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

项目总结/摘要 端粒，人类染色体的末端，在细胞每次分裂时都会缩短。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 染色体末端以抵消端粒缩短。在人体中，端粒酶在干细胞中表达， 让它们在我们的生活中不断增殖。端粒酶功能缺陷导致过早的 衰老性疾病，如先天性角化不良，这是由干细胞群的耗竭引起的。在 此外，端粒酶在约90%的肿瘤中表达，并允许癌细胞无限增殖。是 因此，开发靶向方法来促进或干扰端粒酶活性以治疗 早老疾病和癌症。端粒酶是一种复杂的核糖核蛋白， 端粒酶逆转录酶蛋白（TERT）、端粒酶RNA（TR）和各种辅因子，包括 端粒酶卡哈尔体蛋白1（TCAB 1）。TCAB 1是人类细胞中端粒酶功能所必需的， 其对端粒维持的作用机制仍有争议。最初有人提出， 端粒酶在缺乏TCAB 1的情况下被正确组装并完全活化，导致TCAB 1被激活的模型。 端粒酶运输到端粒所必需的。最近的观察表明，TCAB 1是必需的， 端粒酶催化活性，促进TR折叠，而不影响整体RNP组装。重要的是所有 以前的研究清楚地表明TR在缺乏TCAB 1的细胞中富集在核仁中。我们之前的工作 表明TERT被特异性地排除在核仁之外。因此，我们认为，在没有TCAB 1的情况下， 端粒酶不能组装，因为TR被捕获在核仁中，而TERT被排除在核仁之外。两个核仁 和Cajal小体是相分离的核细胞器。相分离是一种新兴的分子 蛋白质和核酸从液滴中分离出来的现象。在初步实验中，我们 表明TCAB 1含有可以进行液-液相分离的无序区域。我们的中央 假设TCAB 1的特异性相分离特性通过驱动端粒酶组装来促进端粒酶组装 将端粒酶RNA从核仁中排除，促进其与Cajal小体的相互作用。为了验证这一 假设，我们将致力于以下具体目标，（1）确定TCAB 1在端粒酶组装中的作用， （2）确定TCAB 1的相分离特性并分析其在端粒酶组装中的作用。

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.