[show abstract][hide abstract] ABSTRACT: Regulation of the positive transcription elongation factor, P-TEFb, plays a major role in controlling mammalian transcription and this is accomplished in part by controlled release of P-TEFb from the 7SK snRNP that sequesters the kinase in an inactive state. We demonstrate here that a similar P-TEFb control system exists in Drosophila. We show that an RNA previously suggested to be a 7SK homolog is, in fact, associated with P-TEFb, through the action of a homolog of the human HEXIM1/2 proteins (dHEXIM). In addition, a Drosophila La related protein (now called dLARP7) is shown to be the functional homolog of human LARP7. The Drosophila 7SK snRNP (d7SK snRNP) responded to treatment of cells with P-TEFb inhibitors and to nuclease treatment of cell lysates by releasing P-TEFb. Supporting a critical role for the d7SK snRNP in Drosophila development, dLARP7 and dHEXIM were found to be ubiquitously expressed throughout embryos and tissues at all stages. Importantly, knockdown of dHEXIM was embryonic lethal, and reduction of dHEXIM in specific tissues led to serious developmental defects. Our results suggest that regulation of P-TEFb by the d7SK snRNP is essential for the growth and differentiation of tissues required during Drosophila development.
Nucleic Acids Research 02/2012; 40(12):5283-97. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The positive transcription elongation factor, P-TEFb, is required for the production of mRNAs, however the majority of the factor is present in the 7SK snRNP where it is inactivated by HEXIM1. Expression of HIV-1 Tat leads to release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo, but the release mechanisms are unclear.
We developed an in vitro P-TEFb release assay in which the 7SK snRNP immunoprecipitated from HeLa cell lysates using antibodies to LARP7 was incubated with potential release factors. We found that P-TEFb was directly released from the 7SK snRNP by HIV-1 Tat or the P-TEFb binding region of the cellular activator Brd4. Glycerol gradient sedimentation analysis was used to demonstrate that the same Brd4 protein transfected into HeLa cells caused the release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo. Although HEXIM1 binds tightly to 7SK RNA in vitro, release of P-TEFb from the 7SK snRNP is accompanied by the loss of HEXIM1. Using a chemical modification method, we determined that concomitant with the release of HEXIM1, 7SK underwent a major conformational change that blocks re-association of HEXIM1.
Given that promoter proximally paused polymerases are present on most human genes, understanding how activators recruit P-TEFb to those genes is critical. Our findings reveal that the two tested activators can extract P-TEFb from the 7SK snRNP. Importantly, we found that after P-TEFb is extracted a dramatic conformational change occurred in 7SK concomitant with the ejection of HEXIM1. Based on our findings, we hypothesize that reincorporation of HEXIM1 into the 7SK snRNP is likely the regulated step of reassembly of the 7SK snRNP containing P-TEFb.
PLoS ONE 01/2010; 5(8):e12335. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Regulation of the elongation phase of RNA polymerase II transcription by P-TEFb is a critical control point for gene expression. The activity of P-TEFb is regulated, in part, by reversible association with one of two HEXIMs and the 7SK snRNP. A recent proteomics survey revealed that P-TEFb and the HEXIMs are tightly connected to two previously-uncharacterized proteins, the methyphosphate capping enzyme, MEPCE, and a La-related protein, LARP7. Glycerol gradient sedimentation analysis of lysates from cells treated with P-TEFb inhibitors, suggested that the 7SK snRNP reorganized such that LARP7 and 7SK remained associated after P-TEFb and HEXIM1 were released. Immunodepletion of LARP7 also depleted most of the 7SK regardless of the presence of P-TEFb, HEXIM or hnRNP A1 in the complex. Small interfering RNA knockdown of LARP7 in human cells decreased the steady-state level of 7SK, led to an initial increase in free P-TEFb and increased Tat transactivation of the HIV-1 LTR. Knockdown of LARP7 or 7SK ultimately caused a decrease in total P-TEFb protein levels. Our studies have identified LARP7 as a 7SK-binding protein and suggest that free P-TEFb levels are determined by a balance between release from the large form and reduction of total P-TEFb.
Nucleic Acids Research 05/2008; 36(7):2219-29. · 8.28 Impact Factor