, shINTS6, and shINTS11 (Z-score of normalized read counts per row). Column and row orders were determined by unsupervised hierarchical clustering. h , Immunoblot detection of Drosha after siRNA knock-down in HeLa. i , Volcano plot comparing statistical significance and miRNA log 2 fold change between siControl and siDrosha knock-down HeLa cells. Significantly regulated miRNAs are depicted in red. Drosha-independent miRNAs are indicated. j , k , Relative miRNA expression levels in j , HeLa shControl, shINTS6, and shINTS11 cells, or k , HEK293T cells transfected with siControl, siINTS6, siINTS11, or siDrosha. MiRNAs were detected by specific TaqMan probes for the indicated miRNAs and relative miRNA levels were calculated against RNU43 expression and shControl/siControl using ΔΔct method. Mean ± SEM, n = 4. Drosha-independent miRNA examples are indicated in red.

Article Snippet: After transfer on nitrocellulose membranes, we detected our protein of interest using the following antibodies: α-INTS1 (Bethyl Laboratories, #A300-361A), α-INTS3 (Sigma Prestige, HPA074391), α-INTS6 (Novus Biologicals, NB10086990), α-INTS7 (Bethyl Laboratories, A300-271A), α-INTS11 (Sigma Prestige, HPA029025), α-GAPDH (Abcam, ab8245), α-Drosha (Abcam, ab12286), α-Dicer (Abcam, ab14601), α-DGCR8 (Abcam, ab90579), α-Ago1 (Cell Signaling, D84G10), α-Ago2 (Abcam, ab57113), α-Ago3 (Sigma-Aldrich, SAB4200112), α-Ago4 (Cell Signaling, D10F10), α-Lamin B1 (Proteintech, #66095-1-1g).

Techniques: Western Blot, Knockdown, shRNA, Control, Expressing, Transfection

Journal: bioRxiv

Article Title: A PP2A-Integrator complex fine-tunes transcription by opposing CDK9

doi: 10.1101/2020.07.12.199372

Figure Lengend Snippet: A. CRISPR-Cas9 genome-wide CDK9i survival and nascent RNA screens. B. Venn diagrams for significant enriched sgRNAs (CDK9i versus untreated at Tend) in survival screens in THP-1-Cas9 cells (left; different CDK9 inhibitors) and using different genome-wide sgRNA libraries (adjusted p-value < 0.1 for > 3 sgRNAs in 1 or more replicate screens) C. Enriched sgRNAs for replicate nascent RNA screens in THP-1-Cas9 cells (significance relative to T0). D. Enrichment of INTS6 targeting sgRNAs in CDK9i-treated THP-1-Cas9 cells at endpoint relative to T0. E. Comparison of enriched sgRNAs for genome-wide CDK9i nascent RNA and survival screens. F. Overview of sgRNA Competitive proliferation assays. G. Western blot of THP-1-Cas9 cells expressing indicated sgRNAs. H. Competitive proliferation assay for THP-1-Cas9 cells expressing indicated sgRNAs in the presence of CDK9i. I. Annexin-V analysis of THP-1-Cas9 cells expressing indicated SCR and INTS6 targeting sgRNAs treated as indicated for 72 hours. J. Competitive proliferation assay for CFP-Cas9 expressing HeLa sgSCR and mCherry(CH)-Cas9 expressing HeLa sg INTS6 cells treated with CDK9i as indicated for 96 hours. K. Western blot of HeLa cells expressing sgSCR or sgINTS6 (mixed) treated with CDK9i as indicated for 96 hours. L. Annexin-V analysis of D.melanogaster S2-Cas9 expressing indicated SCR and IntS6 targeting sgRNAs and treated as indicated for 72 hours. Blue dots (Figures C and E) represent nominal p-value < 0.01. Figures G-L are representative of 3 independent experiments. Figures H, I, J, and L were analyzed by 2-way ANOVA, ** p < 0.01, **** p<0.0001.

Article Snippet: Human full-length INTS6 ORF in pCMV6-entry vector was purchased from Origene (RC208036).

Techniques: CRISPR, Genome Wide, Comparison, Western Blot, Expressing, Proliferation Assay

Journal: bioRxiv

Article Title: A PP2A-Integrator complex fine-tunes transcription by opposing CDK9

doi: 10.1101/2020.07.12.199372

Figure Lengend Snippet: A. Comparison of enriched sgRNAs for replicate survival screens in THP-1-Cas9 and MV4;11-Cas9 cells (CDK9i versus untreated at Tend) plus enrichment of INTS6 sgRNAs in CDK9i-treated versus untreated cells at Tend. B. Overview of nascent RNA analysis using ClickIT-EU assay and flow cytometry. C. Representative flow cytometry profiles of THP-1 cells treated with CDK9i (170nM) for the indicated time-points (left) or for 24 hours with the indicated dose of CDK9i (right). D. Overview of nascent RNA CRISPR-Cas9 genome-wide screen in THP-1-Cas9 cells; resistant cells were selected by FACS for the maintenance of high-EU signal in the presence of CDK9i. E. Identification of significantly enriched guides in EU-high cells (CDK9i treated relative to T0). F. Enrichment of sgRNAs for Integrator complex subunits in THP-1-Cas9 cells for individual replicate survival screens (significance relative to DMSO-treated cells at Tend) and nascent RNA screens (significance relative to T0). G. Competitive proliferation assay for MV4;11-Cas9 cells expressing indicated INTS6 targeting sgRNAs in the presence of CDK9i. H. Western blot of THP-1 cells treated with 22-533 for 6 hours. I. Competitive proliferation assay for THP-1-Cas9 cells expressing indicated SCR or INTS6 targeting sgRNAs in the presence of 22-533. J. Competitive proliferation assay for THP-1-Cas9 cells expressing indicated SCR or INTS3 targeting sgRNAs in the presence of CDK9i or DMSO. K. Competitive proliferation assay for THP-1-Cas9 cells expressing indicated SCR or INTS11 targeting sgRNAs in the presence of CDK9i or DMSO. L. Competitive proliferation assay for THP-1-Cas9 or MV4;11-Cas9 cells expressing indicated SCR or INTS6 targeting sgRNAs in the presence of DMSO. M. Annexin-V analysis of THP-1-Cas9 cells expressing indicated INTS6 targeting sgRNAs treated as indicated for 72 hours. N. Western blot of THP-1-Cas9 cells expressing SCR or INTS6 -A targeting sgRNAs plus/minus V5-INTS6 overexpression. O. Competitive proliferation assay and P. Annexin-V analysis of THP-1-Cas9 cells expressing indicated SCR or INTS6 -A targeting sgRNAs plus/minus V5-INTS6 overexpression in the presence of A5576 (CDK9i). Q. Representative flow-cytometry profiles of THP-1-Cas9 cells expressing indicated SCR or INTS6 targeting sgRNAs and treated as indicated for 24 hours, plus relative inhibition of nascent RNA production following CDK9i treatment. R. Overview and representative flow cytometry profiles of HeLa cell competitive proliferation assay: cells expressing sgSCR (CFP-Cas9) and sgINTS6 (mCherry-Cas9) were mixed 1:1 and incubated for 96 hours with indicated doses of CDK9i. S. Western blot of D. melanogaster S2-Cas9 sgSCR and sg IntS6 -5D cells plus/minus SBP-IntS6 over-expression. T. Annexin-V analysis of D. melanogaster S2-Cas9 cells expressing indicated SCR or IntS6 targeting sgRNAs plus/minus SBP-IntS6 overexpression treated as indicated for 72 hours. Blue dots (Figures A and E) represent nominal p-value < 0.05 or p-value < 0.01 respectively. Figures G, H, I J, K, L, M, N, O, P, S, Q, T are representative of 3 independent experiments. Figures G, I, J, K, M, O, P, Q and T were analyzed by 2-way ANOVA, ** p < 0.01, *** p< 0.01, **** p<0.0001.

Article Snippet: Human full-length INTS6 ORF in pCMV6-entry vector was purchased from Origene (RC208036).

Techniques: Comparison, Flow Cytometry, CRISPR, Genome Wide, Proliferation Assay, Expressing, Western Blot, Over Expression, Inhibition, Incubation

Journal: bioRxiv

Article Title: A PP2A-Integrator complex fine-tunes transcription by opposing CDK9

doi: 10.1101/2020.07.12.199372

Figure Lengend Snippet: Log2 iBAQ scores of proteins identified in INTS6 IP versus mouse IgG isotype control IP mass spectrometry experiments for A. THP-1 and B. MV4;11 cells. C. Western blot of HeLa cell cytoplasmic, nuclear, and chromatin fractions. D. Co-IP western blot for INTS11 in THP-1-Cas9 sgSCR and sgINTS6-KO cells. E. Volcano plot of differentially enriched proteins in SBP-IntS6 versus SBP (isotype control) streptavidin-IP mass spectrometry experiments in D. melanogaster S2 cells.

Article Snippet: Human full-length INTS6 ORF in pCMV6-entry vector was purchased from Origene (RC208036).

Techniques: Control, Mass Spectrometry, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: A PP2A-Integrator complex fine-tunes transcription by opposing CDK9

doi: 10.1101/2020.07.12.199372

Figure Lengend Snippet: A. Log10 iBAQ scores of proteins identified in INTS6 IP mass spectrometry experiments for THP-1 versus MV4;11 cells (filtered for proteins identified in isotype-control IP experiments). B. Protein-protein interaction network of INTS6, RNAPII, and PP2A interaction partners identified in THP-1 and MV4;11 INTS6 IP experiments. C. Log10 iBAQ scores of proteins identified in INTS6 versus PP2A-C IPs in HeLa cells. Co-IP western blot of D. endogenous V5-tagged PPP2R1A IP in THP-1 cells and E. endogenous PP2A-C, INTS3, and INTS11 IP in HeLa cells. F. Log10 iBAQ scores of proteins identified in PP2A-C IP mass spectrometry experiments in HeLa shLUC versus shINTS6 cells. G. Co-IP western blot for PP2A-C in shLUC, shINTS5, shINTS6, shINTS8, and shINTS12-infected HeLa cells. H. Co-IP western blot for PP2A-C in THP-1-Cas9 sgSCR and sgINTS6-KO cells. I. Relative expression levels of misprocessed UsnRNA from shLUC, shINTS2, or shINTS6-infected HeLa cells. Values normalized by expression of ribosomal 18S and GUSB is used as control (ctrl) mRNA. J. Western blot of glycerol gradient fractions of nuclear extracts from shLUC, shINTS2, or shINTS6-infected HeLa cells. IP mass spectrometry experiments are representative of 3 (THP-1, HeLa) or 2 (MV4;11) independent experiments. Western blots are representative of 3 independent experiments. qPCR (I) was analyzed by one-way ANOVA followed by Tukey’s HSD post-hoc test, **** p<0.0001.

Article Snippet: Human full-length INTS6 ORF in pCMV6-entry vector was purchased from Origene (RC208036).

Techniques: Mass Spectrometry, Control, Co-Immunoprecipitation Assay, Western Blot, Infection, Expressing

Journal: bioRxiv

Article Title: A PP2A-Integrator complex fine-tunes transcription by opposing CDK9

doi: 10.1101/2020.07.12.199372

Figure Lengend Snippet: A. Average profiles of ChIP-seq signal for CDK9, RNAPII, BRD4, INTS6, INTS11, and PPP2R1A around the TSS, plus metagene occupancy heatmaps for indicated proteins in THP-1 cells treated as indicated for 2 hours. B. Representative IGV ChIP-profiles for indicated proteins in THP-1 cells treated as indicated for 2 hours. C. Representative IGV profile for RNAPII and PPP2R1A ChIP signal in THP-1 cells untreated or acutely treated with 5.0µg/mL LPS for 3 hours. Average gene profiles for D. RNAPII and E. PPP2R1A ChIP-seq at LPS-induced genes (n=35) inTHP-1 cells under the same conditions. F. Representative IGV ChIP-profile for indicated proteins in HeLa cells untreated or acutely treated with EGF (0.1µg/mL) for 15 minutes. INTS11 ChIP-seq tracks are from a published dataset . Average gene profiles for G. RNAPII, H. PPP2R1A and I. PP2A-C ChIP-seq at EGF-induced genes (n=50) in HeLa cells under the same conditions. J. Average gene profile at PP2A-occupied genes (n=194) and K. representative IGV ChIP-profiles for PPP2R1A ChIP-seq in shLUC and shINTS6 infected HeLa. Scale bar for C represents 5kb.

Article Snippet: Human full-length INTS6 ORF in pCMV6-entry vector was purchased from Origene (RC208036).

Techniques: ChIP-sequencing, Infection

Journal: The Journal of Clinical Investigation

Article Title: Disrupting integrator complex subunit INTS6 causes neurodevelopmental disorders and impairs neurogenesis and synapse development

doi: 10.1172/JCI191729

Figure Lengend Snippet: ( A ) The distributions of nonsense, frameshift, and splicing variants in INTS 6 identified in NDDs are shown in a protein model and gene model, respectively. ( B ) The distribution of missense variants in INTS6 identified in NDDs is shown in a protein model. Protein tolerance landscape for missense variants in INTS6 was visualized via MetaDome20. All variants in INTS6 are predicted to be “intolerant” for aa substitutions. The density plot of ultrarare missense variants in gnomAD is shown. ( C ) Comparison of the distribution of combined annotation-dependent depletion (CADD) and MPC scores between de novo missense variants in NDDs and ultrarare missense variants in gnomAD database. Data are reported as mean ± SEM. P values were determined from a 2-tailed, unpaired Mann-Whitney test. ( D ) Comparison of SIFT, PolyPhen-2, and AlphaMissense prediction between de novo missense variants in NDDs and ultrarare missense variants in the gnomAD database. SIFT: D (deleterious), T (tolerated); PolyPhen-2: D (probably damaging), P (possibly damaging), B (benign); AlphaMissense: P (likely pathogenic), B (likely benign), A (ambiguous). ( E ) Left: Ribbon diagram of the INTS-PP2A complex bound to paused Pol II (PDB:7PKS). The disease-associated protein INTS6 and its interacting proteins are labeled. Right: Close-up view of NDD-related variants on INTS6 (red spheres), highlighting the importance of these residues in mediating protein-protein interactions or maintaining the structural integrity of INTS6.

Article Snippet: Ints6 flox/flox mice were generated by Cyagen Biotechnology using the CRISPR-Cas9 method, following the strategy outlined in .

Techniques: Comparison, MANN-WHITNEY, Labeling, Protein-Protein interactions

Journal: The Journal of Clinical Investigation

Article Title: Disrupting integrator complex subunit INTS6 causes neurodevelopmental disorders and impairs neurogenesis and synapse development

doi: 10.1172/JCI191729

Figure Lengend Snippet: ( A ) Heatmap and spatial distribution of RNAPII binding at the TSS of genes analyzed by CUT&Tag in WT and cKO E15.5 mice. ( B ) Average distribution profile of RNAPII across gene regions, including TSSs and TESs. ( C ) Spatial distribution and heatmap representation of the distance of RNAPII binding around the TSS of RIP-Seq genes. ( D ) Average distribution profile of RNAPII across coding sequences (CDS) regions of RIP-Seq. The gradient of blue to white color ( A – D ) indicates high to low counts in the corresponding region. ( E ) A Venn diagram illustrating the overlap between DGEs ( P < 0.05) identified in RNA-Seq and CUT&Tag data sets. ( F ) Heatmap analysis of the relative expression levels of 2,374 genes in the overlap of RNA-Seq and CUT&Tag. Upregulated genes are depicted in blue; downregulated genes are shown in red. ( G ) Bar graph depicting enriched KEGG pathways identified from the overlap data. ( H ) Bubble plot depicting enriched GO terms identified from the overlap data. ( I ) Browser tracks of CUT&Tag profiles for the genes related to the cell cycle at E15.5 days of embryonic development, comparing expression levels in WT and INTS6 cKO mice. ( J ) Western blot analysis of total RNAPII and Ser2P in HEK293T cells transfected with either WT, missense variants ( n = 5), or LGD variants ( n = 8). P values were determined from Friedman with Dunnett’s multiple comparisons test. ( K ) Statistical analysis of the effects of CDK9i on the growth of WT ( n = 27 DMSO-treated; n = 14 CDK9i-treated); cHET ( n = 23 DMSO-treated, n = 18 CDK9i-treated) and cKO ( n = 12 DMSO-treated, n = 19 CDK9i-treated) neurosphere. P values were determined from a 2-tailed unpaired t test and Mann-Whitney test. * P < 0.05, ** P < 0.01. CON, control. Data are reported as mean ± SEM.

Article Snippet: Ints6 flox/flox mice were generated by Cyagen Biotechnology using the CRISPR-Cas9 method, following the strategy outlined in .

Techniques: Binding Assay, RNA Sequencing, Expressing, Western Blot, Transfection, MANN-WHITNEY, Control

Journal: The Journal of Clinical Investigation

Article Title: Disrupting integrator complex subunit INTS6 causes neurodevelopmental disorders and impairs neurogenesis and synapse development

doi: 10.1172/JCI191729

Figure Lengend Snippet: ( A ) Heatmaps depicting the movement of WT ( n = 14) or cHET ( n = 14) mice in a 3-chamber social interaction test. Preference scores were calculated as (S – E)/(S + E) for social versus empty interactions and (S2 – S1)/(S2 + S1) for stranger versus original mouse interactions. Data are reported as mean ± SEM. P values were determined from a 2-tailed unpaired t test. ( B ) Morris water maze test of spatial learning and memory in Ints6 cHET mice. Latent time (s) during training trials, time in platform quadrant, and distance traveled are measured ( n = 14). Data are reported as mean ± SEM. P values were determined from 2-way ANOVA with Bonferroni’s multiple comparisons test and a 2-tailed unpaired Mann-Whitney test. ( C ) Elevated cross-maze experiments were performed with WT ( n = 13) and cHET ( n = 14) mice to evaluate anxiety-related behaviors. The experiments statistically analyzed the movement distance and dwell time in both the open and closed arms of the maze. Data are reported as mean ± SEM. P values were determined from a 2-tailed unpaired Mann-Whitney test. ( D ) Path-tracking images from an open field test, showing movement patterns of WT ( n = 14) and cHET ( n = 15) mice. Bar graph representing the distance traveled and the time spent in the central area of the open field over 10 minutes. Data are reported as mean ± SEM. P values were determined from a 2-tailed unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. snRNP, small nuclear ribonucleoprotein.

Article Snippet: Ints6 flox/flox mice were generated by Cyagen Biotechnology using the CRISPR-Cas9 method, following the strategy outlined in .

Techniques: MANN-WHITNEY