Regulation of the elongation phase of RNA polymerase II transcription by P-TEFb is a critical control point for gene expression. The activity of P-TEFb is regulated, in part, by reversible association with one of two HEXIMs and the 7SK snRNP. A recent proteomics survey revealed that P-TEFb and the HEXIMs are tightly connected to two previously-uncharacterized proteins, the methyphosphate capping enzyme, MEPCE, and a La-related protein, LARP7. Glycerol gradient sedimentation analysis of lysates from cells treated with P-TEFb inhibitors, suggested that the 7SK snRNP reorganized such that LARP7 and 7SK remained associated after P-TEFb and HEXIM1 were released. Immunodepletion of LARP7 also depleted most of the 7SK regardless of the presence of P-TEFb, HEXIM or hnRNP A1 in the complex. Small interfering RNA knockdown of LARP7 in human cells decreased the steady-state level of 7SK, led to an initial increase in free P-TEFb and increased Tat transactivation of the HIV-1 LTR. Knockdown of LARP7 or 7SK ultimately caused a decrease in total P-TEFb protein levels. Our studies have identified LARP7 as a 7SK-binding protein and suggest that free P-TEFb levels are determined by a balance between release from the large form and reduction of total P-TEFb.

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...

The exon-junction complex (EJC) functionally links splicing to subsequent mRNA localization, translation and stability. Sequence-independent binding of the EJC core to RNA is ensured by the DEAD-box helicase eIF4AIII. Here, we identified the splicing factor CWC22 as a new eIF4AIII partner in flies and humans. CWC22 coexists with eIF4AIII in large protein complexes distinct from EJCs. Recombinant CWC22 directly contacts eIF4AIII and prevents it from binding RNA. In vitro splicing assays revealed that CWC22 introduces eIF4AIII to spliceosomes before remodeling to facilitate eIF4AIII incorporation into the EJC. Finally, using knockdowns in vivo, we showed that CWC22 is essential for EJC assembly. We elucidated the initial step of EJC assembly and the duality of CWC22 function that hinders eIF4AIII from nonspecifically binding RNA and escorts it to the splicing machinery to promote EJC assembly on mature mRNAs.