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TAP-MS of human Integrator subunits INTS10 and INTS13

ABSTRACT: Using a combination of proteomics, interaction studies and structural characterization we show that the INTS10-13-14 module of INT binds several transcriptional regulators and gene-specific transcription factors, whichcould form the molecular basis for a part of INT's gene specificity.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Kevin Sabath

LAB HEAD: Stefanie Jonas

PROVIDER: PXD041374 | Pride | 2024-06-27

REPOSITORIES: Pride

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			Action	DRS
	20200430_002_S216142_KS_GFP_1_Control.raw	Raw
	20200430_003_S216143_KS_GFP_2_Control.raw	Raw
	20200430_004_S216144_KS_GFP_3_Control.raw	Raw
	20200430_005_S216145_KS_GFP_4_Control.raw	Raw
	20200430_007_S216138_KS_13dC_1_TAP_INTS13.raw	Raw

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Publications

Basis of gene-specific transcription regulation by the Integrator complex.

Sabath Kevin K Nabih Amena A Arnold Christian C Moussa Rim R Domjan David D Zaugg Judith B JB Jonas Stefanie S

Molecular cell 20240620 13

The Integrator complex attenuates gene expression via the premature termination of RNA polymerase II (RNAP2) at promoter-proximal pausing sites. It is required for stimulus response, cell differentiation, and neurodevelopment, but how gene-specific and adaptive regulation by Integrator is achieved remains unclear. Here, we identify two sites on human Integrator subunits 13/14 that serve as binding hubs for sequence-specific transcription factors (TFs) and other transcription effector complexes. ...[more]

PMID: 38906142

Similar Datasets

Integrator is a genome-wide attenuator of non-productive transcription

Project description:Termination of RNA polymerase II (RNAPII) transcription in metazoans relies largely on the Cleavage and Polyadenylation (CPA) and Integrator (INT) complexes originally found to act at the ends of protein-coding and snRNA genes, respectively. Here we monitor CPA- and INT-dependent termination activities genome-wide, including at thousands of previously unannotated transcription units (TUs), producing unstable RNA. We verify the global activity of CPA, occurring at pA sites indiscriminately of their positioning relative to the TU promoter. We also identify a global activity of INT, which is, however, largely sequence-independent and restricted to a ~3 kb promoter-proximal region. Our analyses suggest two functions of genome-wide INT activity; it dampens transcriptional output from weak promoters and it provides quality-control of RNAPII complexes that are unfavorably configured for transcriptional elongation. We suggest that the function of INT in stable snRNA production is an exception from its general cellular role, the attenuation of non-productive transcription.

2021-01-03 | GSE151919 | GEO

TAP-MS of human BRAT1 and WDR73

Project description:Using a combination of proteomics, interaction studies and structural characterization we show that WDR73 and BRAT1 are interactors of the heterodimer INTS9-11 of INT. Both proteins are involved in the stability and formation of the cleavage module of INT consisting of INTS4-9-11.

2024-07-31 | PXD044324 | Pride

iCLIP of TDP-43

Project description:A stable HEK293 FlpIn T-Rex cells expressing TDP-43 with an N-terminal eGFP-tag was generated that allowed inducible physiological expression of the protein (Ling et al. 2010). Duplicate iCLIP experiments were performed using an antibody targeting eGFP (Abcam ab290). Crosslinked RNA-protein complexes were isolated by immuno-precipitation and cDNAs were generated to allow preparation of Illumina compatible DNA libraries as described in Huppertz et al. (2014).

2017-05-01 | E-MTAB-4733 | biostudies-arrayexpress

snRNA 3' end formation requires heterodimeric association of integrator subunits.

Project description:The Integrator Complex is a group of proteins responsible for the endonucleolytic cleavage of primary small nuclear RNA (snRNA) transcripts within the nucleus. Integrator subunits 9 and 11 (IntS9/11) are thought to contain the catalytic activity based on their high sequence similarity to CPSF100 and CPSF73, which have been shown to be components of both the poly(A)(+) and histone pre-mRNA cleavage complex. Here we demonstrate that the specific heterodimeric interaction between IntS9 and IntS11 is mediated by a discrete domain present at the extreme C terminus of IntS9 and within the C terminus of IntS11, adjacent to the predicted active site of this endonuclease. This domain is highly conserved within IntS11 but conspicuously absent in CPSF73. Using a cell-based complementation assay that measures Integrator activity, we determined that the IntS9 interaction domain within IntS11 is required for its ability to restore snRNA 3' end processing after RNA interference (RNAi)-mediated depletion of IntS11. Moreover, overexpression of these interaction domains alone elicits snRNA misprocessing through a dominant-negative titration of endogenous Integrator subunits. These data collectively explain the mechanism by which the IntS11/9 and, by analogy, the CPSF73/100 heterodimeric cleavage factors distinguish themselves from each other and demonstrate that the heterodimeric interaction is functionally required for snRNA 3' end formation.

| S-EPMC3295014 | biostudies-literature

Human mutations in integrator complex subunits link transcriptome integrity to brain development.

Project description:Integrator is an RNA polymerase II (RNAPII)-associated complex that was recently identified to have a broad role in both RNA processing and transcription regulation. Importantly, its role in human development and disease is so far largely unexplored. Here, we provide evidence that biallelic Integrator Complex Subunit 1 (INTS1) and Subunit 8 (INTS8) gene mutations are associated with rare recessive human neurodevelopmental syndromes. Three unrelated individuals of Dutch ancestry showed the same homozygous truncating INTS1 mutation. Three siblings harboured compound heterozygous INTS8 mutations. Shared features by these six individuals are severe neurodevelopmental delay and a distinctive appearance. The INTS8 family in addition presented with neuronal migration defects (periventricular nodular heterotopia). We show that the first INTS8 mutation, a nine base-pair deletion, leads to a protein that disrupts INT complex stability, while the second missense mutation introduces an alternative splice site leading to an unstable messenger. Cells from patients with INTS8 mutations show increased levels of unprocessed UsnRNA, compatible with the INT function in the 3'-end maturation of UsnRNA, and display significant disruptions in gene expression and RNA processing. Finally, the introduction of the INTS8 deletion mutation in P19 cells using genome editing alters gene expression throughout the course of retinoic acid-induced neural differentiation. Altogether, our results confirm the essential role of Integrator to transcriptome integrity and point to the requirement of the Integrator complex in human brain development.

| S-EPMC5466333 | biostudies-literature

Occupancy of Integrator subunits on DNA after hyperosmotic stress

Project description:ChIP-seq was performed using antibodies against Integrator subunits 11 or 3 to measure the occupancy of these proteins on DNA after hyperosmotic stress.

2021-01-04 | GSE159190 | GEO

Molecular basis for the interaction between Integrator subunits IntS9 and IntS11 and its functional importance.

Project description:The metazoan Integrator complex (INT) has important functions in the 3'-end processing of noncoding RNAs, including the uridine-rich small nuclear RNA (UsnRNA) and enhancer RNA (eRNA), and in the transcription of coding genes by RNA polymerase II. The INT contains at least 14 subunits, but its molecular mechanism of action is poorly understood, because currently there is little structural information about its subunits. The endonuclease activity of INT is mediated by its subunit 11 (IntS11), which belongs to the metallo-?-lactamase superfamily and is a paralog of CPSF-73, the endonuclease for pre-mRNA 3'-end processing. IntS11 forms a stable complex with Integrator complex subunit 9 (IntS9) through their C-terminal domains (CTDs). Here, we report the crystal structure of the IntS9-IntS11 CTD complex at 2.1-Å resolution and detailed, structure-based biochemical and functional studies. The complex is composed of a continuous nine-stranded ?-sheet with four strands from IntS9 and five from IntS11. Highly conserved residues are located in the extensive interface between the two CTDs. Yeast two-hybrid assays and coimmunoprecipitation experiments confirm the structural observations on the complex. Functional studies demonstrate that the IntS9-IntS11 interaction is crucial for the role of INT in snRNA 3'-end processing.

| S-EPMC5410846 | biostudies-literature

TMT Screening of Human Samples:SiHa cell

Project description:A screen for APOA1 downstream proteins affecting platinum-based chemoresistance using Tandem Mass Tag revealed 64 differentially expressed proteins in SiHa cells, which were subjected to Gene Ontology (annotation, Kyoto Encyclopedia of Genes and Genomes enrichment, subcellular localization, structural domain annotation and enrichment, clustering, and interaction network analyses

2022-05-20 | PXD030885 | Pride

Correction: Human mutations in integrator complex subunits link transcriptome integrity to brain development.

Project description:[This corrects the article DOI: 10.1371/journal.pgen.1006809.].

| S-EPMC5538630 | biostudies-other

A combinatorial approach to uncover a novel Integrator subunit [RNA-seq]

Project description:The Integrator complex is one of the most elusive transcriptional regulators in metazoans, deemed to be recruited after initiation to help establish and modulate paused RNAPII. Recent structural, biochemical, and functional evidence suggest that Integrator is composed of 14 subunits, assembling, and operating in a modular fashion. We employed proteomics and machine-learning (alphafold2) approaches to discover a novel Integrator subunit, INTS15. We report that INTS15 assembles primarily with the INTS10-INTS13-INTS14 module and interfaces with the Int-PP2A phosphatase module. Functional genomics analysis further reveals a role for INTS15 in modulating RNAPII pausing at a subset of genes in human cell lines.

2023-01-25 | GSE204905 | GEO

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