Functional characterization of the telomere repeat containing RNA, TERRA, in telomere maintenance

哺乳动物的端粒是序列六聚体TTAGGG DNA重复序列，它覆盖了 线形染色体的末端。在结构上，端粒DNA在末端形成套索或T环 以保护线形染色体的末端不受退化和/或 非法重组。6亚基蛋白质复合体保护素结合和保护 具有序列特异性的端粒DNA既能抑制DNA损伤反应 并调节T环的形成。保护素功能缺陷导致端粒不稳定和 会导致过早衰老和癌症的进展。最近这个 端粒末端的保护帽已经扩展到包括端粒重复序列-- 含有RNA的Terra。Terra在几种真核生物中表达，包括植物，酵母， 鱼和哺乳动物。在人类细胞中，Terra是从C-链转录而来的 亚端粒区和转录延伸延伸到端粒重复序列中，产生 转录本在200bp到9kb之间。Terra定位于核内，并与 端粒DNA和端粒相关蛋白。这一长期的非-发现 编码RNA，从一个曾经被认为转录沉默的区域表达，表明 关于端粒维持的机制和这些机制的作用，仍有许多需要了解 机制在癌症的发展中起着作用。在之前的研究中，我们已经证明 Terra是受细胞周期调节的，这种调节驱动着分子开关 在体外协调端粒复制和端粒末端保护。虽然这些研究是 在第一批证明Terra在端粒维持中的功能要求的人中， Terra究竟如何在哺乳动物细胞中调节这些过程仍不清楚。 这些研究在一定程度上受到了无法操纵内源Terra的限制 文字记录水平。为了克服这一挑战，我们定期设计了一个集群 允许我们操纵基因的间隔短回文重复(CRISPR)系统 来自内源Terra启动子的表达。因此，这项建议的目标是 进一步确定Terra在调节端粒稳定性中的功能，并最终， 了解Terra功能缺陷如何导致癌症的发生。

Mammalian telomeres are sequential hexameric TTAGGG DNA repeats that cap the ends of linear chromosomes. Structurally, the telomeric DNA forms a lariat, or T-loop, at the end of each chromosome to shield the ends of linear chromosomes from degradation and/or illegitimate recombination. The six-subunit protein complex shelterin binds and protects telomeric DNA with sequence specificity functioning to both inhibit the DNA damage response and regulate T-loop formation. Defects in shelterin function lead to telomere instability and contribute to both premature aging and the progression towards cancer. Recently this protective cap at telomere ends has expanded beyond shelterin to include the telomere repeat- containing RNA, TERRA. TERRA is expressed in several eukaryotes including plants, yeast, fish, and mammals. In human cells, TERRA is transcribed from the C-strand within the subtelomeric region and transcription elongation extends into the telomere repeats generating a transcript between 200bp and 9kb. TERRA is localized within the nucleus and associates with both the telomeric DNA and telomere associated proteins. The discovery of this long non- coding RNA, expressed from a region once considered transcriptionally silent, suggests that there is still much to learn about the mechanisms of telomere maintenance and the role these mechanisms play in the development of cancer. In previous studies, we have demonstrated that TERRA is cell cycle regulated, and that this regulation drives a molecular switch coordinating telomere replication with telomere end protection in vitro. While these studies were among the first to demonstrate a functional requirement for TERRA in telomere maintenance, exactly how TERRA functions to mediate these process in mammalian cells is still unclear. These studies have been limited, in part, by an inability to manipulate endogenous TERRA transcript levels. To overcome this challenge, we have designed a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system that allows us to manipulate gene expression from the endogenous TERRA promoter. Therefore, the goal of this proposal is to further define the function of TERRA in regulation of telomere stability and ultimately, understand how defects in TERRA function contribute to the development of cancer.