38 research outputs found
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FUS Binds the CTD of RNA Polymerase II and Regulates its Phosphorylation at Ser2
Mutations in the RNA-binding protein FUS (fused in sarcoma)/TLS have been shown to cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS), but the normal role of FUS is incompletely understood. We found that FUS binds the C-terminal domain (CTD) of RNA polymerase II (RNAP2) and prevents inappropriate hyperphosphorylation of Ser2 in the RNAP2 CTD at thousands of human genes. The loss of FUS leads to RNAP2 accumulation at the transcription start site and a shift in mRNA isoform expression toward early polyadenylation sites. Thus, in addition to its role in alternative RNA splicing, FUS has a general function in orchestrating CTD phosphorylation during RNAP2 transcription
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The TORC1 phosphoproteome in C. elegans reveals roles in transcription and autophagy
The protein kinase complex target of rapamycin complex 1 (TORC1) is a critical mediator of nutrient sensing that has been widely studied in cultured cells and yeast, yet our understanding of the regulatory activities of TORC1 in the context of a whole, multi-cellular organism is still very limited. Using Caenorhabditis elegans, we analyzed the DAF-15/Raptor-dependent phosphoproteome by quantitative mass spectrometry and characterized direct kinase targets by in vitro kinase assays. Here, we show new targets of TORC1 that indicate previously unknown regulation of transcription and autophagy. Our results further show that DAF-15/Raptor is differentially expressed during postembryonic development, suggesting a dynamic role for TORC1 signaling throughout the life span. This study provides a comprehensive view of the TORC1 phosphoproteome, reveals more than 100 DAF-15/Raptor-dependent phosphosites that reflect the complex function of TORC1 in a whole, multi-cellular organism, and serves as a rich resource to the field.
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The identification of protein and RNA interactors of the splicing factor Caper in the adult Drosophila nervous system
Post-transcriptional gene regulation is a fundamental mechanism that helps regulate the development and healthy aging of the nervous system. Mutations that disrupt the function of RNA-binding proteins (RBPs), which regulate post-transcriptional gene regulation, have increasingly been implicated in neurological disorders including amyotrophic lateral sclerosis, Fragile X Syndrome, and spinal muscular atrophy. Interestingly, although the majority of RBPs are expressed widely within diverse tissue types, the nervous system is often particularly sensitive to their dysfunction. It is therefore critical to elucidate how aberrant RNA regulation that results from the dysfunction of ubiquitously expressed RBPs leads to tissue specific pathologies that underlie neurological diseases. The highly conserved RBP and alternative splicing factor Caper is widely expressed throughout development and is required for the development of Drosophila sensory and motor neurons. Furthermore, caper dysfunction results in larval and adult locomotor deficits. Nonetheless, little is known about which proteins interact with Caper, and which RNAs are regulated by Caper. Here we identify proteins that interact with Caper in both neural and muscle tissue, along with neural specific Caper target RNAs. Furthermore, we show that a subset of these Caper-interacting proteins and RNAs genetically interact with caper to regulate Drosophila gravitaxis behavior
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The nuclear interactome of DYRK1A reveals a functional role in DNA damage repair
The chromosome 21 encoded protein kinase DYRK1A is essential for normal human development. Mutations in DYRK1A underlie a spectrum of human developmental disorders, and increased dosage in trisomy 21 is implicated in Down syndrome related pathologies. DYRK1A regulates a diverse array of cellular processes through physical interactions with substrates and binding partners in various subcellular compartments. Despite recent large-scale protein-protein interaction profiling efforts, DYRK1A interactions specific to different subcellular compartments remain largely unknown, impeding progress toward understanding emerging roles for this kinase. Here, we used immunoaffinity purification and quantitative mass spectrometry to identify nuclear interaction partners of endogenous DYRK1A. This interactome was enriched in DNA damage repair factors, transcriptional elongation factors and E3 ubiquitin ligases. We validated an interaction with RNF169, a factor that promotes homology directed repair upon DNA damage, and found that DYRK1A expression and kinase activity are required for maintenance of 53BP1 expression and subsequent recruitment to DNA damage loci. Further, DYRK1A knock out conferred resistance to ionizing radiation in colony formation assays, suggesting that DYRK1A expression decreases cell survival efficiency in response to DNA damage and points to a tumor suppressive role for this kinase.
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Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics
Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-Seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0 – 24h), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair.Chemistry and Chemical Biolog
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Suppression of p53 response by targeting p53-Mediator binding with a stapled peptide
DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator, a 26-subunit complex that controls RNA polymerase II activity genome-wide. We sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterize a stapled peptide, with functional mimics of both p53 activation domains, that blocks p53-Mediator binding and selectively inhibits p53-dependent transcription in human cells; importantly, this “bivalent” peptide has negligible impact, genome-wide, on non-p53 target genes. Our proof-of-concept strategy circumvents the TF entirely and targets the TF-Mediator interface instead, with desired functional outcomes (i.e., selective inhibition of p53 activation). Furthermore, these results demonstrate that TF activation domains represent viable starting points for Mediator-targeting molecular probes, as an alternative to large compound libraries. Different TFs bind Mediator through different subunits, suggesting this strategy could be broadly applied to selectively alter gene expression programs.
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Human TFIIH Kinase CDK7 Regulates Transcription-Associated Chromatin Modifications.
CDK7 phosphorylates the RNA polymerase II (pol II) C-terminal domain CTD and activates the P-TEFb-associated kinase CDK9, but its regulatory roles remain obscure. Here, using human CDK7 analog-sensitive (CDK7as) cells, we observed reduced capping enzyme recruitment, increased pol II promoter-proximal pausing, and defective termination at gene 3' ends upon CDK7 inhibition. We also noted that CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5' ends and H3K36me3 was displaced toward gene 3' ends in CDK7as cells. Mass spectrometry identified factors that bound TFIIH-phosphorylated versus P-TEFb-phosphorylated CTD (versus unmodified); capping enzymes and H3K4 methyltransferase complexes, SETD1A/B, selectively bound phosphorylated CTD, and the H3K36 methyltransferase SETD2 specifically bound P-TEFb-phosphorylated CTD. Moreover, TFIIH-phosphorylated CTD stimulated SETD1A/B activity toward nucleosomes, revealing a mechanistic basis for CDK7 regulation of H3K4me3 spreading. Collectively, these results implicate a CDK7-dependent "CTD code" that regulates chromatin marks in addition to RNA processing and pol II pausing
Molecular Architecture of the Human Mediator–RNA Polymerase II–TFIIF Assembly
The authors perform a cryo-EM study of the 1.9 MDa human Mediator-RNA polymerase II-TFIIF assembly, which reveals the structural organization of the human transcription initiation apparatus
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Multiomic Analysis Reveals Disruption of Cholesterol Homeostasis by Cannabidiol in Human Cell Lines
The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines. CBD treatment leads to alterations in the abundance of metabolites, mRNA transcripts, and proteins associated with activation of cholesterol biosynthesis, transport, and storage. We found that CBD rapidly incorporates into cellular membranes, alters cholesterol accessibility, and disrupts cholesterol-dependent membrane properties. Sustained treatment with high concentrations of CBD induces apoptosis in a dose-dependent manner. CBD-induced apoptosis is rescued by inhibition of cholesterol synthesis and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use.
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