The Transcription-Dependent Dissociation of P-TEFb-HEXIM1-7SK RNA Relies upon Formation of hnRNP-7SK RNA Complexes

Abstract

The positive transcription elongation factor P-TEFb controls the elongation of transcription by RNA polymerase II. P-TEFb is inactivated upon binding to HEXIM1 or HEXIM2 proteins associated with a noncoding RNA, 7SK. In response to the inhibition of transcription, 7SK RNA, as well as HEXIM proteins, is released by an unknown mechanism and P-TEFb is activated. New partners of 7SK RNA were searched for as potential players in this feedback process. A subset of heterogeneous ribonuclear proteins, hnRNPs Q and R and hnRNPs A1 and A2, were thus identified as major 7SK RNA-associated proteins. The degree of association of 7SK RNA with these hnRNPs increased when P-TEFb-HEXIM1-7SK was dissociated following the inhibition of transcription or HEXIM1 knockdown. This finding suggested that 7SK RNA shuttles from HEXIM1-P-TEFb complexes to hnRNPs. The transcription-dependent dissociation of P-TEFb-HEXIM1-7SK complexes was attenuated when both hnRNPs A1 and A2 were knocked down by small interfering RNA. As hnRNPs are known to interact transiently with RNA while it is synthesized, hnRNPs released from nascent transcripts may trap 7SK RNA and thereby contribute to the activation of P-TEFb.The positive transcription elongation factor P-TEFb is required to activate the transcription of most class II genes (48). P-TEFb comprises two subunits, CDK9, a cyclin-dependent protein kinase, and its corresponding cyclin T1 or cyclin T2. The activity of P-TEFb is regulated. It increases in response to the inhibition of transcription (45, 63) or cardiac cell hypertrophic stimulation (54). Previous studies have indicated that 7SK RNA associates with an inactive form of P-TEFb. 7SK RNA is an abundant (2 ϫ 10 5 -molecule-per-cell) noncoding nuclear RNA of 331 nucleotides (60, 68). The inhibition of P-TEFb activity relies upon the binding of HEXIM1 or HEXIM2 proteins to cyclin T1 or T2 (5,13,41,64). This process requires the association of 7SK RNA with HEXIM1 or HEXIM2 (40,65). Two hairpins in the 7SK RNA structure are involved in this association (14). 7SK binding to HEXIM proteins promotes a major conformational change allowing the C-terminal domains of the proteins to interact with the Nterminal domains of cyclin T's (41, 55). This transcriptiondependent regulation may constitute a feedback loop finetuning the efficiency of the elongation step in class II gene transcription.The molecular regulatory mechanism remains largely unknown. 7SK RNA is very stable even when it dissociates from HEXIM proteins; its degradation is unlikely to contribute to the dissociation. Posttranslational modifications of protein subunits in P-TEFb-HEXIM-7SK may determine their association. For instance, the phosphorylation of CDK9 on threonine residue T186 is required, but the in vivo regulation of this step has not been established (10,35,47). HEXIM proteins and 7SK RNA may be released when P-TEFb binds to components of the transcriptional machinery, such as transcription factors like NF-B (2), retinoblastoma protein (56), androgen receptor (34), aryl hydrocarbon r...

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

“…Overall, these findings suggest that in addition to the capping of 7SK by BCDIN3 and binding its 3Ј poly(U) tail by LARP7 (17,11), the extensive RNA-protein and protein-protein interactions within core 7SK snRNP guarantee its maintenance. Hence, core 7SK snRNP cannot be disintegrated when exposed to ActD, which explains the stability of 7SK under this cell stress-inducing condition and allows core 7SK snRNP to be a platform for the dynamic and reversible remodeling of 7SK snRNP as observed before (6,24,25).…”

“…S6 C-E). These EDA levels were not stimulated by hnRNP A1, an inhibitory splicing factor (23) present in a distinct 7SK snRNP complex devoid of P-TEFb (24,25), which is most likely also released from this 7SK snRNP upon depletion of LARP7 or BCDIN3. As expected, depletion of hnRNP A1 mRNA enhanced the inclusion of EDA (Fig.…”

Transcription control by long non-coding RNAs

“…83,[92][93][94] 7SK can also function cooperatively with the chromatin regulator HMGA1 to control transcription in both P-TEFb-dependent and -independent modes. 95,96 These cases exemplify a vast range of new mechanistic possibilities by which a regulatory RNA may control transcription by interacting with different protein complexes.…”

An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

“…Although the total amount of 7SK RNA does not vary in live cells, the level of 7SK RNA available for Hexim binding is modulated (10)(11)(12). As a consequence, the overall P-TEFb activity increases on inhibition of transcription (3,4).…”

“…As a consequence, the overall P-TEFb activity increases on inhibition of transcription (3,4). Thus, in addition to other more specific functions (13)(14)(15), 7SK RNA serves as a sensor of transcriptional activity through a feedback loop, fine-tuning P-TEFb activity (10). Understanding how this noncoding RNA mechanistically regulates the activity of P-TEFb, a central transcription factor, remains challenging.…”