Structure-Function Relationship Study of Telomerase Reverse Transcriptase's Non-Canonical Activities

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Literature Telomerase reverse transcriptase (TERT) is a ribonucleoprotein reverse transcriptase responsible for the de novo addition of telomeric DNA repeats to human chromosomes. In some specialized cells that require extended proliferation lifespans, such as stem cells and germ-line cells, telomerase activity is necessary for telomere repair to extend the proliferative capacity of these cell types. New evidence suggests that constitutive TERT expression in immortal human cells also promotes cell survival under stressful growth conditions through multiple mechanisms. These survival advantages provided by TERT are mediated through cellular functions not related to telomere repair, but the precise mechanism and TERT's structural form responsible for these activities remained largely unknown. Recent Progress My laboratory reported that recombinant expression of telomerase confers cellular growth advantages and promotes anchorage-independent growth, in a manner that is unrelated to telomere-length maintenance. TERT expression also protects against genotoxicity caused by DNA-damaging agents. We showed that TERT variants and alternatively spliced (truncated protein) forms, devoid of telomere-synthesis activity, also exhibited the same protection against multiple genotoxic stimuli, suggesting that telomerase engaged in structurally, as well as functionally distinct complexes for its canonical (telomere synthesis-related) and non-canonical (telomere-synthesis-independent) activities. Hypothesis Telomerase promotes cell survival following cytotoxic and metabolic stress, and abets growth and proliferation through participation in multiple structural complexes. Objectives and Methods Aim 1: Biochemical characterization of a minimal TERT domain in its DNA damage repair activities We will map the minimal TERT domain responsible for its DNA damage repair/protection activity, and generate short-peptide spanning the identified domain sequence, and test these peptides as a strategy to disrupt this TERT activity. Aim 2: Affinity purification and mass-spectrometry identification of distinct TERT complexes. We will perform tandem-affinity-tag (TAP) purification and LC-MS-MS analysis of purified TERT-complexes, from ALT cells expressing FL-TERT or truncated TERT variants. The biological functions of selected non-canonical TERT protein partners will be evaluated with genetic and/or chemical means. Impact Our functional and biochemical characterization of the different TERT structural forms and their membership in unique biological complexes will further the understanding on the plethora of telomerase activities in human biology. This work will also support 7 HQP, including 2 senior graduate students and 5 UGs. Our proposal is in agreement with NSERC's 2020 Research Strategic Plan to build a diversify and competitive research base by providing cutting-edge biochemical, protein science and molecular genetics training for the next generation of researchers.