Project Summary Overview: The ribonucleoprotein enzyme telomerase maintains telomere length homeostasis – a shared hallmark between aging and carcinogenesis. Insufficient telomerase leads to telomere dysfunctions and replicative senescence in stem cells, precipitating short-telomere diseases. Conversely, telomerase is exploited by ~90% of cancers for growth immortality. Activating promoter mutations in the reverse transcriptase (TERT) gene ranks the most frequent non-coding driver mutations. We have made contributions to an understanding of how telomerase and telomere can be modulated. 1) we uncovered that TCAB1, a non-catalytic subunit of telomerase and a Cajal body (CB) trafficking protein, functions as an activity switch during telomerase catalysis. Using next-gen RNA structural profiling, we found that TCAB1-bound RNPs are endowed with an RNA conformation favorable for TERT-TR engagement, linking an RNA quality-control with RNP trafficking and catalysis; 2) we showed that the 5’ TR cap hypermethylation plays a negative role in telomerase RNA accumulation and activity. We can induce robust telomere elongation by genetic inactivation and chemical inhibition of the cap hypermethylase TGS1; 3) we also discovered a crosstalk between snRNA 5’ and 3’ PTM critical for global splicing fidelity and motor neuron viability in multiple organisms. 4) we uncovered a Toxic telomerase RNP that provokes acute and selective cancer genotoxicity at telomeres, unlike the conventional delayed killing by direct telomerase inhibition. Goals: We will test a model that RNP assembly and/or activity can be limited by phase-separated CB. We have found that dismantlement of the RNA and protein components of CB both led to GOF of telomerase. We are addressing: 1) Can CB contribute to the molecular determinant of telomere length set points? we are testing this with a novel optogenetic pipeline to manipulate phase separation of a subset of cellular CBs and monitor a single telomere elongation; 2) What is the RNA basis for telomerase phase separation at CB, at telomeres, or when mislocalized to nucleoli? We will improve the current icSHAPE-seq to determine RNA structure at subcellular locations; 3) We aim to elucidate the mechanism by which phase separation sequesters telomerase RNPs, focusing on the dynamic interplay between TCAB1 and Coilin; 4) we aim to understand the genotoxic mechanism underly the Toxic TERT. We also use this as a tractable system to identify suppressors as potential new factors in RNP assembly and targeting. Vision: we will engineer tools to study structure-function of RNP within phase-separated bodies. We will identify additional telomerase-like RNPs that are similarly governed by phase-separation, RNA QC, and RNA PTMs. Our long-term goal is to develop novel chemical matters that can boost telomerase to improve cell therapy, such as making exhaustion-resistant CAR-T; we aim to further develop our bifunctional telomerase-targeting molecule that induces r...