HEXIM1 is a promiscuous double-stranded RNA-binding protein and interacts with RNAs in addition to 7SK in culture cells

Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA.
Nucleic Acids Research (Impact Factor: 9.11). 03/2007; 35(8):2503-12. DOI: 10.1093/nar/gkm150
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P-TEFb regulates eukaryotic gene expression at the level of transcription elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein HEXIM1 or HEXIM2. In an effort to determine the minimal region of 7SK needed to interact with HEXIM1 in vitro, we found that an oligo comprised of nucleotides 10-48 sufficed. A bid to further narrow down the minimal region of 7SK led to a surprising finding that HEXIM1 binds to double-stranded RNA in a sequence-independent manner. Both dsRNA and 7SK (10-48), but not dsDNA, competed efficiently with full-length 7SK for HEXIM1 binding in vitro. Upon binding dsRNA, a large conformational change was observed in HEXIM1 that allowed the recruitment and inhibition of P-TEFb. Both subcellular fractionation and immunofluorescence demonstrated that, while most HEXIM1 is found in the nucleus, a significant fraction is found in the cytoplasm. Immunoprecipitation experiments demonstrated that both nuclear and cytoplasmic HEXIM1 is associated with RNA. Interestingly, the one microRNA examined (mir-16) was found in HEXIM1 immunoprecipitates, while the small nuclear RNAs, U6 and U2, were not. Our study illuminates novel properties of HEXIM1 both in vitro and in vivo, and suggests that HEXIM1 may be involved in other nuclear and cytoplasmic processes besides controlling P-TEFb.

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Available from: Gary Altwerger, Jun 25, 2015
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    • "This comparison revealed that there is an evolutionally conserved region from positions 150 to 165, in which the serine at position 158 (S158) lies in a canonical PKC phosphorylation site (RxxS/TxK/R, Figure 1A) (41). Since this highly basic region also interacts with 7SK snRNA (42–45), the addition of a negative charge by phosphorylation of S158 should decrease the ability of HEXIM1 to bind to RNA. "
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    ABSTRACT: The positive transcription elongation factor b (P-TEFb) regulates RNA polymerase II elongation. In cells, P-TEFb partitions between small active and larger inactive states. In the latter, HEXIM1 binds to 7SK snRNA and recruits as well as inactivates P-TEFb in the 7SK snRNP. Several stimuli can affect this P-TEFb equilibrium. In this study, we demonstrate that protein kinase C (PKC) phosphorylates the serine at position158 (S158) in HEXIM1. This phosphorylated HEXIM1 protein neither binds to 7SK snRNA nor inhibits P-TEFb. Phorbol esters or the engagement of the T cell antigen receptor, which activate PKC and the expression of the constitutively active (CA) PKCθ protein, which is found in T cells, inhibit the formation of the 7SK snRNP. All these stimuli increase P-TEFb-dependent transcription. In contrast, the kinase-negative PKCθ and the mutant HEXIM1 (S158A) proteins block effects of these PKC-activating stimuli. These results indicate that the phosphorylation of HEXIM1 by PKC represents a major regulatory step of P-TEFb activity in cells.
    Nucleic Acids Research 07/2012; 40(18):9160-9170. DOI:10.1093/nar/gks682 · 9.11 Impact Factor
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    • "Another possible elongation repression mechanism was suggested by the finding that HEXIM1 could bind to TAR [23] (Figure 4, bottom). The interaction of TAR with HEXIM1 would trigger the conformational change needed for P-TEFb binding [54] and, therefore, would act as a P-TEFb repressor [23]. The effects of reducing or increasing the level of HEXIM1 on expression from the HIV LTR has been interpreted as being mediated through the sequestration of P-TEFb by the 7SK snRNP [55–58]. "
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    ABSTRACT: HIV-1 usurps the RNA polymerase II elongation control machinery to regulate the expression of its genome during lytic and latent viral stages. After integration into the host genome, the HIV promoter within the long terminal repeat (LTR) is subject to potent downregulation in a postinitiation step of transcription. Once produced, the viral protein Tat commandeers the positive transcription elongation factor, P-TEFb, and brings it to the engaged RNA polymerase II (Pol II), leading to the production of viral proteins and genomic RNA. HIV can also enter a latent phase during which factors that regulate Pol II elongation may play a role in keeping the virus silent. HIV, the causative agent of AIDS, is a worldwide health concern. It is hoped that knowledge of the mechanisms regulating the expression of the HIV genome will lead to treatments and ultimately a cure.
    10/2011; 2011(20):726901. DOI:10.4061/2011/726901
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    • "(C–G) EMSA of mutated HP1-long (1–108) variants at increasing concentrations (0.1–1.2 µM) of full-length HEXIM1. The concentration of HEXIM1 was maintained low to avoid non-specific binding, a known tendency of HEXIM (70), that leads to the appearance of multiple bands. (H) Recapitulation of full length HEXIM1 binding of all HP1-long mutants. "
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    ABSTRACT: 7SK snRNA, an abundant RNA discovered in human nucleus, regulates transcription by RNA polymerase II (RNAPII). It sequesters and inhibits the transcription elongation factor P-TEFb which, by phosphorylation of RNAPII, switches transcription from initiation to processive elongation and relieves pauses of transcription. This regulation process depends on the association between 7SK and a HEXIM protein, neither isolated partner being able to inhibit P-TEFb alone. In this work, we used a combined NMR and biochemical approach to determine 7SK and HEXIM1 elements that define their binding properties. Our results demonstrate that a repeated GAUC motif located in the upper part of a hairpin on the 5'-end of 7SK is essential for specific HEXIM1 recognition. Binding of a peptide comprising the HEXIM Arginine Rich Motif (ARM) induces an opening of the GAUC motif and stabilization of an internal loop. A conserved proline-serine sequence in the middle of the ARM is shown to be essential for the binding specificity and the conformational change of the RNA. This work provides evidences for a recognition mechanism involving a first event of induced fit, suggesting that 7SK plasticity is involved in the transcription regulation.
    Nucleic Acids Research 11/2010; 38(21):7749-63. DOI:10.1093/nar/gkq660 · 9.11 Impact Factor
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