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. Telomerase activity is low or undetectable in most somatic cells, while high levels of telomerase activity are essential for viability of most cancer cells. All telomerases contain a large RNA (TR) (451 nt in humans) that includes an RNA template and a unique telomerase reverse transcriptase (TERT), as well as other proteins important for function in vivo. The 5'half of vertebrate TR contains the RNA elements essential for catalysis and TERT binding, while the 3'half contains the elements essential for localization, processing, TR accumulation, and binding the H/ACA RNP proteins. The 5'half of vertebrate TR includes the core domain and the CR4/CR5 domain which, together with TERT, comprise the essential elements of telomerase for catalysis. To address the molecular basis of telomerase catalytic activity, in this grant application we are proposing structural and functional studies of the catalytic core of vertebrate telomerase RNA and its interaction with TERT. Studies will focus on both human telomerase as well as telomerase from medaka fish, a model organism which has the smallest known vertebrate TR. Our overall goals are to determine the global fold and structure of the TR core and CR4/CR5 domains that comprise the catalytic core of telomerase, and provide a view of how they interact with hTERT. We will primarily use NMR methods for structure determination and dynamics, complemented by small angle X-ray scattering (SAXS), electron microscopy (EM), X-ray crystallography, and SHAPE chemistry, to obtain a structural and dynamical view of how telomerase RNA contributes to catalysis and interacts with TERT. Based on the structural data, we will make nucleotide substitutions in the TR to test our hypotheses on the role of the structure in catalysis (nucleotide addition, telomere repeat addition, translocation, processivity, primer binding) and TERT binding.
Our specific aims are: (1) Determine the structure and investigate the role of dynamics in the function of the vertebrate TR core domain. The position of the template relative to the pseudoknot will be determined using paramagnetic relaxation enhancement, and dynamics studies will be studied using NMR relaxation measurements and RDCs;(2) Determine the structure and investigate the role of dynamics in the function of the CR4/CR5 domain;and (3) Determine the global fold and interactions between the core and CR4/CR5 domains and hTERT that form the catalytic core of vertebrate telomerase, using a combination of SAXS, EM, and NMR data. These experiments will reveal the structure of the catalytic core of telomerase and the conserved elements important for function. The results of these investigations will provide a molecular basis for understanding telomerase activity as well as how mutations linked to disease affect activity, and for designing drugs that target TR.

Public Health Relevance

Telomerase is a multimeric complex, composed of several proteins and a single RNA, that is essential for replication of the ends of 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 telomerase function, we propose to investigate the structure and dynamics of vertebrate telomerase RNA and its association with telomerase reverse transcriptase using biophysical approaches including NMR spectroscopy combined with small angle X-ray scattering, electron microscopy, and X-ray crystallography.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048123-20
Application #
8327121
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
1992-08-01
Project End
2015-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
20
Fiscal Year
2012
Total Cost
$376,950
Indirect Cost
$132,177
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
Miracco, Edward J; Jiang, Jiansen; Cash, Darian D et al. (2014) Progress in structural studies of telomerase. Curr Opin Struct Biol 24:115-24
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
Eichhorn, Catherine D; Kang, Mijeong; Feigon, Juli (2014) Structure and function of preQ1 riboswitches. Biochim Biophys Acta 1839:939-950
Kang, Mijeong; Eichhorn, Catherine D; Feigon, Juli (2014) Structural determinants for ligand capture by a class II preQ1 riboswitch. Proc Natl Acad Sci U S A 111:E663-71
Hartman, Elon; Wang, Zhonghua; Zhang, Qi et al. (2013) Intrinsic dynamics of an extended hydrophobic core in the S. cerevisiae RNase III dsRBD contributes to recognition of specific RNA binding sites. J Mol Biol 425:546-62
Cash, Darian D; Cohen-Zontag, Osnat; Kim, Nak-Kyoon et al. (2013) Pyrimidine motif triple helix in the Kluyveromyces lactis telomerase RNA pseudoknot is essential for function in vivo. Proc Natl Acad Sci U S A 110:10970-5
Singh, Mahavir; Choi, Charles P; Feigon, Juli (2013) xRRM: a new class of RRM found in the telomerase La family protein p65. RNA Biol 10:353-9
Hong, Kyungah; Upton, Heather; Miracco, Edward J et al. (2013) Tetrahymena telomerase holoenzyme assembly, activation, and inhibition by domains of the p50 central hub. Mol Cell Biol 33:3962-71
Jiang, Jiansen; Miracco, Edward J; Hong, Kyungah et al. (2013) The architecture of Tetrahymena telomerase holoenzyme. Nature 496:187-92
Hengesbach, Martin; Kim, Nak-Kyoon; Feigon, Juli et al. (2012) Single-molecule FRET reveals the folding dynamics of the human telomerase RNA pseudoknot domain. Angew Chem Int Ed Engl 51:5876-9

Showing the most recent 10 out of 25 publications