ints6 gfp alone  (Addgene inc)

Journal: Molecular cell

Article Title: IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events.

doi: 10.1016/j.molcel.2023.10.035

Figure Lengend Snippet: Figure 1. Integrator activity at a subset of reporter genes is lost upon over-expression of IntS6 (A) eGFP-based reporters to examine Integrator activity at protein-coding genes (left) and snRNAs (right). The promoter and 50 UTR of each indicated protein- coding gene were cloned upstream of eGFP (left). The snRNA promoter, coding sequence, and downstream region were cloned upstream of eGFP, thereby enabling eGFP production when Integrator fails to process the snRNA 30 end between the stem loop and 30 box sequences (right). (B) Each individual reporter plasmid was transfected into DL1 cells that had been treated with the indicated dsRNAs (left) or was co-transfected with 100 ng of a plasmid that over-expresses a FLAG-tagged Integrator subunit from the Ubi-p63e promoter (right). A plasmid containing a multi-cloning site (MCS) driven by the Ubi-p63e promoter was used as a control for the over-expression experiments. CuSO4 was added for the last 14 h only when measuring eGFP production from

Article Snippet: Plasmids expressing IntS6 (pMtnA FLAG-IntS6 puro, Addgene #195076), IntS7 (pMtnA FLAG-IntS7 puro, Addgene #208405), IntS12 (pMtnA FLAG-IntS12 puro, Addgene #195077), IntS13 (pMtnA FLAG-IntS13 puro, Addgene #208406), or IntS14 (pMtnA FLAG-IntS14 puro, Addgene #208407) under the control of the Metallothionein A promoter were generated using the previously described pMT FLAGMCS puro plasmid.27 The reporter plasmid expressing eGFP downstream of the U4:39B snRNA gene was previously described.44 An analogous reporter expressing eGFP downstream of U5:34A snRNA (Hy_U5:34A eGFP SV40, Addgene #195064) was generated from Hy_pPepck1 eGFP SV40.

Techniques: Activity Assay, Over Expression, Clone Assay, Sequencing, Plasmid Preparation, Transfection, Cloning, Control

Journal: Molecular cell

Article Title: IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events.

doi: 10.1016/j.molcel.2023.10.035

Figure Lengend Snippet: Figure 2. Over-expression of IntS6 does not affect Integrator activity at endogenous Drosophila snRNA loci (A) Parental DL1 cells or DL1 cells stably maintaining IntS6 or IntS12 transgenes driven by the copper-inducible MtnA promoter were grown for 3 days. 500 mM CuSO4 was added for the last 24 h prior to total RNA isolation from three independent biological replicates. rRNA-depleted RNA-seq libraries were then generated, sequenced, and analyzed. (B) Cell lines were seeded in 12-well plates (5 3 105 cells per well) and grown for 3 days. As indicated, a final concentration of 500 mM CuSO4 was added to cells for the last 24 h prior to harvesting total protein. Western blot analysis was then performed using antibodies that recognize FLAG, IntS6, IntS8, IntS11, IntS12, mts, Rbp1 phosphorylated at Ser2, Ser5, or Ser7, and Rbp1 (C-terminal domain). * denotes non-specific band. IIo denotes hyperphosphorylated Rbp1, whereas IIa denotes hypophosphorylated Rbp1. a-Tubulin was used as a loading control. Subunit expression data were normalized to the parental DL1 cells without CuSO4 treatment and are shown as mean ± SD, n = 3. *p < 0.05. (C) To quantify readthrough transcription downstream of endogenous snRNAs, the levels of RNA-seq fragments that map to the 3 kb downstream of mature snRNA 30 ends were normalized to the levels of fragments that map to mature snRNA sequences. (D and E) Normalized values of endogenous snRNA readthrough among (D) CuSO4-treated parental DL1 cells and DL1 cells stably maintaining IntS6 or IntS12 transgenes, and (E) DL1 cells subjected to mock, control (b-gal) dsRNA, or IntS4 dsRNA treatments. Center lines represent medians, boxes represent interquartile ranges (IQRs), and whiskers represent extreme data points within 1.53 IQRs. Black points were outliners exceeding 1.53 IQRs. p values were calculated by Wilcoxon signed-rank test. **p < 0.01; ***p < 0.001; n.s., not significant. (F) UCSC genome browser tracks depicting exemplar snRNA loci. IntS1 and IntS12 ChIP-seq profiles in DL1 cells (GEO: GSE114467) are shown in black. RNA-seq data generated from DL1 cells treated for 3 days with control (b-gal), IntS4, or IntS6 dsRNAs are shown in blue. RNA-seq data generated from parental

Article Snippet: Plasmids expressing IntS6 (pMtnA FLAG-IntS6 puro, Addgene #195076), IntS7 (pMtnA FLAG-IntS7 puro, Addgene #208405), IntS12 (pMtnA FLAG-IntS12 puro, Addgene #195077), IntS13 (pMtnA FLAG-IntS13 puro, Addgene #208406), or IntS14 (pMtnA FLAG-IntS14 puro, Addgene #208407) under the control of the Metallothionein A promoter were generated using the previously described pMT FLAGMCS puro plasmid.27 The reporter plasmid expressing eGFP downstream of the U4:39B snRNA gene was previously described.44 An analogous reporter expressing eGFP downstream of U5:34A snRNA (Hy_U5:34A eGFP SV40, Addgene #195064) was generated from Hy_pPepck1 eGFP SV40.

Techniques: Over Expression, Activity Assay, Stable Transfection, Isolation, RNA Sequencing, Generated, Concentration Assay, Western Blot, Control, Expressing, ChIP-sequencing

Journal: Molecular cell

Article Title: IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events.

doi: 10.1016/j.molcel.2023.10.035

Figure Lengend Snippet: Figure 4. IntS6 over-expression titrates the catalytic subunit of PP2A and causes it to be limiting for Integrator activity (A) Cryo-EM structure (PDB: 7PKS) of the human Integrator complex,14 highlighting the positions of the RNA endonuclease IntS11 (green), IntS6 (orange), and PP2A subunits (teal and pink). There are direct contacts between IntS6 and the PP2A subunits.

Article Snippet: Plasmids expressing IntS6 (pMtnA FLAG-IntS6 puro, Addgene #195076), IntS7 (pMtnA FLAG-IntS7 puro, Addgene #208405), IntS12 (pMtnA FLAG-IntS12 puro, Addgene #195077), IntS13 (pMtnA FLAG-IntS13 puro, Addgene #208406), or IntS14 (pMtnA FLAG-IntS14 puro, Addgene #208407) under the control of the Metallothionein A promoter were generated using the previously described pMT FLAGMCS puro plasmid.27 The reporter plasmid expressing eGFP downstream of the U4:39B snRNA gene was previously described.44 An analogous reporter expressing eGFP downstream of U5:34A snRNA (Hy_U5:34A eGFP SV40, Addgene #195064) was generated from Hy_pPepck1 eGFP SV40.

Techniques: Over Expression, Activity Assay, Cryo-EM Sample Prep

Journal: Molecular cell

Article Title: IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events.

doi: 10.1016/j.molcel.2023.10.035

Figure Lengend Snippet: Figure 5. The phosphatase module is differentially required for Integrator activity across the genome (A) Means of normalized values of endogenous snRNA readthrough. Readthrough values (see Figure 2C) were calculated from RNA-seq data of DL1 cells subjected to a mock treatment or treatment with b-gal, mts, Pp2A-29B, IntS6, or IntS4 dsRNAs (3 independent biological replicates). Center lines represent medians, boxes represent interquartile ranges (IQRs), and whiskers represent extreme data points within 1.53 IQRs. Black points were outliners exceeding 1.53 IQRs. p values were calculated by Wilcoxon signed-rank test. **p < 0.01; ***p < 0.001; n.s., not significant. (B) ChIP-seq and RNA-seq tracks at the U5:34A snRNA locus. IntS1 and IntS12 ChIP-seq profiles in DL1 cells (GEO: GSE114467) are shown in black. RNA-seq data generated from DL1 cells treated for 3 days with control (b-gal), IntS4, IntS6, mts, or Pp2A-29B dsRNAs are shown in blue. Green arrow, transcription start site (TSS). (C) RNA-seq was used to define genes that were up- or down-regulated (|log2(fold-change)| > 0.585 and adjusted p < 0.001) upon IntS4, IntS6, mts, or Pp2A-29B depletion using RNAi or upon IntS6 over-expression (top). These gene lists were then stratified by ChIP-seq data that identified 3,932 protein-coding genes with peaks of IntS1 and/or IntS12 binding located ±1 kb of gene bodies in DL1 cells (green, middle). For genes bound by Integrator subunits and differentially regulated upon IntS4 or IntS6 depletion, the effect of depleting PP2A subunits on their expression is graphed (bottom). See also Tables S1, S2, S3, and S4.

Article Snippet: Plasmids expressing IntS6 (pMtnA FLAG-IntS6 puro, Addgene #195076), IntS7 (pMtnA FLAG-IntS7 puro, Addgene #208405), IntS12 (pMtnA FLAG-IntS12 puro, Addgene #195077), IntS13 (pMtnA FLAG-IntS13 puro, Addgene #208406), or IntS14 (pMtnA FLAG-IntS14 puro, Addgene #208407) under the control of the Metallothionein A promoter were generated using the previously described pMT FLAGMCS puro plasmid.27 The reporter plasmid expressing eGFP downstream of the U4:39B snRNA gene was previously described.44 An analogous reporter expressing eGFP downstream of U5:34A snRNA (Hy_U5:34A eGFP SV40, Addgene #195064) was generated from Hy_pPepck1 eGFP SV40.

Techniques: Activity Assay, RNA Sequencing, ChIP-sequencing, Generated, Control, Over Expression, Binding Assay, Expressing