RESUMO
How transcription affects genome 3D organization is not well understood. We found that during influenza A (IAV) infection, rampant transcription rapidly reorganizes host cell chromatin interactions. These changes occur at the ends of highly transcribed genes, where global inhibition of transcription termination by IAV NS1 protein causes readthrough transcription for hundreds of kilobases. In these readthrough regions, elongating RNA polymerase II disrupts chromatin interactions by inducing cohesin displacement from CTCF sites, leading to locus decompaction. Readthrough transcription into heterochromatin regions switches them from the inert (B) to the permissive (A) chromatin compartment and enables transcription factor binding. Data from non-viral transcription stimuli show that transcription similarly affects cohesin-mediated chromatin contacts within gene bodies. Conversely, inhibition of transcription elongation allows cohesin to accumulate at previously transcribed intragenic CTCF sites and to mediate chromatin looping and compaction. Our data indicate that transcription elongation by RNA polymerase II remodels genome 3D architecture.
Assuntos
Cromatina/metabolismo , Genoma Humano , Virus da Influenza A Subtipo H5N1/metabolismo , Sítios de Ligação , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Flavonoides/farmacologia , Humanos , Interferon beta/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperidinas/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , CoesinasRESUMO
INTS11 and CPSF73 are metal-dependent endonucleases for Integrator and pre-mRNA 3'-end processing, respectively. Here, we show that the INTS11 binding partner BRAT1/CG7044, a factor important for neuronal fitness, stabilizes INTS11 in the cytoplasm and is required for Integrator function in the nucleus. Loss of BRAT1 in neural organoids leads to transcriptomic disruption and precocious expression of neurogenesis-driving transcription factors. The structures of the human INTS9-INTS11-BRAT1 and Drosophila dIntS11-CG7044 complexes reveal that the conserved C terminus of BRAT1/CG7044 is captured in the active site of INTS11, with a cysteine residue directly coordinating the metal ions. Inspired by these observations, we find that UBE3D is a binding partner for CPSF73, and UBE3D likely also uses a conserved cysteine residue to directly coordinate the active site metal ions. Our studies have revealed binding partners for INTS11 and CPSF73 that behave like cytoplasmic chaperones with a conserved impact on the nuclear functions of these enzymes.
Assuntos
Núcleo Celular , Citoplasma , Proteínas de Drosophila , Ligação Proteica , Humanos , Animais , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Citoplasma/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Endonucleases/metabolismo , Endonucleases/genética , Células HEK293 , Neurogênese/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/genética , Domínio CatalíticoRESUMO
The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3' processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3' end-processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cromatina , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Proteínas de Domínio MADS , RNA Polimerase II , Terminação da Transcrição Genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimologia , Cromatina/metabolismo , Cromatina/genética , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Histonas/metabolismo , Histonas/genética , Histona DesacetilasesRESUMO
N6-methyladenosine (m6A) is a crucial RNA modification that regulates diverse biological processes in human cells, but its co-transcriptional deposition and functions remain poorly understood. Here, we identified the RNA helicase DDX21 with a previously unrecognized role in directing m6A modification on nascent RNA for co-transcriptional regulation. DDX21 interacts with METTL3 for co-recruitment to chromatin through its recognition of R-loops, which can be formed co-transcriptionally as nascent transcripts hybridize onto the template DNA strand. Moreover, DDX21's helicase activity is needed for METTL3-mediated m6A deposition onto nascent RNA following recruitment. At transcription termination regions, this nexus of actions promotes XRN2-mediated termination of RNAPII transcription. Disruption of any of these steps, including the loss of DDX21, METTL3, or their enzymatic activities, leads to defective termination that can induce DNA damage. Therefore, we propose that the R-loop-DDX21-METTL3 nexus forges the missing link for co-transcriptional modification of m6A, coordinating transcription termination and genome stability.
Assuntos
Adenosina , Adenosina/análogos & derivados , RNA Helicases DEAD-box , Exorribonucleases , Instabilidade Genômica , Metiltransferases , Estruturas R-Loop , RNA Polimerase II , Terminação da Transcrição Genética , Humanos , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Adenosina/metabolismo , Adenosina/genética , Exorribonucleases/metabolismo , Exorribonucleases/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Células HEK293 , Cromatina/metabolismo , Cromatina/genética , Dano ao DNA , Células HeLa , RNA/metabolismo , RNA/genética , Transcrição Gênica , Metilação de RNARESUMO
Bacterial small RNAs (sRNAs) have been implicated in various aspects of post-transcriptional gene regulation. Here, we demonstrate that sRNAs also act at the level of transcription termination. We use the rpoS gene, which encodes a general stress sigma factor σ(S), as a model system, and show that sRNAs DsrA, ArcZ, and RprA bind the rpoS 5'UTR to suppress premature Rho-dependent transcription termination, both in vitro and in vivo. sRNA-mediated antitermination markedly stimulates transcription of rpoS during the transition to the stationary phase of growth, thereby facilitating a rapid adjustment of bacteria to global metabolic changes. Next generation RNA sequencing and bioinformatic analysis indicate that Rho functions as a global "attenuator" of transcription, acting at the 5'UTR of hundreds of bacterial genes, and that its suppression by sRNAs is a widespread mode of bacterial gene regulation.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Pequeno RNA não Traduzido/metabolismo , Fator sigma/metabolismo , Terminação da Transcrição Genética , Regiões 5' não TraduzidasRESUMO
Integrator is a metazoan-specific protein complex capable of inducing termination at all RNAPII-transcribed loci. Integrator recognizes paused, promoter-proximal RNAPII and drives premature termination using dual enzymatic activities: an endonuclease that cleaves nascent RNA and a protein phosphatase that removes stimulatory phosphorylation associated with RNAPII pause release and productive elongation. Recent breakthroughs in structural biology have revealed the overall architecture of Integrator and provided insights into how multiple Integrator modules are coordinated to elicit termination effectively. Furthermore, functional genomics and biochemical studies have unraveled how Integrator-mediated termination impacts protein-coding and noncoding loci. Here, we review the current knowledge about the assembly and activity of Integrator and describe the role of Integrator in gene regulation, highlighting the importance of this complex for human health.
Assuntos
Regulação da Expressão Gênica , RNA Polimerase II , Animais , Humanos , Fosfoproteínas Fosfatases/genética , Fosforilação , RNA Polimerase II/metabolismo , Transcrição Gênica , Proteínas/genética , Proteínas/metabolismoRESUMO
Genomic DNA is a crowded track where motor proteins frequently collide. It remains underexplored whether these collisions carry physiological function. In this work, we develop a single-molecule assay to visualize the trafficking of individual E. coli RNA polymerases (RNAPs) on DNA. Based on transcriptomic data, we hypothesize that RNAP collisions drive bidirectional transcription termination of convergent gene pairs. Single-molecule results show that the head-on collision between two converging RNAPs is necessary to prevent transcriptional readthrough but insufficient to release the RNAPs from the DNA. Remarkably, co-directional collision of a trailing RNAP into the head-on collided complex dramatically increases the termination efficiency. Furthermore, stem-loop structures formed in the nascent RNA are required for collisions to occur at well-defined positions between convergent genes. These findings suggest that physical collisions between RNAPs furnish a mechanism for transcription termination and that programmed genomic conflicts can be exploited to co-regulate the expression of multiple genes.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/metabolismo , DNA/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.
Assuntos
Inativação Gênica , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Transcrição Gênica , Genoma/genética , RNARESUMO
The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif (SLiM) in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit RNAPII termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. We find that ZC3H4, in turn, forms a direct connection to the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.
Assuntos
Proteínas Nucleares , Transcrição Gênica , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNARESUMO
The transcriptional termination of unstable non-coding RNAs (ncRNAs) is poorly understood compared to coding transcripts. We recently identified ZC3H4-WDR82 ("restrictor") as restricting human ncRNA transcription, but how it does this is unknown. Here, we show that ZC3H4 additionally associates with ARS2 and the nuclear exosome targeting complex. The domains of ZC3H4 that contact ARS2 and WDR82 are required for ncRNA restriction, suggesting their presence in a functional complex. Consistently, ZC3H4, WDR82, and ARS2 co-transcriptionally control an overlapping population of ncRNAs. ZC3H4 is proximal to the negative elongation factor, PNUTS, which we show enables restrictor function and is required to terminate the transcription of all major RNA polymerase II transcript classes. In contrast to short ncRNAs, longer protein-coding transcription is supported by U1 snRNA, which shields transcripts from restrictor and PNUTS at hundreds of genes. These data provide important insights into the mechanism and control of transcription by restrictor and PNUTS.
Assuntos
RNA Polimerase II , Transcrição Gênica , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Núcleo Celular/metabolismo , RNA não Traduzido/genética , Proteínas Cromossômicas não Histona/genéticaRESUMO
Transcription termination pathways mitigate the detrimental consequences of unscheduled promiscuous initiation occurring at hundreds of thousands of genomic cis-regulatory elements. The Restrictor complex, composed of the Pol II-interacting protein WDR82 and the RNA-binding protein ZC3H4, suppresses processive transcription at thousands of extragenic sites in mammalian genomes. Restrictor-driven termination does not involve nascent RNA cleavage, and its interplay with other termination machineries is unclear. Here we show that efficient termination at Restrictor-controlled extragenic transcription units involves the recruitment of the protein phosphatase 1 (PP1) regulatory subunit PNUTS, a negative regulator of the SPT5 elongation factor, and Symplekin, a protein associated with RNA cleavage complexes but also involved in cleavage-independent and phosphatase-dependent termination of noncoding RNAs in yeast. PNUTS and Symplekin act synergistically with, but independently from, Restrictor to dampen processive extragenic transcription. Moreover, the presence of limiting nuclear levels of Symplekin imposes a competition for its recruitment among multiple transcription termination machineries, resulting in mutual regulatory interactions. Hence, by synergizing with Restrictor, Symplekin and PNUTS enable efficient termination of processive, long-range extragenic transcription.
Assuntos
RNA Polimerase II , Transcrição Gênica , Animais , RNA Polimerase II/metabolismo , Sequências Reguladoras de Ácido Nucleico , Proteínas de Ligação a RNA/metabolismo , Processamento de Proteína Pós-Traducional , Mamíferos/genéticaRESUMO
RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination.
Assuntos
RNA Polimerases Dirigidas por DNA , RNA , RNA/genética , RNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Gênica , DNA , Bactérias/genética , Bactérias/metabolismo , Conformação de Ácido NucleicoRESUMO
Most eukaryotic messenger RNAs (mRNAs) are processed at their 3' end by the cleavage and polyadenylation specificity factor (CPF/CPSF). CPF mediates the endonucleolytic cleavage of the pre-mRNA and addition of a polyadenosine (poly(A)) tail, which together define the 3' end of the mature transcript. The activation of CPF is highly regulated to maintain the fidelity of RNA processing. Here, using cryo-EM of yeast CPF, we show that the Mpe1 subunit directly contacts the polyadenylation signal sequence in nascent pre-mRNA. The region of Mpe1 that contacts RNA also promotes the activation of CPF endonuclease activity and controls polyadenylation. The Cft2 subunit of CPF antagonizes the RNA-stabilized configuration of Mpe1. In vivo, the depletion or mutation of Mpe1 leads to widespread defects in transcription termination by RNA polymerase II, resulting in transcription interference on neighboring genes. Together, our data suggest that Mpe1 plays a major role in accurate 3' end processing, activating CPF, and ensuring timely transcription termination.
Assuntos
Precursores de RNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Poliadenilação e Clivagem de mRNA , Sequência de Aminoácidos , Microscopia Crioeletrônica , Poliadenilação , Ligação Proteica , Estrutura Terciária de Proteína , Precursores de RNA/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
The exonuclease torpedo Xrn2 loads onto nascent RNA 5'-PO4 ends and chases down pol II to promote termination downstream from polyA sites. We report that Xrn2 is recruited to preinitiation complexes and "travels" to 3' ends of genes. Mapping of 5'-PO4 ends in nascent RNA identified Xrn2 loading sites stabilized by an active site mutant, Xrn2(D235A). Xrn2 loading sites are approximately two to 20 bases downstream from where CPSF73 cleaves at polyA sites and histone 3' ends. We propose that processing of all mRNA 3' ends comprises cleavage and limited 5'-3' trimming by CPSF73, followed by handoff to Xrn2. A similar handoff occurs at tRNA 3' ends, where cotranscriptional RNase Z cleavage generates novel Xrn2 substrates. Exonuclease-dead Xrn2 increased transcription in 3' flanking regions by inhibiting polyA site-dependent termination. Surprisingly, the mutant Xrn2 also rescued transcription in promoter-proximal regions to the same extent as in 3' flanking regions. eNET-seq revealed Xrn2-mediated degradation of sense and antisense nascent RNA within a few bases of the TSS, where 5'-PO4 ends may be generated by decapping or endonucleolytic cleavage. These results suggest that a major fraction of pol II complexes terminates prematurely close to the start site under normal conditions by an Xrn2-mediated torpedo mechanism.
Assuntos
Poli A , RNA Polimerase II , RNA Polimerase II/genética , Núcleo Celular , Exonucleases , RNA AntissensoRESUMO
Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Traditional models of transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryoelectron microscopy (cryo-EM) structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific "pre-termination complex" (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC. RNA exiting RNAP interacts with NusA before entering the central channel of Rho from the distal C-terminal side of the ring. We map the principal interactions in the PTC and demonstrate their critical role in termination. Our results support a mechanism in which the formation of a persistent PTC is a prerequisite for termination.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Alongamento de Peptídeos/química , Fatores de Transcrição/química , Terminação da Transcrição Genética , Fatores de Elongação da Transcrição/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
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 small nuclear RNA (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 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.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerase II/metabolismo , RNA Nuclear Pequeno/biossíntese , Terminação da Transcrição Genética , Fator de Especificidade de Clivagem e Poliadenilação/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Poliadenilação , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Nuclear Pequeno/genéticaRESUMO
Transcription elongation has emerged as a regulatory hub in gene expression of metazoans. A major control point occurs during early elongation before RNA polymerase II (Pol II) is released into productive elongation. Prior research has linked BRD4 with transcription elongation. Here, we use rapid BET protein and BRD4-selective degradation along with quantitative genome-wide approaches to investigate direct functions of BRD4 in Pol II transcription regulation. Notably, as an immediate consequence of acute BRD4 loss, promoter-proximal pause release is impaired, and transcriptionally engaged Pol II past this checkpoint undergoes readthrough transcription. An integrated proteome-wide analysis uncovers elongation and 3'-RNA processing factors as core BRD4 interactors. BRD4 ablation disrupts the recruitment of general 3'-RNA processing factors at the 5'-control region, which correlates with RNA cleavage and termination defects. These studies, performed in human cells, reveal a BRD4-mediated checkpoint and begin to establish a molecular link between 5'-elongation control and 3'-RNA processing.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Nucleares/fisiologia , Elongação da Transcrição Genética/fisiologia , Fatores de Transcrição/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Expressão Gênica , Histonas/metabolismo , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/fisiologia , Fatores de Transcrição/metabolismo , Terminação da Transcrição Genética/fisiologia , Transcrição Gênica/genética , Transcrição Gênica/fisiologiaRESUMO
Heat shock instantly reprograms transcription. Whether gene and enhancer transcription fully recover from stress and whether stress establishes a memory by provoking transcription regulation that persists through mitosis remained unknown. Here, we measured nascent transcription and chromatin accessibility in unconditioned cells and in the daughters of stress-exposed cells. Tracking transcription genome-wide at nucleotide-resolution revealed that cells precisely restored RNA polymerase II (Pol II) distribution at gene bodies and enhancers upon recovery from stress. However, a single heat exposure in embryonic fibroblasts primed a faster gene induction in their daughter cells by increasing promoter-proximal Pol II pausing and by accelerating the pause release. In K562 erythroleukemia cells, repeated stress refined basal and heat-induced transcription over mitotic division and decelerated termination-coupled pre-mRNA processing. The slower termination retained transcripts on the chromatin and reduced recycling of Pol II. These results demonstrate that heat-induced transcriptional memory acts through promoter-proximal pause release and pre-mRNA processing at transcription termination.
Assuntos
Mitose/genética , Regiões Promotoras Genéticas/genética , Estresse Fisiológico/genética , Transcrição Gênica/genética , Linhagem Celular Tumoral , Cromatina/genética , Fibroblastos/fisiologia , Regulação da Expressão Gênica/genética , Genoma/genética , Resposta ao Choque Térmico/genética , Humanos , Células K562 , RNA Polimerase II/genética , RNA Mensageiro/genéticaRESUMO
Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly because of technical limitations in purifying authentic nascent transcripts. We present a new approach to characterize nascent RNA, called polymerase intact nascent transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5' ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano), and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5' P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that co-transcriptional splicing either occurs immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.
Assuntos
Nanotecnologia , RNA Polimerase II/metabolismo , Precursores de RNA/biossíntese , Splicing de RNA , RNA Mensageiro/biossíntese , RNA-Seq , Transcrição Gênica , Exorribonucleases/genética , Exorribonucleases/metabolismo , Células HCT116 , Células HeLa , Humanos , Cinética , Poliadenilação , Capuzes de RNA , RNA Polimerase II/genética , Precursores de RNA/genética , RNA Mensageiro/genéticaRESUMO
Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here we report that Integrator subunit 8 (IntS8) is critical for transcription repression and required for association with protein phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the RNA Pol II C-terminal domain and Spt5, preventing the transition to productive elongation. Thus, blocking PP2A association with Integrator stimulates pause release and gene activity. These results reveal a second catalytic function associated with Integrator-mediated transcription termination and indicate that control of productive elongation involves active competition between transcriptional kinases and phosphatases.