Eradication of the latent HIV reservoir remains a major barrier to curing AIDS. However, the mechanisms that direct viral persistence in the host are not well understood. Studying a model system of ...postintegration latency, we found that viral integration into the actively transcribed host genes led to transcriptional interference (TI) caused by the elongating RNA polymerase II (RNAPII) transcribing through the viral promoter. The resulting physical exclusion of preinitiation complex formation on the 5' long terminal repeat (LTR) promoted the silencing of HIV transcription. This block could be counteracted by inhibiting the upstream transcription or cooperatively activating viral transcription initiation and elongation. Importantly, PCR-based analysis, which detects host transcription through the 5'LTR independently of the viral integration site, revealed substantial levels of this transcription in HIV-infected primary CD4(+) T cells. Collectively, our findings suggest that TI contributes significantly to HIV latency and should be considered when attempting to purge the latent reservoir.
DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 ...(RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.
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•iCLIP identifies a genotoxic-stress-enhanced interaction of RBM7 with 7SK snRNA•RBM7 interacts with core subunits of 7SK snRNP to activate P-TEFb•Induced Pol II transcription through P-TEFb is a hallmark of the DNA damage response•The RBM7-P-TEFb axis promotes viability of genotoxic-stressed cells
Bugai et al. report that stimulation of Pol II pause release is central to the DNA damage response. Mechanistic studies uncover the vital role of RBM7 in the genotoxic-stress-induced release of P-TEFb from the inhibitory 7SK snRNP, which enables a transcriptional response that is crucial to the survival of stressed cells.
The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in ...high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.
Promoter-proximal pausing of RNAPII coincides with the formation of the cap structure at the 5′ end of pre-mRNA, which is bound by the cap-binding protein complex (CBC). Although the positive ...transcription elongation factor b (P-TEFb) stimulates the release of RNAPII from pausing and promotes transcription elongation and alternative splicing by phosphorylating the RNAPII C-terminal domain at Ser2 (S2-P RNAPII), it is unknown whether CBC facilitates these events. In this study, we report that CBC interacts with P-TEFb and transcriptionally engaged RNAPII and is globally required for optimal levels of S2-P RNAPII. Quantitative nascent RNA immunoprecipitation and ChIP experiments reveal that depletion of CBC attenuates HIV-1 Tat transactivation and impedes transcription elongation of investigated CBC-dependent endogenous genes by decreasing the levels of P-TEFb and S2-P RNAPII, leading to accumulation of RNAPII in the body of these genes. Finally, CBC is essential for the promotion of alternative splicing through facilitating P-TEFb, S2-P RNAPII, and splicing factor 2/alternative splicing factor occupancy at a splicing minigene. These findings disclose a vital role of CBC in connecting pre-mRNA capping to transcription elongation and alternative splicing via P-TEFb.
Hexamethylene bisacetamide (HMBA) is a potent inducer of cell differentiation and HIV production in chronically infected cells. However, its mechanism of action remains poorly defined. In this study, ...we demonstrate that HMBA activates transiently the PI3K/Akt pathway, which leads to the phosphorylation of HEXIM1 and the subsequent release of active positive transcription elongation factor b (P-TEFb) from its transcriptionally inactive complex with HEXIM1 and 7SK small nuclear RNA (snRNA). As a result, P-TEFb is recruited to the HIV promoter to stimulate transcription elongation and viral production. Despite the continuous presence of HMBA, the released P-TEFb reassembles rapidly with 7SK snRNA and HEXIM1. In contrast, a mutant HEXIM1 protein that cannot be phosphorylated and released from P-TEFb and 7SK snRNA via the PI3K/Akt pathway antagonizes this HMBA-mediated induction of viral production. Thus, our studies reveal how HIV transcription is induced by HMBA and suggest how modifications in the equilibrium between active and inactive P-TEFb could contribute to cell differentiation.
Eukaryotic gene expression is commonly controlled at the level of RNA polymerase II (RNAPII) pausing subsequent to transcription initiation. Transcription elongation is stimulated by the positive ...transcription elongation factor b (P-TEFb) kinase, which is suppressed within the 7SK small nuclear ribonucleoprotein (7SK snRNP). However, the biogenesis and functional significance of 7SK snRNP remain poorly understood. Here, we report that LARP7, BCDIN3, and the noncoding 7SK small nuclear RNA (7SK) are vital for the formation and stability of a cell stress-resistant core 7SK snRNP. Our functional studies demonstrate that 7SK snRNP is not only critical for controlling transcription elongation, but also for regulating alternative splicing of pre-mRNAs. Using a transient expression splicing assay, we find that 7SK snRNP disintegration promotes inclusion of an alternative exon via the increased occupancy of P-TEFb, Ser2-phosphorylated (Ser2-P) RNAPII, and the splicing factor SF2/ASF at the minigene. Importantly, knockdown of larp7 or bcdin3 orthologues in zebrafish embryos destabilizes 7SK and causes severe developmental defects and aberrant splicing of analyzed transcripts. These findings reveal a key role for P-TEFb in coupling transcription elongation with alternative splicing, and suggest that maintaining core 7SK snRNP is essential for vertebrate development.
Promoter-proximal pausing of RNA polymerase II (RNAPII) across the genome has renewed our attention to the early transcriptional events that control the establishment of pausing and the release of ...RNAPII into a productive transcription elongation. Here, we review our current understanding of the transcriptional cycle by RNAPII with a particular emphasis on the mechanisms that stimulate transcription elongation and cotranscriptional pre-mRNA splicing through an essential transcriptional kinase, the positive transcription elongation factor b (P-TEFb). We illustrate that by targeting a limited set of transcription elongation factors and paused RNAPII molecule during an early phase of transcription, P-TEFb unleashes an extensive crosstalk between transcription apparatus, RNA processing factors and chromatin for optimal production of mRNA.
The HIV-1 Tat protein promotes viral transcription elongation by recruiting P-TEFb to RNA element TAR on the viral mRNA. Recent work from D'Orso and Frankel uncovers unexpected aspects of this ...process.
The generation of messenger RNA (mRNA) in eukaryotes is achieved by transcription from the DNA template and pre‐mRNA processing reactions of capping, splicing, and polyadenylation. Although RNA ...polymerase II (RNAPII) catalyzes the synthesis of pre‐mRNA, it also serves as a principal coordinator of the processing reactions in the course of transcription. In this review, we focus on the interplay between transcription and cotranscriptional pre‐mRNA maturation events, mediated by the recruitment of RNA processing factors to differentially phosphorylated C‐terminal domain of Rbp1, the largest subunit of RNAPII. Furthermore, we highlight the bidirectional nature of the interplay by discussing the impact of RNAPII kinetics on pre‐mRNA processing as well as how the processing events reach back to different phases of gene transcription. WIREs RNA 2013, 4:139–154. doi: 10.1002/wrna.1148
This article is categorized under:
RNA Processing > Capping and 5' End Modifications
RNA Processing > Splicing Regulation/Alternative Splicing
RNA Processing > 3' End Processing
Positive transcription elongation factor b (P-TEFb) is the crucial player in RNA polymerase II (Pol II) pause release that has emerged as a promising target in cancer. Because single-agent therapy ...may fail to deliver durable clinical response, targeting of P-TEFb shall benefit when deployed as a combination therapy. We screened a comprehensive oncology library and identified clinically relevant antimetabolites and Mouse double minute 2 homolog (MDM2) inhibitors as top compounds eliciting p53-dependent death of colorectal cancer cells in synergy with selective inhibitors of P-TEFb. While the targeting of P-TEFb augments apoptosis by anti-metabolite 5-fluorouracil, it switches the fate of cancer cells by the non-genotoxic MDM2 inhibitor Nutlin-3a from cell-cycle arrest to apoptosis. Mechanistically, the fate switching is enabled by the induction of p53-dependent pro-apoptotic genes and repression of P-TEFb-dependent pro-survival genes of the PI3K-AKT signaling cascade, which stimulates caspase 9 and intrinsic apoptosis pathway in BAX/BAK-dependent manner. Finally, combination treatments trigger apoptosis of cancer cell spheroids. Together, co-targeting of P-TEFb and suppressors of intrinsic apoptosis could become a viable strategy to eliminate cancer cells.
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