P-TEFb-Mediated Phosphorylation of hSpt5 C-Terminal Repeats Is Critical for Processive Transcription Elongation

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan.
Molecular Cell (Impact Factor: 14.02). 02/2006; 21(2):227-37. DOI: 10.1016/j.molcel.2005.11.024
Source: PubMed


Human DSIF, a heterodimer composed of hSpt4 and hSpt5, plays opposing roles in transcription elongation by RNA polymerase II (RNA Pol II). Here, we describe an evolutionarily conserved repetitive heptapeptide motif (consensus = G-S-R/Q-T-P) in the C-terminal region (CTR) of hSpt5, which, like the C-terminal domain (CTD) of RNA Pol II, is highly phosphorylated by P-TEFb. Thr-4 residues of the CTR repeats are functionally important phosphorylation sites. In vitro, Thr-4 phosphorylation is critical for the elongation activation activity of DSIF, but not to its elongation repression activity. In vivo, Thr-4 phosphorylation is critical for epidermal growth factor (EGF)-inducible transcription of c-fos and for efficient progression of RNA Pol II along the gene. We consider this phosphorylation to be a switch that converts DSIF from a repressor to an activator. We propose the "mini-CTD" hypothesis, in which phosphorylated CTR is thought to function in a manner analogous to phosphorylated CTD, serving as an additional code for active elongation complexes.

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    • "DSIF, which consists of Spt4 and Spt5, promotes pausing in conjunction with NELF and also serves as a factor required for increasing the catalytic rate of transcription elongation by Pol II. P-TEFb is a kinase that phosphorylates the C-terminal domain (CTD) of RPB1, the largest subunit of Pol II, the C-terminal domain of SPT5, and the NELF-E subunit to promote the transition to transcription elongation (Fujinaga et al., 2004; Marshall et al., 1996; Yamada et al., 2006). The RPB1 CTD contains a heptad repeat (consensus Tyr1- Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) that is subject to extensive post-translational modifications, most notably phosphorylation at Ser2 and Ser5 residues (Smith and Shilatifard, 2013). "
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    ABSTRACT: The control of promoter-proximal pausing and the release of RNA polymerase II (Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify that Pol-II-associated factor 1 (PAF1) possesses an evolutionarily conserved function in metazoans in the regulation of promoter-proximal pausing. Reduction in PAF1 levels leads to an increased release of paused Pol II into gene bodies at thousands of genes. PAF1 depletion results in increased nascent and mature transcripts and increased levels of phosphorylation of Pol II's C-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase super elongation complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing PAF1 as a regulator of promoter-proximal pausing by Pol II. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cell 08/2015; 162(5). DOI:10.1016/j.cell.2015.07.042 · 32.24 Impact Factor
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    • "Recruitment of P-TEFb at TAR enables CDK9 to phosphorylate NELF-E and force its dissociation from TAR RNA and the paused polymerase complex (Fujinaga et al., 2004; Jadlowsky et al., 2014; Yamaguchi et al., 2002; Zhang et al., 2007). CDK9 also extensively phosphorylates the CTD of RNAPII mainly at Ser2 and Ser 5 residues of the heptad repeats (Czudnochowski et al., 2012), and homologous heptapeptide repeats (G-S-Q/R-T-P) of hSpt5 at Thr4 residues which converts it into a stimulatory elongation factor (Bourgeois et al., 2002; Yamada et al., 2006). Therefore, the overall effect Tat and P-TEFb is to remove the blocks to elongation imposed by NELF and DSIF and to stimulate efficient elongation and cotranscriptional processing of proviral transcripts. "
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    Virology 02/2014; 454-455(1). DOI:10.1016/j.virol.2014.02.008 · 3.32 Impact Factor
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    • "P-TEFb triggers elongation of RNA polymerase II (RNAPII) transcription by phosphorylating the negative elongation factor (NELF) and the DRB-sensitivity inducing complex (DSIF/Spt4/Spt5), thus promoting the release of NELF and also phosphorylating Ser-2 residues in RNAPII CTD (reviewed in [9]). Upon the dissociation of NELF, DSIF becomes a positive elongation factor [11] and increased the processivity RNAPII [12]. Although P-TEFb travels with the elongation complex, its CTD kinase activity is no longer required once the complex is released from the pause [10]. "
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    ABSTRACT: HIV-1 transcription is regulated by CDK9/cyclin T1, which, unlike a typical cell cycle-dependent kinase, is regulated by associating with 7SK small nuclear ribonuclear protein complex (snRNP). While the protein components of this complex are well studied, the mechanism of the complex formation is still not fully understood. The association of CDK9/cyclin T1 with 7SK snRNP is, in part, regulated by a reversible CDK9 phosphorylation. Here, we present a comprehensive review of the kinases and phosphatases involved in CDK9 phosphorylation and discuss their role in regulation of HIV-1 replication and potential for being targeted for drug development. We propose a novel pathway of HIV-1 transcription regulation via CDK9 Ser-90 phosphorylation by CDK2 and CDK9 Ser-175 dephosphorylation by protein phosphatase-1.
    01/2014; 2014(2):964964. DOI:10.1155/2014/964964
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