RESUMO
CDK9 is a kinase critical for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. We also confirm the splicing factor SF3B1 as a target of CDK9 and show that SF3B1 in complex with polyadenylation factors is lost from chromatin after CDK9 inhibition. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.
Assuntos
Fator B de Elongação Transcricional Positiva , RNA Polimerase II , Cromatina/genética , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , RNA , RNA Polimerase II/metabolismo , Fatores de Processamento de RNA/genética , Transcrição Gênica , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
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
OBJECTIVES: Transcription of eukaryotic protein-coding genes by RNA polymerase II (pol II) is a highly regulated process. Most human genes have multiple poly(A) sites, which define different possible mRNA ends, suggesting the existence of mechanisms that regulate which poly(A) site is used. Poly(A) site selection may be mediated by cleavage factor I (CFIm), which is part of the cleavage and polyadenylation (CPA) complex. CFIm comprises CFIm25, CFIm59 and CFim68 subunits. It has been documented that the CPA complex also regulates pol II transcription at the start of genes. We therefore investigated whether CFIm, in addition to its role in poly(A) site selection, is involved in the regulation of pol II transcription. DATA DESCRIPTION: We provide genome-wide data of the effect of reducing by 90% expression of the CFIm25 constituent of CFIm, which is involved in pre-mRNA cleavage and polyadenylation, on pol II transcription in human cells. We performed pol II ChIP-seq in the presence or absence of CFIm25 and with or without an inhibitor of the cyclin-dependent kinase (CDK)9, which regulates the entry of pol II into productive elongation.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/genética , RNA Polimerase II/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Células HEK293 , HumanosRESUMO
Termination of RNA Polymerase II (Pol II) activity serves a vital cellular role by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast Saccharomyces cerevisiae, Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance. In particular, it is unclear how much crosstalk occurs between CPF-CF and NNS complexes and how Pol II attenuation is modulated during stress adaptation. In this study, we have identified an attenuator in the DEF1 DNA repair gene, which includes a portion of the 5'-untranslated region (UTR) and upstream open reading frame (ORF). Using a plasmid-based reporter gene system, we conducted a genetic screen of 14 termination mutants and their ability to confer Pol II read-through defects. The DEF1 attenuator behaved as a hybrid terminator, relying heavily on CPF-CF and Sen1 but without Nrd1 and Nab3 involvement. Our genetic selection identified 22 cis-acting point mutations that clustered into four regions, including a polyadenylation site efficiency element that genetically interacts with its cognate binding-protein Hrp1. Outside of the reporter gene context, a DEF1 attenuator mutant increased mRNA and protein expression, exacerbating the toxicity of a constitutively active Def1 protein. Overall, our data support a biologically significant role for transcription attenuation in regulating DEF1 expression, which can be modulated during the DNA damage response.
Assuntos
Proteínas Cromossômicas não Histona/genética , DNA Helicases/metabolismo , Reparo do DNA/genética , Poliadenilação/genética , RNA Helicases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Terminação da Transcrição Genética , Transcrição Gênica , Sequência de Bases , Proteínas Cromossômicas não Histona/metabolismo , Códon/genética , Genes Reporter , Mutação/genética , Fases de Leitura Aberta/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
In Saccharomyces cerevisiae, short noncoding RNA (ncRNA) generated by RNA polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3, and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is also generally required for NRD-dependent transcription termination through the action of its C-terminal domain (CTD)-interacting domain (CID). Pcf11 localizes downstream from Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore, mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA, and restricted Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar, as both accumulate RNA:DNA hybrids and display Pol II pausing downstream from NRD terminators. We predict a mechanism by which the exchange of Nrd1 and Pcf11 on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo.