Controlling Cellular P-TEFb Activity by the HIV-1 Transcriptional Transactivator Tat

The human immunodeficiency virus 1 (HIV-1) transcriptional transactivator (Tat) is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II). Tat recruits the host positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter through binding to the transactivator RNA (TAR) at the 5′-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA) and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5′-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR

Strigolactones play an important role in shaping exodermal morphology via a KAI2-dependent pathway

The majority of land plants have two suberized root barriers: the endodermis and the hypodermis (exodermis). Both barriers bear non-suberized passage cells that are thought to regulate water and nutrient exchange between the root and the soil. We learned a lot about endodermal passage cells, whereas our knowledge on hypodermal passage cells (HPCs) is still very scarce. Here we report on factors regulating the HPC number in Petunia roots. Strigolactones exhibit a positive effect, whereas supply of abscisic acid (ABA), ethylene, and auxin result in a strong reduction of the HPC number. Unexpectedly the strigolactone signaling mutant d14/dad2 showed significantly higher HPC numbers than the wild-type. In contrast, its mutant counterpart max2 of the heterodimeric receptor DAD2/MAX2 displayed a significant decrease in HPC number. A mutation in the Petunia karrikin sensor KAI2 exhibits drastically decreased HPC amounts, supporting the hypothesis that the dimeric KAI2/MAX2 receptor is central in determining the HPC number

Caractérisation structurale et fonctionnelle de la ribonucléoparticule 7SK chez les eucaryotes supérieurs

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Perturbations de la transcription liées à une dérégulation de P-TEFb : cancer, Sida et hypertrophie cardiaque

Le facteur P-TEFb stimule l’élongation de la transcription par l’ARN pol II. Dans les cellules humaines, le taux de P-TEFb disponible pour activer la transcription est contrôlé par un ARN non codant, l’ARN 7SK. Cet ARN s’associe à une fraction de P-TEFb qu’il séquestre au sein d’un complexe inactif. Un équilibre dynamique entre les formes active et inactive de P-TEFb permet d’adapter sa disponibilité aux besoins transcriptionnels de la cellule. Des perturbations de cet équilibre sont associées au développement de certaines tumeurs et à l’hypertrophie cardiaque. Ce système de régulation est également ciblé par le VIH pour augmenter la fraction active de P-TEFb dans les cellules infectées

7SK small nuclear RNA, a multifunctional transcriptional regulatory RNA with gene-specific features

The 7SK small nuclear RNA is a multifunctional transcriptional regulatory RNA that controls the nuclear activity of the positive transcription elongation factor b (P-TEFb), specifically targets P-TEFb to the promoter regions of selected protein-coding genes and promotes transcription of RNA polymerase II-specific spliceosomal small nuclear RNA genes

Cooperative dimerization of the POU domain protein Brn-2 on a new motif activates the neuronal promoter of the human aromatic l-amino acid decarboxylase gene

International audienc

Regulation of Polymerase II Transcription by 7SK snRNA: Two Distinct RNA Elements Direct P-TEFb and HEXIM1 Binding

The positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1/T2, stimulates transcription by phosphorylating RNA polymerase II. The 7SK small nuclear RNA, in cooperation with HEXIM1 protein, functions as a general polymerase II transcription regulator by sequestering P-TEFb into a large kinase-inactive 7SK/HEXIM1/P-TEFb complex. Here, determination and characterization of the functionally essential elements of human 7SK snRNA directing HEXIM1 and P-TEFb binding led to a new model for the assembly of the 7SK/HEXIM1/P-TEFb regulatory complex. We demonstrate that two structurally and functionally distinct protein binding elements located in the 5′- and 3′-terminal hairpins of 7SK support the in vivo recruitment of HEXIM1 and P-TEFb. Consistently, a minimal regulatory RNA composed of the 5′ and 3′ hairpins of 7SK can modulate polymerase II transcription in HeLa cells. HEXIM1 binds independently and specifically to the G24-C48/G60-C87 distal segment of the 5′ hairpin of 7SK. Binding of HEXIM1 is a prerequisite for association of P-TEFb with the G302-C324 apical region of the 3′ hairpin of 7SK that is highly reminiscent of the human immunodeficiency virus transactivation-responsive RNA

Chromatin Structure Is Implicated in “Late” Elongation Checkpoints on the U2 snRNA and β-Actin Genes▿ §

The negative elongation factor NELF is a key component of an early elongation checkpoint generally located within 100 bp of the transcription start site of protein-coding genes. Negotiation of this checkpoint and conversion to productive elongation require phosphorylation of the carboxy-terminal domain of RNA polymerase II (pol II), NELF, and DRB sensitivity-inducing factor (DSIF) by positive transcription elongation factor b (P-TEFb). P-TEFb is dispensable for transcription of the noncoding U2 snRNA genes, suggesting that a NELF-dependent checkpoint is absent. However, we find that NELF at the end of the 800-bp U2 gene transcription unit and RNA interference-mediated knockdown of NELF causes a termination defect. NELF is also associated 800 bp downstream of the transcription start site of the β-actin gene, where a “late” P-TEFb-dependent checkpoint occurs. Interestingly, both genes have an extended nucleosome-depleted region up to the NELF-dependent control point. In both cases, transcription through this region is P-TEFb independent, implicating chromatin in the formation of the terminator/checkpoint. Furthermore, CTCF colocalizes with NELF on the U2 and β-actin genes, raising the possibility that it helps the positioning and/or function of the NELF-dependent control point on these genes