The human 7SK small nuclear RNP (snRNP) is a dynamic assembly of the abundant 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 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 core 7SK RNP comprises the 331 nt RNAPIII-transcribed non-coding 7SK RNA, an unusual methyl capping enzyme called MePCE that methylates the ?-phosphate on the RNA 5'terminus, and the La related protein group 7, hLARP7, that associates with the terminal UUU-3'. In the active 7SK snRNP, HEXIM and P-TEFb bind the core snRNP;interaction of P-TEFb in this complex inactivates it by sequestering its active site. Recent studies have highlighted the importance of hLARP7 and MePCE in 7SK RNA stability and P-TEFb assembly;however, the structural basis of these interactions has not been established. We propose to investigate the structural characteristics and assembly of the core 7SK snRNP and its interactions with HEXIM1 and P-TEFb to form the "active" snRNP using a combination of NMR spectroscopy, X-ray crystallography, electron microscopy, and biochemical methods.
Our specific aims are: (1) Determine how the La related protein hLARP7 interacts with 7SK RNA;(2) Determine how MePCE interacts with 7SK RNA and hLARP7;(3) Determine the requirements for assembly of the core and "active" 7SK RNPs;and (4) Determine the architecture of the "active" 7SK RNP. Improper P-TEFb regulation by the 7SK snRNP plays a role in myriad diseases including cardiac hypertrophy, leukemia, lymphoma, cervical cancer, breast cancer, and gastric cancer. The results of the experiments proposed in this grant application will lead to an understanding of how the core 7SK snRNP assembles and ultimately binds to and inactivates P-TEFb. This in turn will provide fundamental information on transcription regulation, HIV-1 replication, mechanism of cancer progression, and dynamic non-coding RNA-directed cellular function.
P-TEFb is an essential transcription elongation factor for transcription of eukaryotic mRNAs by RNA polymerase and is required in HIV replication. The 7SK ribonucleoprotein (RNP) complex directly regulates the activity of P-TEFb, and improper P-TEFb regulation or loss of 7SK RNP function can lead to cardiac hypertrophy, leukemia, lymphoma, cervical cancer, and gastric cancer. It is therefore critical to understand how the 7SK RNP assembles and binds P-TEFb in order to understand the basis of cancer biogenesis and HIV replication.