Structural biology of 7SK RNP and its interaction with HIV-1 Tat

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PROJECT SUMMARY/ABSTRACT The human 7SK RNP is a dynamic assembly of the long non-coding 7SK RNA and cellular proteins that regulates the activity of positive transcription elongation factor b (P-TEFb). P-TEFb is an essential eukaryotic transcription factor for mRNA transcription elongation, which regulates the transition from promoter proximal paused RNA polymerase II (RNAPII) into productive elongation. P-TEFb is also an essential human cofactor for HIV-1 Tat transactivation and therefore viral replication. The human 7SK core RNP comprises the 331 nt RNAPIII-transcribed non-coding 7SK RNA, an unusual methyl capping enzyme called MePCE that methylates the γ phosphate on the RNA 5' terminus, and the La related protein 7, Larp7, that associates with the terminal hairpin and UUU-3'OH. In the active 7SK snRNP, Hexim and P-TEFb, a heterodimer of Cyclin T1 and the kinase Cdk9, bind the 7SK core RNP; interaction of P-TEFb in this complex inactivates it by sequestering its active site. Despite the central role of 7SK in transcription regulation of mRNA, other RNAPII RNAs, and HIV-1 transcription, relatively little is known at a structural or mechanistic level about how cellular proteins assemble with 7SK RNA to form a functional 7SK RNP or how Tat interacts with it to ultimately release P-TEFb. We will employ a combination of NMR spectroscopy, X-ray crystallography, and cryo electron microscopy along with biochemical methods to investigate the structures and assembly of the 7SK core RNP (MePCE–7SK–Larp7) and 7SK core RNP plus Hexim and P-TEFb (the `active' 7SK RNP) in order to achieve an atomic-level understanding of this important host RNP for HIV-1 viral replication. These structural studies will lay the groundwork for elucidating the molecular mechanisms of Tat-Hexim competition in the context of 7SK RNP. We aim to dissect the potential intermediate steps (i.e. Tat-bound 7SK RNP) that lead to P-TEFb hijacking from 7SK RNP into the HIV-1 viral super-elongation complex. The results of these experiments will provide fundamental molecular insights into and a structural basis for drug targeting of this largely structurally uncharacterized RNP that is essential for HIV-1 transcription and therefore escape from latency.