Structure and function of Tetrahymena thermophila telomerase RNA Public Deposited

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  • March 21, 2019
  • Legassie, Jason Donald
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
  • Telomerase is a specialized reverse transcriptase, which synthesizes telomeric repeats to the 3' ends of linear chromosomes. By helping to maintain adequate telomere length, telomerase ensures chromosomal and genetic stability. Telomerase represents a possible universal cancer target as its expression and activity are upregulated in >85% of all cancers. This thesis describes two major areas of investigation of important structural aspects of the RNA subunit of a model telomerase enzyme from the unicellular ciliate, Tetrahymena thermophila. In the first major area of research, we engineered two mutant forms of the Tetrahymena telomerase RNA, tTER, that contain DNA only in the templating region. These chimeric template telomerase mutants were able to extend telomeric DNA primers, though with reduced efficiency compared to wild type. Additionally, the DNA dependent telomerases were RNase sensitive confirming that non-template portions of tTER are critical for maintaining activity of the assembled telomerase complex. The second major study utilized a novel chemical footprinting approach termed SHAPE for Selective 2'-Hydroxyl Acylation analyzed by Primer Extension. SHAPE employs the small molecule N-methylisatoic anhydride, which selectively acylates the 2'- hydroxyl of unconstrained or non-base paired ribonucleotides. This approach was employed to analyze the solution structure of the essential stem IV of tTER. The SHAPE chemistry of stem IV exhibited excellent correlation with high resolution NMR structures. Stem IV mutants were also SHAPE analyzed and their proposed structures were confirmed. SHAPE chemistry was further employed to analyze the structure of tTER in solution and in the telomerase complex. SHAPE was able to recapitulate most of the phylogenetically predicted secondary structure of tTER. However, SHAPE chemistry suggests that the solution structure of tTER deviates substantially within the pseudoknot region, which instead forms a 12-base pair stem with a 4-base bulge and 6-member apical loop. SHAPE analysis of tTER in the telomerase complex suggests the formation of a pseudoknot that is similar to the phylogenetically predicted structure. This study allows us to propose a model for telomerase assembly where the RNA undergoes one or more structural reorganizations upon holoenzyme maturation. These studies illustrate the complexity of structure and function of tTER. The novel investigative approaches and intimate knowledge generated of the Tetrahymena telomerase RNA should guide future research of more clinically relevant telomerase enzymes.
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  • Jarstfer, Michael
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  • University of North Carolina at Chapel Hill
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