Investigating Telomerase Mechanism by Exploring the RNA

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After studying transcriptional silencing and DMA replication during my PhD thesis research with Rolf Sternglanz at Stony Brook University, I moved to Tom Cech's lab at the University of Colorado for postdoctoral work. My goal has been to deepen my knowledge of biochemistry and specifically to learn more about RNA, drawing on the expertise of the Cech lab and the interactive RNA community at CU-Boulder. My research concerns telomerase, which replicates the ends of eukaryotic chromosomes and is composed of an RNA as well as several protein subunits. Telomerase plays a prominent role in human diseases such as cancer. Already in my postdoctoral work I have developed a secondary structure model for the 1157-nt yeast telomerase RNA, TLC1, and the first in vitro reconstituted yeast telomerase activity assay. I now propose to use these tools to examine the flexibility of RNA scaffolding in the yeast telomerase complex and to begin to define the roles of important accessory proteins in the enzyme mechanism. In specific aim #1, I plan to investigate structural requirements for yeast telomerase RNA function, in part by determining if Ku is flexibly scaffolded by the RNA by moving its binding site and then testing functionality. I will also test the absolute limitations of TLC1 RNA size and the proposed consensus for telomerase RNA secondary structure. My career development plan involves learning how RNA structure is predicted by computational means, which will facilitate this research. Specific aim #2 describes, in part, testing the hypothesis that yeast telomerase core enzyme is nonprocessive in vitro because factors that iteratively recruit and remove the enzyme from its substrate in vivo are absent. Collaboration with other labs has been undertaken to acquire the necessary purified proteins to test in the assay. Lastly, in specific aim #3, I plan to examine and exploit phenotypes of cells expressing the miniaturized telomerase RNA, Mini-T, and propose to characterize a related suppressor that I have already identified. By testing these hypotheses and learning new research skills through coursework and a mini-sabbatical, as well as gaining a degree of teaching and other relevant training, I expect to attain my goal of becoming prepared to subsequently assume a faculty position. Relevance: Telomerase is an enzyme that must be carefully regulated to perform appropriately, or else it can play a pivotal role in causing diseases such as cancer. Because abnormal overproduction of telomerase is thought to be critical for unlimited proliferation of ~90% of human cancer cells, telomerase is an attractive drug target, although it has been very difficult to find an appropriate inhibitor and therefore more information about telomerase function is needed. Here I describe experiments to deduce the minimal requirements of yeast telomerase function, a genetically and biochemically advantageous system that is expected to continue to illuminate features of the human enzyme.