In this application, we propose to develop a novel technique, namely, RNA-guided RNA 2'-O-methylation, for regulating telomerase activity in vivo. We also propose to understand how 2'-O-methylation, as a naturally occurring process, is adopted by cells to trigger the cessation of growth under certain conditions. Given the close relationship between telomerase activity and aging, we believe that our proposed work (under three specific aims, see below), once accomplished, will significantly advance our understanding of telomerase regulation, aging, and human diseases.
Specific Aim 1 --Targeting of nucleotides in the conserved pseudoknot region of TLC1 Using box C/D RNA-guided 2'-O-methylation, we have artificially targeted TLC1 at six sites within the conserved pseudoknot region, and identified at least three sites where modification led to altered telomerase activity (two led to enhancement and one to reduction). Building on these results, we plan to expand our modification targets to cover each and every site in this important pseudoknot region. Any sites at which 2'-O- methylation affects telomerase activity will be identified and selected (individully or in combination) for further telomerase activity analyses, telomerase RNP assembly and recruitment assays. Our studies will allow us to understand the basis of effects, thus offering a variety of choices to influence telomerase activity and aging.
Specific Aim 2 --Global screening of important 2'-OH groups in TLC1 via unbiased 2'-O-methylation Given that TLC1 is a long RNA molecule and has at least several important domains, we believe that some nucleotides (and their 2'-OH groups) outside of the pseudoknot region are important for function as well. To obtain a complete spectrum of the effects of 2'-O-methylation, we propose an unbiased full screen throughout the TLC1 sequence. Specifically, we will use TLC1 cDNA to construct an artificial box C/D guide RNA library, directing 2'-O-methylation to each and every nucleotide of TLC1. A growth phenotype screen assay will be carried out to identify important sites where 2'-O-methylation negatively impacts telomerase activity.
Specific Aim 3 --Identification of enzyme responsible for induced TLC1 2'-O-methylation Our preliminary results also showed that upon entry into the stationary phase, TLC1 was 2'-O-methylated (~50%) at several sites, including a site where modification reduces telomerase activity. This remarkable result prompted us to explore further the mechanism of this inducible 2'-O-methylation. Specifically, we propose to identify the enzyme responsible for this induced modification. To achieve this goal, we plan to conduct a series of experiments, including screening using yeast ORF libraries (if the activity is orchestrated by protein only) or a yeast box C/D guide RNA library (if a box C/D RNP is involved). Identification of the enzyme responsible for induced 2'-O-methylation will eventually lead to the elucidation of the mechanism of this modification, and will thus significantly advance our understanding of the regulation of telomerase activity, aging and diseases.

Public Health Relevance

Relevance In this application, we propose to develop a novel approach, namely, RNA-guided RNA 2'-O-methylation, for regulating telomerase activity and aging, and to investigate naturally-occurring 2'-O-methylation that we have recently discovered in telomerase RNA. We believe that our proposed work will greatly enhance our understanding of how telomerase activity can be regulated in the cell, both artificially and naturally.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AG039559-01A1
Application #
8242209
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Guo, Max
Project Start
2012-08-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
1
Fiscal Year
2012
Total Cost
$193,125
Indirect Cost
$68,125
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
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Huang, Chao; Wu, Guowei; Yu, Yi-Tao (2016) Purification and Functional Reconstitution of Box H/ACA Ribonucleoprotein Particles. Methods Mol Biol 1421:97-109
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Karijolich, John; Yi, Chengqi; Yu, Yi-Tao (2015) Transcriptome-wide dynamics of RNA pseudouridylation. Nat Rev Mol Cell Biol 16:581-5
Adachi, Hironori; Yu, Yi-Tao (2014) Insight into the mechanisms and functions of spliceosomal snRNA pseudouridylation. World J Biol Chem 5:398-408
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Karijolich, John; Yu, Yi-Tao (2014) Therapeutic suppression of premature termination codons: mechanisms and clinical considerations (review). Int J Mol Med 34:355-62

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