Human immunodeficiency virus type 1 (HIV-1) transcriptional transactivator (Tat) recruits the positive transcription elongation factor b (P-TEFb) to the viral promoter. Consisting of cyclin dependent kinase 9 (Cdk9) and cyclin T1, P-TEFb phosphorylates RNA polymerase II and the negative transcription elongation factor to stimulate the elongation of HIV-1 genes. A major fraction of nuclear P-TEFb is sequestered into a transcriptionally inactive 7SK small nuclear ribonucleoprotein (snRNP) by the coordinated actions of the 7SK small nuclear RNA (snRNA) and hexamethylene bisacetamide (HMBA) induced protein 1 (HEXIM1). In this study, we demonstrate that Tat prevents the formation of and also releases P-TEFb from the 7SK snRNP in vitro and in vivo. This ability of Tat depends on the integrity of its N-terminal activation domain and stems from the high affinity interaction between Tat and cyclin T1, which allows Tat to directly displace HEXIM1 from cyclin T1. Furthermore, we find that in contrast to the Tat-independent activation of the HIV-1 promoter, Tat-dependent HIV-1 transcription is largely insensitive to the inhibition by HEXIM1. Finally, primary blood lymphocytes display a reduced amount of the endogenous 7SK snRNP upon HIV-1 infection. All these data are consistent with the model that Tat not only recruits but also increases the active pool of P-TEFb for efficient HIV-1 transcription.

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. The reservoir of HIV in infected people remains an insurmountable problem in the era of highly active antiretroviral therapy. Thus, the virus persists despite the best possible treatment. HIV hides in many cells and tissues, where its genome is not expressed. Thus, neither drugs nor the immune system can eradicate it from the body. One hope is to activate the production of HIV in these reservoirs in the presence of optimal treatment. Strategies aimed at activating hematopoetic cells and thus viral replication have been tried and failed. In this report, we targeted a specific host transcriptional complex that is essential for the transcription of HIV genome. Its activation should not lead to generalized stimulation of the immune system. Indeed, paradoxically, hexamethylene bisacetamide (HMBA) and related compounds lead to cellular differentiation and apoptosis. By studying properties of these differentiation agents, we discovered that they activate transiently transcription of HIV, be it in stable cell lines or in primary infected cells. Thus, compounds related to HMBA, some of which have now been approved for clinical use, could be tried to diminish or eliminate the reservoir of HIV in optimally treated infected individuals.

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.

Release of RNA polymerase II (Pol II) from promoter-proximal pausing has emerged as a critical step regulating gene expression in multicellular organisms. The transition of Pol II into productive elongation requires the kinase activity of positive transcription elongation factor b (P-TEFb), which is itself under a stringent control by the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. Here, we provide an overview on stimulating Pol II pause release by P-TEFb and on sequestering P-TEFb into 7SK snRNP. Furthermore, we highlight mechanisms that govern anchoring of 7SK snRNP to chromatin as well as means that release P-TEFb from the inhibitory complex, and propose a unifying model of P-TEFb activation on chromatin. Collectively, these studies shine a spotlight on the central role of RNA binding proteins (RBPs) in directing the inhibition and activation of P-TEFb, providing a compelling paradigm for controlling Pol II transcription with a non-coding RNA.

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.

Transcription elongation of eukaryotic genes by RNA polymerase II depends on the positive transcription elongation factor b (P‐TEFb). When sequestered into the large complex, P‐TEFb kinase activity is inhibited by the coordinate actions of 7SK small nuclear RNA (7SK snRNA) and hexamethylene bisacetamide (HMBA)‐induced protein 1 (HEXIM1). We found that the basic region in HEXIM1 directs its nuclear import via two monopartite and two bipartite nuclear localization sequences. Moreover, the arginine‐rich motif within it is essential for its binding to 7SK snRNA, P‐TEFb, and inhibition of transcription. Notably, the basic region interacts with the adjacent acidic regions in the absence of RNA. The removal of the positive or negative charges from these regions in HEXIM1 leads to its sequestration into the large complex and inhibition of transcription independently of the arginine‐rich motif. Finally, the removal of the negative charges from HEXIM1 results in its subnuclear localization into nuclear speckles. We propose a model where the interplay between 7SK snRNA and oppositely charged regions in HEXIM1 direct its binding to P‐TEFb and subcellular localization that culminates in the inhibition of transcription.