A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat.

BACKGROUND: The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. RESULTS: Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. CONCLUSIONS: Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription.

Ubiquitination of HEXIM1 by HDM2.

Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) is an inhibitor of the positive transcription elongation factor b (P-TEFb), which controls RNA polymerase II transcription and human immunodeficiency virus Tat transactivation. In cells, more than half of P-TEFb is associated with HEXIM1 resulting in the inactivation of P-TEFb. Recently, we found that nucleophosmin (NPM), a key factor involved in p53 signaling pathway, interacts with HEXIM1 and activates P-TEFb-dependent transcription. Here we report that human double minute-2 protein (HDM2), a p53-specific E3 ubiquitin ligase, specifically ubiquitinates HEXIM1 through the lysine residues located within the basic region of HEXIM1. However, the HDM2-induced HEXIM1 ubiquitination does not lead to proteasome-mediated protein degradation. Fusion of ubiquitin to HEXIM1 demonstrates stronger inhibition on P-TEFb-dependent transcription. Our results demonstrate that HDM2 functions as a specific E3 ubiquitin ligase for HEXIM1, suggesting a possible role for HEXIM1 ubiquitination in the regulation of P-TEFb activity.

7SK RNA, a non-coding RNA regulating P-TEFb, a general transcription factor.

Human 7SK RNA is an abundant 331 nt nuclear transcript generated by RNA polymerase III. Binding of 7SK RNA to HEXIM1/2 turns these proteins into inhibitors of P-TEFb (Positive Transcriptional Elongation Factor b). P-TEFb is required for RNA polymerase II transcription elongation. 7SK RNA is released from P-TEFb/HEXIM/7SK complexes upon an arrest in transcription and physiological stimulations such as cardiac hypertrophy, leading to P-TEFb activation. The released 7SK RNA associates a subset of heterogeneous nuclear ribonucleoproteins (hnRNP). 7SK RNA has been evolutionary conserved in vertebrates and homologues are found in annelid, mollusc and insect genomes. 7SK RNA folds into several hairpins that serve as specific platforms for binding proteins. It is stabilized by mono-methylation of its 5'-triphosphate group and binding of a specific La-Related protein, LARP7 at its 3' end. As the likely best characterized example, 7SK RNA is a paradigm for non-coding RNAs regulating transcription.