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--一个共同的 衰老和致癌之间的标志。端粒酶不足导致端粒功能障碍和 干细胞中的复制性衰老，导致短端粒疾病。相反，端粒酶被利用。 由~90%的癌症长生不老。激活逆转录酶(TERT)启动子突变 基因是最常见的非编码驱动突变。我们为理解 端粒酶和端粒是如何调节的。1)我们发现了TCAB1，一个非催化亚基 端粒酶和Cajal小体(CB)运输蛋白在端粒酶催化过程中起着活性开关的作用。 利用下一代RNA结构图谱，我们发现TCAB1结合的RNPs具有RNA 有利于TERT-TR参与的构象，将RNA质量控制与RNP贩运和 2)我们发现5‘TRCAP甲基化在端粒酶RNA中起着负性作用 积累和活动。我们可以通过基因失活和化学物质诱导强大的端粒延长 抑制帽子超甲基化酶TGS1；3)我们还发现了SnRNA5‘和3’PTM之间的串扰 对多种生物体的全球剪接保真度和运动神经元活性至关重要。我们发现了一种有毒物质 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 通过直接抑制端粒酶而延迟杀伤。 目标：我们将测试一个模型，即RNP的组装和/或活动可以受到相分离CB的限制。我们有 发现CB的RNA和蛋白质组分的拆除都会导致端粒酶的GOF。我们是 解决：1)Cb是否对端粒长度设定点的分子决定因素起作用？我们正在测试这一点 利用一种新型的光遗传管道来操纵细胞CB子集的相分离并监测单个 端粒延长；2)端粒酶在CB、端粒或端粒时相分离的RNA基础是什么？ 错误定位到核仁？我们将对现有的icshape-seq进行改进，以确定亚细胞的rna结构。 我们的目标是阐明相分离隔离端粒酶RNPs的机制， 重点研究了TCAB1和Colin之间的动态相互作用；4)我们旨在了解其遗传毒性机制 在有毒的TERT之下。我们还将其作为一种易于处理的系统来识别潜在的新因素 在RNP组装和靶向方面。 愿景：我们将设计工具来研究RNP在相分离体内的结构和功能。我们将确定 附加的端粒酶样RNPs，类似于由相分离、RNA QC和RNA PTM控制的RNPs。我们的 长期目标是开发能够促进端粒酶的新型化学物质，以改善细胞治疗，例如 研制抗疲劳CAR-T；我们的目标是进一步开发我们的双功能端粒酶靶向分子 将快速和选择性的癌症细胞毒性作用转化为泛癌药物。

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.