PROJECT SUMMARY/ABSTRACT Telomeres, the ends of human chromosomes, shorten every time a cell divides. When telomeres become too short, human cells stop dividing or undergo programmed cell death. Telomerase adds telomeric DNA onto chromosome ends to counteract telomere shortening. In the human body telomerase is expressed in stem cells, allowing them to continuously proliferate throughout our lives. Defects in telomerase function lead to premature aging diseases such as Dyskeratosis Congenita, which are caused by the depletion of stem cell populations. In addition, telomerase is expressed in ~90% of tumors and allows cancer cells to proliferate indefinitely. It is therefore critical to develop targeted approaches to promote or interfere with telomerase activity to treat premature aging diseases and cancer, respectively. Telomerase is a complex ribonucleoprotein that contains the telomerase reverse transcriptase protein (TERT), the telomerase RNA (TR), and various co-factors including the telomerase cajal body protein 1 (TCAB1). TCAB1 is required for telomerase function in human cells but the mechanism underlying its contribution to telomere maintenance is controversial. Initially it was suggested that telomerase is properly assembled and fully active in the absence of TCAB1, leading to the model that TCAB1 is required for telomerase trafficking to telomeres. More recent observations suggest that TCAB1 is required for telomerase catalytic activity by promoting TR folding without affecting overall RNP assembly. Importantly, all previous studies clearly demonstrate that TR is enriched in nucleoli in cells lacking TCAB1. Our prior work has shown that TERT is specifically excluded from nucleoli. We therefore believe that in the absence of TCAB1, telomerase fails to assemble because TR is trapped in nucleoli and TERT is excluded from nucleoli. Both nucleoli and Cajal bodies are phase separated nuclear organelles. Phase separation is an emerging molecular phenomenon by which proteins and nucleic acids from liquid like droplets. In preliminary experiments we have shown that TCAB1 contains disordered regions that can undergo liquid-liquid phase separation. Our central hypothesis is that the specific phase separation properties of TCAB1 facilitate telomerase assembly by driving the exclusion of the telomerase RNA from nucleoli and promoting its interaction with Cajal bodies. To test this hypothesis, we will address the following specific aims, (1) Determine the role of TCAB1 in telomerase assembly, and (2) Define the phase separation properties of TCAB1 and dissect their contribution to telomerase assembly.