Telomerase is a large ribonucleoprotein complex responsible for replicating the G-rich strand of telomeres, the physical ends of chromosomes. It is critical for maintaining telomere length and preventing chromosome instability, and is a critica determinant of cellular aging, stem cell renewal, and tumorigenesis. Telomerase activity is low or undetectable in most somatic cells, while telomerase activation is important for the immortal phenotype of most cancer cells. Despite the importance of telomerase in human health and longevity, a detailed understanding of the structural biology of telomerase is still lacking. Telomerase contains two components required for catalytic activity, a unique eukaryotic reverse transcriptase (telomerase reverse transcriptase, TERT) and a large RNA (telomerase RNA, TER) that includes a template sequence used for copying the telomere repeat, plus other species-specific proteins required for function in vivo. To address the molecular basis of telomerase catalytic activity, in this grant application we are proposing to: (1) Determine a high resolution cryoelectron microscopy (cryoEM) structure of telomerase from the model organism Tetrahymena to provide a detailed view of the TERT-TER interactions in the catalytic core; (2) Use the information from the cryoEM structure to model the catalytic core of human telomerase and investigate the functional importance of TER and TERT domain interactions observed in cryoEM maps using NMR, some X- ray crystallography, and biochemistry; (3) Determine the pathway of the telomeric DNA and TER on telomerase during the catalytic cycle using cryoEM and biochemical methods; and (4) Develop stable cell lines for purifying human telomerase for structural studies to determine the subunit organization. These experiments will reveal the structure of the catalytic core of telomerase and provide insight into the roles of TERT and TER in catalysis and the catalytic mechanism. The results of these investigations will provide fundamental insights into the contributions of domains of TERT and TER to activity, an understanding of the molecular basis for human telomerase activity, insights into how TERT and TER mutations linked to disease affect activity, and a basis for structure based design of drugs that target the catalytic core of telomerase.

Public Health Relevance

Telomerase is a multimeric complex, composed of several proteins and a single RNA, that is essential for replication of the 3'ends of eukaryotic chromosomes. Telomerase activity is correlated with cellular aging, cancer, and various inherited diseases of the haemopoeitic system, and it is thus an important drug target. To further our understanding of human telomerase function, we propose to study the structure of the catalytic core of telomerase from the model organism Tetrahymena telomerase by cryoelectron microscopy and NMR spectroscopy and correlate structure and function to the human enzyme.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM048123-23
Application #
8965184
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter
Project Start
1992-08-01
Project End
2019-08-31
Budget Start
2015-09-01
Budget End
2016-08-31
Support Year
23
Fiscal Year
2015
Total Cost
$489,472
Indirect Cost
$143,412
Name
University of California Los Angeles
Department
Genetics
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Jiang, Jiansen; Wang, Yaqiang; SuĊĦac, Lukas et al. (2018) Structure of Telomerase with Telomeric DNA. Cell 173:1179-1190.e13
Chan, Henry; Wang, Yaqiang; Feigon, Juli (2017) Progress in Human and Tetrahymena Telomerase Structure Determination. Annu Rev Biophys 46:199-225
Wang, Yaqiang; Feigon, Juli (2017) Structural biology of telomerase and its interaction at telomeres. Curr Opin Struct Biol 47:77-87
Cash, Darian D; Feigon, Juli (2017) Structure and folding of the Tetrahymena telomerase RNA pseudoknot. Nucleic Acids Res 45:482-495
Upton, Heather E; Chan, Henry; Feigon, Juli et al. (2017) Shared Subunits of Tetrahymena Telomerase Holoenzyme and Replication Protein A Have Different Functions in Different Cellular Complexes. J Biol Chem 292:217-228
Wang, Yaqiang; Yesselman, Joseph D; Zhang, Qi et al. (2016) Structural conservation in the template/pseudoknot domain of vertebrate telomerase RNA from teleost fish to human. Proc Natl Acad Sci U S A 113:E5125-34
Feigon, Juli; Chan, Henry; Jiang, Jiansen (2016) Integrative structural biology of Tetrahymena telomerase - insights into catalytic mechanism and interaction at telomeres. FEBS J 283:2044-50
Jiang, Jiansen; Chan, Henry; Cash, Darian D et al. (2015) Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions. Science 350:aab4070
Singh, Mahavir; Wang, Zhonghua; Cascio, Duilio et al. (2015) Structure and interactions of the CS domain of human H/ACA RNP assembly protein Shq1. J Mol Biol 427:807-23
Kim, Nak-Kyoon; Zhang, Qi; Feigon, Juli (2014) Structure and sequence elements of the CR4/5 domain of medaka telomerase RNA important for telomerase function. Nucleic Acids Res 42:3395-408

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