Phosphorylated Positive Transcription Elongation Factor b (P-TEFb) Is Tagged for Inhibition through Association with 7SK snRNA

Abstract

The positive transcription elongation factor b (PTEFb), comprising CDK9 and cyclin T, stimulates transcription of cellular and viral genes by phosphorylating RNA polymerase II. A major portion of nuclear P-TEFb is sequestered and inactivated by the coordinated actions of the 7SK snRNA and the HEXIM1 protein, whose induced dissociation from P-TEFb is crucial for stressinduced transcription and pathogenesis of cardiac hypertrophy. The 7SK⅐P-TEFb interaction, which can occur independently of HEXIM1 and does not by itself inhibit P-TEFb, recruits HEXIM1 for P-TEFb inactivation. To study the control of this interaction, we established an in vitro system that reconstituted the specific interaction of P-TEFb with 7SK but not other snRNAs. Using this system, together with an in vivo binding assay, we show that the phosphorylation of CDK9, on possibly the conserved Thr-186 in the T-loop, was crucial for the 7SK⅐P-TEFb interaction. This phosphorylation was not caused by CDK9 autophosphorylation or the general CDK-activating kinase CAK, but rather by a novel HeLa nuclear kinase. Furthermore, the stress-induced disruption of the 7SK⅐P-TEFb interaction was not caused by any prohibitive changes in 7SK but by the dephosphorylation of P-TEFb, leading to the loss of the key phosphorylation important for 7SK binding. Thus, the phosphorylated P-TEFb is tagged for inhibition through association with 7SK. We discuss the implications of this mechanism in controlling P-TEFb activity during normal and stress-induced transcription.7SK is an abundant (2 ϫ 10 5 copies/cell) and evolutionarily conserved small nuclear RNA (snRNA) 1 of ϳ330 nucleotides (1-4). It is transcribed by RNA polymerase (Pol) III from one or more genes belonging to a family of interspersed repeats in the mammalian genome (2, 5). The high conservation and abundance of 7SK suggest an important physiological function of this RNA. In searching for nuclear factors that can bind to and regulate the activity of human positive transcription elongation factor b (P-TEFb), we and others (6, 7) have identified 7SK as a specific P-TEFb-associated factor. Consisting of CDK9 and cyclin T1 (CycT1) (8, 9), P-TEFb strongly enhances the processivity of RNA Pol II by phosphorylating the C-terminal domain (CTD) of Pol II and antagonizing the actions of negative elongation factors (10 -13). Studies employing RNA interference in Caenorhabditis elegans and a pharmacological inhibitor of CDK9 in mammalian cells implicate P-TEFb as essential for expression of most protein-coding genes (10, 14).P-TEFb also functions as a specific host cellular cofactor for the HIV-1 Tat protein. Stimulation of Pol II elongation is essential for HIV transcription, during which P-TEFb is recruited to the nascent mRNA by Tat (15, 16). Tat binds to CycT1 and recruits P-TEFb through formation of a stable ternary complex containing P-TEFb, Tat, and the HIV TAR RNA structure located at the 5Ј-end of the nascent viral transcript. Once recruited, P-TEFb phosphorylates the CTD and stimulates the production of the full-l...

Phosphatase PPM1A Regulates Phosphorylation of Thr-186 in the Cdk9 T-loop

“…Overexpression of PPM1A and PPM1B also greatly reduced the amount of the HEXIM1 protein that co-immunoprecipitated with FLAG-Cdk9. The reduction in HEXIM1 associated with Cdk9 was expected as the association of HEXIM1 and 7SK RNA with P-TEFb requires phosphorylation of Thr-186 in the Cdk9 T-loop (21). We also observed in another co-immunoprecipitation experiment that overexpression of PPM1A reduced the association of 7SK RNA and HEXIM1 with a transfected FLAG-Cdk9 protein (data not shown).…”

“…Cyclin-dependent kinases contain a conserved threonine in their T-loop, and phosphorylation of this residue induces a conformational change that allows Cdk substrates to access the catalytic core of the enzyme (19,20). In the case of Cdk9, the association of 7SK with P-TEFb has been shown to be dependent on Thr-186 phosphorylation (21). A recent study reported that under conditions of stress, either UV irradiation or treatment with high concentrations of hexamethylene bisacetamide, the phosphatases PP2B and PP1␣ act cooperatively to disrupt P-TEFb from the 7SK small nuclear ribonucleoprotein through the dephosphorylation of Thr-186 (22).…”

Deep sequencing of the mouse lung transcriptome reveals distinct long non-coding RNAs expression associated with the high virulence of H5N1 avian influenza virus in mice

“…These two mechanisms share similar regulatory logistics and cooperate in part by influencing the activity of the binding sites in target genes. Some lncRNAs, such as 7SK, can directly affect the loading and activity of general TFs and to further affect the production of mRNAs[54]. Moreover, another lncRNA, NRON, can directly serve as either co-factors or inhibitors to regulate the activity of nuclear factor of activated T cells (NFAT) proteins which manipulate gene expression in many cell types[55].…”

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“…CmRBP50 C-terminal Phosphorylation Is Necessary for Interaction with Phloem Proteins-RNA-binding protein phosphorylation has recently been reported as a requirement for RNP complex formation (37,38). To further investigate the role of CmRBP50 phosphorylation in protein-protein interaction, we next conducted co-IP and protein overlay assays using proteins contained within pumpkin phloem sap (30,35,39) (Fig.…”