The ribonucleoprotein complex, telomerase, functions to counteract the gradual telomere shortening that dividing cells experience when the conventional DNA replication machinery fails to fully replicate chromosome ends. Telomerase is inactive in somatic cells, but its activity may contribute to the continued proliferation of stem cells. Furthermore, telomerase is reactivated in over 85% of cancer cell lines and is essential for the perpetual replicative capacity of these cells. Hence, the study of the function and regulation of telomerase activity has significant implications for our understanding of mechanisms underlying aging and cancer. In the budding yeast Saccharomyces cerevisiae, telomerase contains a catalytic reverse transcriptase, Est2p, an RNA moiety that contains the template for nucleotide addition, TLC1, and two regulatory proteins, Est1p and Est3p. Est1p is a critical determinant of telomerase assembly and function: its cell cycle-regulated degradation precludes telomerase assembly in G1 phase of the yeast cell cycle;it likely interacts directly with both the TLC1 RNA and with Est2p;it stimulates the association of Est3p with the telomerase complex;and it recruits telomerase to telomeres through its interaction with the single-stranded end-binding protein, Cdc13p. Despite the clear importance of Est1p function, how these activities are coordinated with telomerase biogenesis both temporally and spatially is very poorly understood. Est1p over expression reveals that it can localize to the nucleus independent of its interactions with other telomerase components (Teixeira et al. 2002). Moreover, investigation of the amino acid sequence of Est1p indicates that the region between residues 107 and 123 constitutes a putative nuclear localization sequence (NLS). Mutations in this region were previously reported to cause telomere shortening and reduced association with TLC1 (Evans and Lundblad 2002), phenotypes that could be interpreted as defects in Est1p localization and association with telomerase. Indeed, preliminary experiments show that the telomere length defect can be suppressed by addition of the SV40 NLS to the mutant proteins. I propose that the sub cellular localization of Est1p as medicated through its NLS functions as an additional mechanism for the regulation of telomerase assembly and activity. Experiments proposed here aim to 1) determine whether Est1p contains an NLS that is important for telomerase function at the telomere and 2) use Est1p's NLS to investigate how its sub cellular localization modulates the sub cellular localization and assembly of the other components of telomerase. These proposed experiments detail the first investigations into telomerase biogenesis that monitor the localization of both protein and RNA components. Because Est1p is conserved in humans, these studies have implications for understanding the pathways through which telomerase assembly and activity are regulated and may lead to the discovery of mechanistic determinants of telomerase activity in human cells.
The goal of the proposed research is to identify mechanisms that regulate the biogenesis of yeast telomerase and that integrate telomerase assembly and activity within the cell cycle. Such interactions and trafficking are important for the activity of human telomerase. Thus, my research may have implications for understanding the mechanisms of telomerase activation in cancer.
|Hawkins, Charlene; Friedman, Katherine L (2014) Normal telomere length maintenance in Saccharomyces cerevisiae requires nuclear import of the ever shorter telomeres 1 (Est1) protein via the importin alpha pathway. Eukaryot Cell 13:1036-50|