Journal: Science Advances

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

doi: 10.1126/sciadv.adf0597

Figure Lengend Snippet: ( A ) smRNA-seq profile at the canonical miRNA locus miR-17. The y -axis represents counts per million (CPM). ( B ) Box and violin plot depicting the log 2 fold change of 205 expressed miRNAs determined by smRNA-seq in the indicated knockdown or uninduced shControl HeLa cells, calculated against induced shControl cells. *** P < 0.001, one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. n.s., not significant. Gray dotted lines indicate 1.5× threshold. ( C ) Global average smRNA-seq profiles around 112 5p-miRNAs aligned at their start site. ( D and E ) Volcano plot comparing statistical significance and miRNA log 2 fold change between control and knockdown cells. P adj refers to adjusted P value as calculated by DESeq2. (D) Sh INTS6 . (E) Sh INTS11 . ( F and G ) smRNA-seq profiles at DROSHA-independent miRNA loci. The y -axis represents CPM. (F) 5′-Capped miR-320a locus. (G) Mirtron miR-877 locus. ( H ) Northern blot miRNA detection from uninduced and induced [+1 μg/ml doxycycline (Dox)] shControl, sh INTS6 , and sh INTS11 HeLa cells. Twenty micrograms of RNA was loaded on a 15% tris-borate EDTA (TBE)–urea gel and probed for let-7d-5p, miR-17-5p, 5 S ribosomal RNA, and RNU43. ( I ) Quantification of Northern blot signal intensity relative to shControl + Dox ( n = 3). ** P < 0.01 and *** P < 0.001, unpaired t test.

Article Snippet: After transfer on nitrocellulose membranes, we detected our protein of interest using the following antibodies: INTS1 (Bethyl Laboratories, #A300-361A), INTS3 (Sigma Prestige, #HPA074391), INTS6 (Novus Biologicals, #NB10086990), INTS7 (Bethyl Laboratories, #A300-271A), INTS11 (Sigma Prestige, #HPA029025), glyceraldehyde-3-phosphate dehydrogenase (Abcam, #ab8245), DROSHA (Abcam, #ab12286), DICER (Abcam, #ab14601), DGCR8 (Abcam, #ab90579), AGO1 (Cell Signaling, #D84G10), AGO2 (Abcam, #ab57113), AGO3 (Sigma-Aldrich, #SAB4200112), AGO4 (Cell Signaling, #D10F10), and lamin B1 (Proteintech, #66095-1-1g).

Techniques: Northern Blot

Journal: Science Advances

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

doi: 10.1126/sciadv.adf0597

Figure Lengend Snippet: ( A and B ) Volcano plot of statistical significance against log 2 fold change between shControl and sh INTS11 cells quantifying 112 pri-miRNAs in (A) PRO-seq (transcriptional elongation) or (B) total RNA-seq. Significantly regulated pri-miRNAs are depicted in red. P adj refers to adjusted P value as calculated by DESeq2. ( C ) Box and violin plot depicting the log 2 fold change of pri-miRNA expression obtained by total RNA-seq in the indicated knockdown cells, calculated against pri-miRNA levels in shControl or siControl cells. One hundred twelve pri-miRNAs were extracted from Ensembl or newly annotated transcripts based on si DROSHA RNA-seq (see Materials and Methods for details). ( D ) Cumulative total RNA-seq read densities across annotated pre-miRNAs ± 100 bp for siControl and si DROSHA samples. ( E ) Example total RNA-seq profiles of indicated knockdowns depicting pre-miRNA excision at the miR-21 locus. Mature miR-21 are indicated in red, and the annotated precursor is indicated in light blue. The y -axis represents CPM. ( F ) Cumulative total RNA-seq read densities across annotated pre-miRNAs ± 100 bp for shControl, sh INTS6 , and sh INTS11 samples. ( G ) Mean length nucleotide (nt) percentage per miRNA ranging from 18 to 30 nucleotides detected in smRNA-seq of shControl, sh INTS6 , and sh INTS11 cells. Means ± SEM.

Article Snippet: After transfer on nitrocellulose membranes, we detected our protein of interest using the following antibodies: INTS1 (Bethyl Laboratories, #A300-361A), INTS3 (Sigma Prestige, #HPA074391), INTS6 (Novus Biologicals, #NB10086990), INTS7 (Bethyl Laboratories, #A300-271A), INTS11 (Sigma Prestige, #HPA029025), glyceraldehyde-3-phosphate dehydrogenase (Abcam, #ab8245), DROSHA (Abcam, #ab12286), DICER (Abcam, #ab14601), DGCR8 (Abcam, #ab90579), AGO1 (Cell Signaling, #D84G10), AGO2 (Abcam, #ab57113), AGO3 (Sigma-Aldrich, #SAB4200112), AGO4 (Cell Signaling, #D10F10), and lamin B1 (Proteintech, #66095-1-1g).

Techniques: RNA Sequencing Assay, Expressing

Journal: Science Advances

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

doi: 10.1126/sciadv.adf0597

Figure Lengend Snippet: ( A ) Scheme of INTS knockdown and s4U labeling. D0, day 0. ( B ) Steady state (unlabeled and T>C labeled) of miRNA expression over time. n = 126. ( C to E ) T>C-labeled miRNA abundance over time, separated for 32 guide or 32 passenger miRNAs. Means ± SEM. * P < 0.05 and ** P < 0.01, Mann-Whitney-Wilcoxon test. (C) ShControl. (D) Sh INTS6 . (E) Sh INTS11 . ( F ) Combined T>C-labeled miRNA abundance. ( G ) Example of linear regression on shControl guide or passenger miRNAs. miRNA biogenesis rates ( k bio ) determined from 15 min to 1 hour or accumulation rates ( k accu ) from 1 to 6 hours. Slope ± SE is indicated. ( H ) Histogram of k bio and k accu . Means ± SEM. ** P < 0.01, one-way ANOVA, followed by Tukey’s post hoc test on single miRNAs with k bio or k accu > 0. ( I ) Single exponential saturation kinetics to calculate median half-life t 1/2 (hours) as depicted in the table below including 95% confidence interval (CI). Shades indicate SEM.

Article Snippet: After transfer on nitrocellulose membranes, we detected our protein of interest using the following antibodies: INTS1 (Bethyl Laboratories, #A300-361A), INTS3 (Sigma Prestige, #HPA074391), INTS6 (Novus Biologicals, #NB10086990), INTS7 (Bethyl Laboratories, #A300-271A), INTS11 (Sigma Prestige, #HPA029025), glyceraldehyde-3-phosphate dehydrogenase (Abcam, #ab8245), DROSHA (Abcam, #ab12286), DICER (Abcam, #ab14601), DGCR8 (Abcam, #ab90579), AGO1 (Cell Signaling, #D84G10), AGO2 (Abcam, #ab57113), AGO3 (Sigma-Aldrich, #SAB4200112), AGO4 (Cell Signaling, #D10F10), and lamin B1 (Proteintech, #66095-1-1g).

Techniques: Labeling, Expressing, MANN-WHITNEY

Journal: Science Advances

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

doi: 10.1126/sciadv.adf0597

Figure Lengend Snippet: ( A ) Steady-state miRNA abundance from AGO2 RIP after 24 hours + s4U. n = 122. ( B ) T>C miRNA percentage. Means ± SEM. ( C ) miRNA TaqMan qPCR before and after AGO2 overexpression (see fig. S4D). miRNA levels relative to ath-miR-159a spike-in and shControl. Means ± SEM, n = 3. ** P < 0.01 and *** P < 0.001, one-way ANOVA, followed by Dunnett’s multiple comparisons test. ( D ) Scatterplot of the miRNA log 2 fold change in sh INTS6 and sh INTS11 from Fig. 1 (D and E) . Integrator-unregulated miRNAs are indicated by orange shaded area. ( R , Spearman correlation coefficient). ( E ) ShControl miRNA abundance for unregulated (No) and down-regulated (Down) miRNAs. * P < 0.05, Welch two-sample t test. Black circles indicate mean. ( F ) Percentage of guide and passenger miRNAs. Absolute miRNA numbers are indicated. * P < 0.05, Fisher’s exact test.

Article Snippet: After transfer on nitrocellulose membranes, we detected our protein of interest using the following antibodies: INTS1 (Bethyl Laboratories, #A300-361A), INTS3 (Sigma Prestige, #HPA074391), INTS6 (Novus Biologicals, #NB10086990), INTS7 (Bethyl Laboratories, #A300-271A), INTS11 (Sigma Prestige, #HPA029025), glyceraldehyde-3-phosphate dehydrogenase (Abcam, #ab8245), DROSHA (Abcam, #ab12286), DICER (Abcam, #ab14601), DGCR8 (Abcam, #ab90579), AGO1 (Cell Signaling, #D84G10), AGO2 (Abcam, #ab57113), AGO3 (Sigma-Aldrich, #SAB4200112), AGO4 (Cell Signaling, #D10F10), and lamin B1 (Proteintech, #66095-1-1g).

Techniques: Over Expression

Journal: Science Advances

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

doi: 10.1126/sciadv.adf0597

Figure Lengend Snippet: ( A ) Global average of INTS11, CPSF73, IgG, and size-matched input eCLIP profiles around 112 5p-miRNAs aligned at their start site. ( B ) Scatterplot of the log 2 fold change of 205 miRNAs in after INTS6 ( y -axis) or INTS11 ( x -axis) depletion (see also Fig. 4D ) with concomitant labeling of INTS11-bound miRNAs (detected by eCLIP, n = 90). ( C to F ) Classification according miRNA abundance. (C) Two hundred five detected miRNAs from shControl smRNA-seq were separated by tertiles to define “low-,” “mid-,” and “high-”expression miRNAs. (D) Same as in (C), calculated with the subset of 90 miRNAs also detected from INTS11 eCLIP. (E) Abundance of the 90 miRNAs subset detected from AGO2 RIP, according to previously defined expression classes. (F) Abundance of miRNAs detected in eCLIP. ( G to J ) Classification according miRNA type (guide or passenger miRNA). (G) Abundance of 92 miRNA couples detected in shControl smRNA-seq and separated by type. (H) Same as (G), calculated with the subset of 47 miRNAs and also detected from INTS11 eCLIP. (I) Abundance separated by type of the 47 miRNA subset detected from AGO2 RIP. (J) INTS11 eCLIP miRNA abundance separated by type. Red circles indicate mean. Statistics were calculated using either one-way ANOVA followed by Tukey’s post hoc test (C to F) or Welch two-sample t test (G to J). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: After transfer on nitrocellulose membranes, we detected our protein of interest using the following antibodies: INTS1 (Bethyl Laboratories, #A300-361A), INTS3 (Sigma Prestige, #HPA074391), INTS6 (Novus Biologicals, #NB10086990), INTS7 (Bethyl Laboratories, #A300-271A), INTS11 (Sigma Prestige, #HPA029025), glyceraldehyde-3-phosphate dehydrogenase (Abcam, #ab8245), DROSHA (Abcam, #ab12286), DICER (Abcam, #ab14601), DGCR8 (Abcam, #ab90579), AGO1 (Cell Signaling, #D84G10), AGO2 (Abcam, #ab57113), AGO3 (Sigma-Aldrich, #SAB4200112), AGO4 (Cell Signaling, #D10F10), and lamin B1 (Proteintech, #66095-1-1g).

Techniques: Labeling, Expressing

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