Interplay between 7SK snRNA and oppositely charged regions in HEXIM1 direct the inhibition of P-TEFb

Barborič, Matjaž; Kohoutek, Jiří; Price, John D.; Blažek, Dalibor; Price, David H.; Peterlin, B. Matija

doi:10.1038/sj.emboj.7600883

Cited by 98 publications

(120 citation statements)

References 27 publications

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“…The inhibitory effect of N-terminally truncated Hexim1 200-359 was indeed as potent as full-length Hexim1 bound to 7SK snRNA, supporting previous observations that the N-terminal part of Hexim1 could be self-inhibitory 29 . At concentrations of 100 nM P-TEFb, half-maximal inhibitory concentration (IC 50 ) values of 67 nM for Hexim1 200-359 and 67 nM for Hexim1/7SK, respectively, were determined compared with an IC 50 of 339 nM for fulllength Hexim1 (Fig.…”

Section: S S S Y S P T P S Y S P T P S Y S P T P Ssupporting

confidence: 85%

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

2012

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Phosphorylation of RnA polymerase II carboxy-terminal domain (CTD) in hepta-repeats YsPTsPs regulates eukaryotic transcription. Whereas ser5 is phosphorylated in the initiation phase, ser2 phosphorylation marks the elongation state. Here we show that the positive transcription elongation factor P-TEFb is a ser5 CTD kinase that is unable to create ser2/ser5 double phosphorylations, while it exhibits fourfold higher activity on a CTD substrate prephosphorylated at ser7 compared with the consensus hepta-repeat or the YsPTsPK variant. mass spectrometry reveals an equal number of phosphorylations to the number of heptarepeats provided, yet the mechanism of phosphorylation is distributive despite the repetitive nature of the substrate. Inhibition of P-TEFb activity is mediated by two regions in Hexim1 that act synergistically on Cdk9 and Cyclin T1. HIV-1 Tat/TAR abrogates Hexim1 inhibition to stimulate transcription of viral genes but does not change the substrate specificity. Together, these results provide insight into the multifaceted pattern of CTD phosphorylation.

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Section: S S S Y S P T P S Y S P T P S Y S P T P Ssupporting

confidence: 85%

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

2012

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“…A large fraction of the active form interacts with the bromodomain 4 (Brd4) recruiting factor which is necessary for its general transcription activity, 20,21 and ~ 50% of P-TEFb in the cell is present in inhibitory complexes containing HEXIM proteins (HEXIM1 or 2) bound to 7SK, an abundant small nuclear RNA. [22][23][24][25][26][27][28][29] P-TEFb contains the cyclin-dependent kinase CDK9 and a cyclin partner, either cyclin T1, T2a, T2b, or K. 30-32 The T cyclins (T1, T2a,b) are exceptionally long cyclins and share a highly homologous cyclin domain at their N termini which binds CDK9 and various cellular transcription modulators that recruit P-TEFb to specific promoters. 31,33-44 Their long C termini are divergent but contain a histidine-rich regulatory domain essential for binding RNA polymerase II and other CDK9 substrates and transcription modulators.…”

Section: Introductionmentioning

confidence: 99%

Developmental Regulators Containing the I-mfa Domain Interact with T cyclins and Tat and Modulate Transcription

Wang

Young

Mathews

et al. 2007

Journal of Molecular Biology

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SummaryPositive transcription elongation factor b (P-TEFb) complexes, composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1 or T2, are engaged by many cellular transcription regulators that activate or inhibit transcription from specific promoters. The related I-mfa (inhibitor of MyoD family a) and HIC (human I-mfa-domain-containing) proteins function in myogenic differentiation and embryonic development by participating in the Wnt signaling pathway. We report that I-mfa is a novel regulator of P-TEFb. Both HIC and I-mfa interact through their homologous I-mfa domains with cyclin T1 and T2 at two binding sites. One site is the regulatory histidine-rich domain that interacts with CDK9 substrates including RNA polymerase II. The second site contains a lysine-and arginine-rich motif that is highly conserved between the two T cyclins. This site overlaps and includes the previously identified Tat/TAR recognition motif of cyclin T1 required for activation of human immunodeficiency virus type 1 (HIV-1) transcription. HIC and I-mfa can serve as substrates for PTEFb. Their I-mfa domains also bind the activation domain of HIV-1 Tat and inhibit Tat-and PTEFb-dependent transcription from the HIV-1 promoter. This transcriptional repression is cell-type specific and can operate via Tat and cyclin T1. Genomic and sequence comparisons indicate that the I-mf and HIC genes, as well as flanking genes, diverged from a duplicated chromosomal region. Our findings link I-mfa and HIC to viral replication and suggest that P-TEFb is modulated in the Wnt signaling pathway.

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“…A transcriptionally inactive complex [referred to herein as 7SK small nuclear ribonucleoprotein (7SK snRNP)] contains P-TEFb, hexamethylene bisacetamide-induced protein (HEXIM) 1 (HEXIM1) and/or HEXIM2, the noncoding 7SK small nuclear RNA (7SK) and the La-related protein 7 (LARP7) (6)(7)(8)(9)(10)(11)(12). Therein, 7SK is a molecular scaffold, which binds HEXIM1, HEXIM2, and LARP7 directly, enabling the sequestration and repression of P-TEFb.…”

mentioning

confidence: 99%

7SK snRNP/P-TEFb couples transcription elongation with alternative splicing and is essential for vertebrate development

Barborič

Lenasi

Chen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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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.C hallenging the once predominantly held view that RNA polymerase II (RNAPII) recruitment to promoters is the rate-limiting event in regulating eukaryotic transcription, several recent studies indicate that RNAPII pauses subsequently to transcription initiation at a large number of genes in Drosophila and in several cell lineages in humans (1). A paradigm for this type of control is the regulation of HIV-1 transcription, which is paused because of concerted actions of the negative transcription elongation factors (N-TEFs) (1, 2). The block is reversed by the positive transcription elongation factor b (P-TEFb), consisting of a heterodimer between the cyclin-dependent kinase 9 (Cdk9) and one of the four C-type cyclins (CycT1/T2a(b)/K) (2), which phosphorylates Ser2 within the multiple heptapeptide repeats YSPTSPS of the carboxy-terminal domain (CTD) of the Rpb1 subunit of RNAPII, as well as components of N-TEFs. Of note, P-TEFb is critical for expressing early embryonic genes in C. elegans (3), and experiments in yeast and Drosophila demonstrate that it is also vital for proper 3Ј end pre-mRNA processing (4, 5). Thus, regulating transcription elongation is a key checkpoint for modulating eukaryotic gene expression.P-TEFb exists in 2 mutually exclusive complexes that differ in their kinase activity (2). A transcriptionally inactive complex [referred to herein as 7SK small nuclear ribonucleoprotein (7SK snRNP)] contains P-TEFb, hexamethylene bisacetamide-induced protein (HEXIM) 1 (HEXIM1) and/or HEXIM2, the noncoding 7SK small nuclear RNA (7SK) and the La-related protein 7 (LARP7) (6-12). Therein, 7SK is a molecular scaffold, which binds HEXIM1, HEXIM2, and LARP7 d...

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Interplay between 7SK snRNA and oppositely charged regions in HEXIM1 direct the inhibition of P-TEFb

Cited by 98 publications

References 27 publications

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

Developmental Regulators Containing the I-mfa Domain Interact with T cyclins and Tat and Modulate Transcription

7SK snRNP/P-TEFb couples transcription elongation with alternative splicing and is essential for vertebrate development

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