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: 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