The role of the TLC1 RNA flexible scaffold in yeast telomerase function
Lebo, Kevin J.
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In eukaryotic cells, the ends of the chromosomes are capped by structures known as telomeres, comprised of repeated DNA segments bound by proteins. Many organisms employ a ribonucleoprotein (RNP) complex, telomerase, to counteract the telomere DNA shortening that occurs during each cell cycle. Telomerase is minimally composed of a telomerase reverse transcriptase (TERT) and a telomerase RNA component, although other accessory protein subunits are required for telomere maintenance in vivo. In the budding yeast Saccharomyces cerevisiae, the telomerase RNA is the 1157-nt TLC1. TLC1 forms a Y-shaped overall secondary structure, with three long arms radiating out from a central catalytic core. Yeast TERT (Est2) localizes to the central core, while accessory proteins, including Est1, Ku, and Sm7, each bind to the tip of a different RNA arm. TLC1 organizes the telomerase RNP by serving as a flexible scaffold, in which accessory protein binding sites can be relocated on the RNA with retention of function. My research has focused on understanding how the TLC1 flexible scaffold regulates telomerase function. By designing a version of TLC1 with stiffened RNA arms, I have demonstrated that physical flexibility in the RNA is not required for telomerase function. This suggests that rather than allowing the proteins to “flex to position,” TLC1 organizes the RNP as “beads on a string,” without specific orientations required for accessory protein function. Furthermore, I identified a Second Essential Est1-arm Domain (SEED) in the Est1-binding arm of TLC1, which has an essential function independent of Est1 protein. This indicates that portions of the flexible scaffold RNA have roles beyond passive scaffolding. Next, while designing a version of TLC1 that would fold competently for function both in vivo and in vitro, I uncovered a previously unknown role for the Ku-binding arm RNA in RNA structure and abundance. Finally, I identified a region of the terminal arm of TLC1 that helps to regulate telomerase RNA levels in the cell. Overall, my research has advanced the flexible scaffold model for telomerase RNA, and suggests that the flexible scaffold RNP may best be viewed as a series of functional modules, which includes both proteins and RNA domains, all tethered together by intervening segments of RNA in non-specific positions within the RNP holoenzyme.