Defining the role of TCAB1 and its phase separation in telomerase assembly.

项目摘要/摘要 端粒是人类染色体的末端，细胞每次分裂时端粒都会变短。当端粒变得太 简而言之，人类细胞停止分裂或经历程序性细胞死亡。端粒酶将端粒DNA添加到 染色体末端的作用是抵消端粒缩短。在人体内，端粒酶在干细胞中表达， 让它们在我们的生活中不断繁殖。端粒酶功能缺陷导致早产 老年性疾病，如先天性角化不良，由干细胞种群枯竭引起。在……里面 此外，端粒酶在约90%的肿瘤中表达，并允许癌细胞无限增殖。它是 因此至关重要的是开发有针对性的方法来促进或干扰端粒酶活性来治疗 分别是早衰病和癌症。端粒酶是一种复杂的核糖核蛋白，含有 端粒酶逆转录酶蛋白(TERT)、端粒酶RNA(TR)和各种辅助因子，包括 端粒酶Cajal体蛋白1(TCAB1)。TCAB1是人类细胞端粒酶功能所必需的，但 其在端粒维持中的作用机制存在争议。最初有人建议说 在没有TCAB1的情况下，端粒酶被正确组装并完全活跃，导致了TCAB1是 端粒酶运输到端粒所必需的。最近的观察表明，TCAB1是 在不影响整个RNP组装的情况下，通过促进TRR折叠来催化端粒酶活性。重要的是，所有 以前的研究清楚地表明，在缺乏TCAB1的细胞中，TR在核仁中丰富。我们之前的工作是 表明TERT被特异性地排除在核仁之外。因此，我们认为，在没有TCAB1的情况下， 端粒酶不能组装是因为TR被困在核仁中，而TERT被排除在核仁之外。两个核仁 Cajal小体为相分离的核细胞器。相分离是一种新兴的分子 蛋白质和核酸从液体中滴出的现象。在初步实验中，我们有 结果表明，TCAB1含有可发生液-液相分离的无序区。我们的中央 假设TCAB1的特定相分离特性通过驱动促进端粒酶的组装 从核仁中排除端粒酶RNA并促进其与卡哈尔小体的相互作用。为了测试这一点 假设，我们将解决以下具体目标，(1)确定TCAB1在端粒酶组装中的作用， (2)定义了TCAB1的相分离性质，分析了它们对端粒酶组装的贡献。

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.