Light-gated integrator for highlighting kinase activity in living cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1138651 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29912142_1.fastq.gz | Fastqsanger.gz | |||
| SRR29912142_2.fastq.gz | Fastqsanger.gz | |||
| SRR29912143_1.fastq.gz | Fastqsanger.gz | |||
| SRR29912143_2.fastq.gz | Fastqsanger.gz | |||
| SRR29912144_1.fastq.gz | Fastqsanger.gz |
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Project description:Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into “permanent” fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA activity distribution in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of GαsR201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.
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Project description:Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV Integration Targeting-Sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3’ end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disrupting histone mRNA processing, and leading to polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites. HITseq was used to interrogate chromatin binding sites of of proteins in the complex (including INIP, INTS3, INTS9, INTS11, NABP1, NABP2, NELFA, NELFB, NELFCD, NELFE, SPT5). These proteins were fused to LEDGF-IBD and expressed in mouse LEDGF-/-MEF cells. The fusion proteins then target HIV integration to the chromatin binding sites. The HIV integration sites were identified using linker-mediated PCR and highthroughput sequencing and serve as surrogate for chromatin binding sites. Mapping was done using mouse genome mm9.
2015-01-20 | E-GEOD-65090 | biostudies-arrayexpress
Project description:The Integrator complex is primarily known to attenuate transcription by inducing premature termination of paused RNA polymerase II (RNAPII) and restraining pause release through its endonuclease and phosphatase activities. However, the mechanisms that govern Integrator recruitment to chromatin and direct gene-specific transcriptional programs in response to external stimuli and diverse biological contexts remain poorly understood. In particular, the regulatory functions of the Integrator auxiliary or arm module (INTS10/13/4/15) are the least characterized. Here, we identify ZBTB26 as an interactor of the Integrator auxiliary module via INTS10 and INTS13, and show that it co-occupies select promoters and enhancers with Integrator and active histone markers across the genome. We demonstrate that ZBTB26 is required for the recruitment of Integrator to its target loci, including genes involved in stimulus response, development, and differentiation. Importantly, the ZBTB26-Integrator axis sustains the active state of specific promoters and enhancers and drives defined gene transcriptional programs. Our findings reveal a transcription factor-like mechanism of targeted gene regulation mediated by ZBTB26-Integrator binding, extending the functional paradigm of Integrator beyond its canonical catalytic roles.
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Project description:MicroRNA (miRNA) homeostasis is crucial for the post-transcriptional regulation of their target genes and miRNA dysregulation has been linked to multiple diseases, including cancer. The mechanistic steps of miRNA biogenesis are well understood at molecular level1,2. Subsequent miRNA duplex loading into members of the Argonaute (Ago) protein family, representing the core of the RNA-induced silencing complex (RISC), is pivotal to miRNA-mediated gene silencing3-5. The Integrator complex has been previously identified as important regulator of RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination, processes dependent on Integrator’s catalytical activity6-9. Here we report that absence of the Integrator complex leads to a global loss of mature miRNAs. By incorporating 4-Thiouridine (s4U) in nascent transcripts, we traced miRNA fate from biogenesis to stabilization and identified Integrator to be essential for proper miRNA assembly into RISC. Indeed, Integrator enhances miRNA-Ago2 interaction in vitro, which functionally translates to amplified miRNA target cleavage. These findings demonstrate for the first time cytoplasmic Integrator complex functions and stress an indirect role of Integrator in transcription regulation through modulation of miRNA abundance and stability.
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Project description:Mutations in BRAT1, encoding BRCA1-associated ATM activator 1, have been associated with neurodevelopmental and neurodegenerative disorders characterized by heterogeneous phenotypes with varying levels of clinical severity. However, the underlying molecular mechanisms of disease pathology remain poorly understood. Here, we show that BRAT1 is a functional component of the RNA processing protein complex, Integrator. BRAT1 interacts with the INTS9/INTS11 heterodimer, the core Integrator cleavage complex that processes the 3’ ends of various non-coding RNAs and pre-mRNAs, and we find that Integrator integrity and function are disrupted by BRAT1 deletion. In particular, defects in BRAT1 impede proper 3’ end processing of UsnRNAs, snoRNAs, and replication-dependent histone mRNAs, and alter expression of protein-coding genes. Importantly, defects in Integrator integrity and function are also evident in patient-derived cells from BRAT1 related neurological disease. Collectively, these data identify BRAT1 as a functional component of Integrator and link defects in this critical endonuclease complex with hereditary neurodegenerative disease.
2022-08-30 | PXD028591 | Pride
Project description:Cellular homeostasis requires transcriptional outputs to be coordinated, and many events post transcription initiation can dictate the levels and functions of mature transcripts. To systematically identify regulators of inducible gene expression, we performed high-throughput RNAi screening of the Drosophila Metallothionein A (MtnA) promoter. This revealed that the Integrator complex, which has a well-established role in 3' end processing of small nuclear RNAs (snRNAs), attenuates MtnA transcription during copper stress. Integrator complex subunit 11 (IntS11) endonucleolytically cleaves MtnA transcripts, resulting in premature transcription termination and degradation of the nascent RNAs by the RNA exosome, a complex also identified in the screen. Using RNA-seq, we then identified >400 additional Drosophila protein-coding genes whose expression increases upon Integrator depletion. We focused on a subset of these genes and confirmed that Integrator is bound to their 5' ends and negatively regulates their transcription via IntS11 endonuclease activity. Many non-catalytic Integrator subunits, which are largely dispensable for snRNA processing, also have regulatory roles at these protein-coding genes, possibly by controlling Integrator recruitment or RNA polymerase II dynamics. Altogether, our results suggest that attenuation via Integrator cleavage limits production of many full-length mRNAs, allowing precise control of transcription outputs.
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