Telomerase RNP Prisonbreaks from Phase-Separated Nuclear Body

项目摘要 概述：核糖核蛋白酶端粒酶维持端粒长度的稳态-一个共享的 衰老和致癌之间的标志端粒酶不足导致端粒功能障碍， 干细胞的复制性衰老，引发短端粒疾病。相反地， 90%的癌症都是为了长生不老。逆转录酶（TERT）中的激活启动子突变 基因排列最频繁的非编码驱动突变。我们为理解 端粒酶和端粒是如何被调节的。1)我们发现TCAB 1，一种非催化亚基， 端粒酶和Cajal小体（CB）运输蛋白，在端粒酶催化过程中起活性开关的作用。 使用下一代RNA结构分析，我们发现TCAB 1结合的RNP被赋予了一种RNA结构， 有利于TERT-TR接合的构象，将RNA质量控制与RNP运输联系起来， 2）我们发现5'端TR帽的超甲基化在端粒酶RNA中起负作用， 积累和活动。我们可以通过基因失活和化学方法诱导端粒延长 抑制帽超甲基化酶TGS 1; 3）我们还发现snRNA 5'和3' PTM之间的串扰 对于多种生物体中的整体剪接保真度和运动神经元活力至关重要。4)我们发现了一种有毒的 端粒酶RNP在端粒引起急性和选择性癌症遗传毒性，不同于传统的 通过直接端粒酶抑制延迟杀伤。 目标：我们将测试RNP组装和/或活性可能受到相分离CB限制的模型。我们有 发现CB的RNA和蛋白组分的缺失都导致端粒酶的GOF。我们 解决：1）CB是否有助于端粒长度设定点的分子决定因素？我们正在测试 利用一种新颖的光遗传学管道来操纵细胞CB子集的相分离并监测单个 端粒延长; 2）端粒酶在CB，端粒或何时发生相分离的RNA基础是什么？ 错误定位到了核仁我们将改进目前的icSHAPE-seq，以确定亚细胞RNA结构， 位置; 3）我们的目的是阐明相分离隔离端粒酶RNP的机制， 重点研究TCAB 1和Coilin之间的动态相互作用; 4）我们旨在了解遗传毒性机制 是有毒的叔硫基转移酶的基础我们也将此作为一个易于处理的系统来识别抑制因子作为潜在的新因子 在RNP组装和定位方面。 愿景：我们将设计工具来研究RNP在相分离体内的结构-功能。我们将确定 类似地由相分离、RNA QC和RNA PTM控制的另外的端粒酶样RNP。我们 长期目标是开发新的化学物质，可以提高端粒酶，以改善细胞治疗，如 我们的目标是进一步开发我们的双功能端粒酶靶向分子， 诱导快速和选择性的癌症细胞毒性成为泛癌症药物。

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 rapid and selective cancer cytotoxicity into a pan-cancer drug.