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.Cyclic phosphorylation of the tandemly repeated YSPTSPS heptapeptide motif in the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is crucial for stimulating mRNA production (9, 14). For example, during the early stage of transcription, phosporylation of the CTD at serine 2 by the positive transcription elongation factor b (PTEFb) is essential for the transition from abortive to productive transcription elongation (5,7,30,35). P-TEFb is a general transcription factor that facilitates the production of fulllength mRNAs of most, if not all, protein-coding genes and also stimulates the Pol II-mediated synthesis of human immunodeficiency virus (HIV) transcripts from the 5Ј long terminal repeat of the integrated proviral genome (5, 29). P-TEFb is composed of a cyclin-dependent kinase, Cdk9, and the regulatory subunit cyclin (Cyc) T1, T2, or K (22, 33, 41; reviewed in reference 23). In human HeLa cells, about half of P-TEFb is associated with large ribonucleoprotein (RNP) complexes which also contain the 7SK small nuclear RNA (snRNA) and the HEXIM1 or HEXIM2 protein (3,17,20,36,37,39). In contrast to its free form, the 7SK/HEXIM1-associated fraction of P-TEFb shows little CTD kinase activity, indicating that the 7SK snRNA, in collaboration with HEXIM1, functions as an inhibitory factor of P-TEFb. Association of P-TEFb with 7SK/HEXIM1 is specific and reversible. Inhibition of transcription by chemical or UV treatment induces dissociation of P-TEFb from the kinase-inactive 7SK/ HEXIM1/P-TEFb complex (17,20,36,37). Consequently, increased accumulation of free P-TEFb facilitates CTD phosphorylation and mRNA production. Likewise, depletion of 7SK snRNA increases the CTD kinase activity of P-TEFb and stimulates transcription from Pol II-specific promoters, including the HIV long terminal repeat...

The 7SK small nuclear RNA (snRNA) regulates RNA polymerase II transcription elongation by controlling the protein kinase activity of the positive transcription elongation factor b (P-TEFb). In cooperation with HEXIM1, the 7SK snRNA sequesters P-TEFb into the kinase-inactive 7SK/HEXIM1/P-TEFb small nuclear ribonucleoprotein (snRNP), and thereby, controls the nuclear level of active P-TEFb. Here, we report that a fraction of HeLa 7SK snRNA that is not involved in 7SK/HEXIM1/P-TEFb formation, specifically interacts with RNA helicase A (RHA), heterogeneous nuclear ribonucleoprotein A1 (hnRNP), A2/B1, R and Q proteins. Inhibition of cellular transcription induces disassembly of 7SK/HEXIM1/P-TEFb and at the same time, increases the level of 7SK snRNPs containing RHA, hnRNP A1, A2/B1, R and Q. Removal of transcription inhibitors restores the original levels of the 7SK/HEXIM1/P-TEFb and '7SK/hnRNP' complexes. 7SK/HEXIM1/P-TEFb snRNPs containing mutant 7SK RNAs lacking the capacity for binding hnRNP A1, A2, R and Q are resistant to stressinduced disassembly, indicating that recruitment of the novel 7SK snRNP proteins is essential for disruption of 7SK/HEXIM1/P-TEFb. Thus, we propose that the nuclear level of active P-TEFb is controlled by dynamic and reversible remodelling of 7SK snRNP.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.The U1 small nuclear RNA (snRNA)-in the form of the U1 spliceosomal Sm small nuclear ribonucleoprotein particle (snRNP) that contains seven Sm and three U1-specific RNP proteins-has a crucial function in the recognition and removal of pre-messenger RNA introns. Here, we show that a fraction of human U1 snRNA specifically associates with the nuclear RNA-binding protein TBP-associated factor 15 (TAF15). We show that none of the known protein components of the spliceosomal U1-Sm snRNP interacts with the newly identified U1-TAF15 snRNP. In addition, the U1-TAF15 snRNP tightly associates with chromatin in an RNA-dependent manner and accumulates in nucleolar caps upon transcriptional inhibition. The Sm-binding motif of U1 snRNA is essential for the biogenesis of both U1-Sm and U1-TAF15 snRNPs, suggesting that the U1-TAF15 particle is produced by remodelling of the U1-Sm snRNP. A demonstration that human U1 snRNA forms at least two structurally distinct snRNPs supports the idea that the U1 snRNA has many nuclear functions.