Most noncoding RNAs assemble with proteins to regulate diverse cellular processes including DNA replication, RNA transcription, RNA processing, and translation. These RNA-protein complexes (RNP) are often dynamic assemblies that include a core RNP plus other proteins that bind transiently or form complexes with different functions. We will use an integrative structural biology approach combining NMR spectroscopy, X-ray crystallography, and electron microscopy along with rigorous biochemistry and molecular biology to investigate structure, assembly, dynamics, and function of two regulatory RNPs, telomerase and 7SK RNP. Telomerase extends the 3'-ends of linear chromosomes by repetitively synthesizing the short telomere repeat sequence (TTAGGG in humans) using an RNA template that is part of its integral telomerase RNA (TER) and its specialized telomerase reverse transcriptase (TERT). It is a highly-regulated determinant of aging, tumorigenesis, and stem cell renewal. Our laboratory has pioneered structural studies of telomerase, most recently determining a 4.8 cryo-electron microscopy structure of active Tetrahymena telomerase with DNA. To elucidate the complete mechanism of telomere repeat synthesis and how telomerase is recruited to and regulated at telomeres, we propose to obtain (1) atomic resolution structures of Tetrahymana telomerase at each step in the catalytic cycle and correlate structure with activity and disease mutations in human telomerase and (2) investigate the structure and function of telomeric DNA-associated proteins in telomerase and at telomeres. These studies will provide fundamental insights into telomerase mechanism and regulation, how TERT and TER mutations linked to disease affect activity, and a structural basis for designing drugs to target telomerase activity. Human 7SK is an abundant nuclear long noncoding RNA that regulates RNA polymerase II (RNAPII) transcription, primarily by assembling with proteins to form an RNP that sequesters and inactivates the positive transcription elongation factor b (P-TEFb). P-TEFb is an integral component of the super elongation complex that phosphorylates negative transcription elongation factors and the RNAPII CTD to stimulate productive elongation of mRNA transcripts. 7SK also regulates the RNAPII transcription of small nuclear RNAs, enhancer RNAs, and axon maintenance through its interaction with hnRNP R. To understand the structural basis of 7SK regulation of P-TEFb activity, we propose to (1) determine the mechanism of assembly and structure of the stress-resistant core 7SK RNP, comprising 7SK, methylphosphate capping enzyme (MePCE), and La related protein group 7 (Larp7), and (2) determine how Hexim and P-TEFb interact with 7SK core RNP and each other to form the ?active? 7SK RNP. Diseases linked to P-TEFb misregulation include cardiac hypertrophy, cancers, and primordial dwarfism, and P-TEFb is a host cofactor for HIV replication. These studies will provide atomic-level information on the structures, interactions, and mechanisms of telomerase and 7SK RNPs that is critical to understanding their myriad effects on health and disease.
Telomerase and 7SK RNP are noncoding RNA-protein complexes that regulate synthesis of the telomeric DNA at the ends of chromosomes and transcription of eukaryotic mRNAs, some small nuclear and small nucleolar RNAs, and the HIV-1 genome (via hijacking), respectively. Both telomerase and 7SK RNP have multiple roles in the cell, and mutations that affect their activity or regulation have profound effects on human health, including aging, tumorigenesis, stem cell renewal, HIV-1 replication, heart disease, and development. We propose to use an integrative structural and molecular biology approach to investigate their mechanisms of action and interactions, which should reveal the structural basis of mutations leading to disease and for drug targeting.
