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

哺乳动物端粒是连续的六聚体TTAGGG DNA重复序列， 线性染色体的末端。从结构上讲，端粒DNA在末端形成一个环状结构， 以保护线性染色体的末端免受降解和/或 非法重组六亚基蛋白复合物shelterin结合并保护 具有序列特异性的端粒DNA既能抑制DNA损伤反应， 调节T环的形成shelterin功能缺陷导致端粒不稳定， 导致过早衰老和癌症的发展。最近这 端粒末端的保护帽已经扩大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， 鱼类和哺乳动物。在人类细胞中，TERRA是从C链转录的， 亚端粒区和转录延伸到端粒重复序列中， 200 bp ~ 9 kb的转录本。TERRA位于细胞核内， 端粒DNA和端粒相关蛋白。这一长期的非- 从一个曾经被认为是转录沉默的区域表达的编码RNA表明， 关于端粒维持的机制以及这些机制的作用， 在癌症的发展中起作用。在以前的研究中，我们已经证明了 TERRA受细胞周期调控，这种调控驱动着一个分子开关， 在体外协调端粒复制和端粒末端保护。虽然这些研究 他们是第一批证明TERRA在端粒维护中的功能需求的人， TERRA如何在哺乳动物细胞中介导这些过程的确切功能尚不清楚。 这些研究受到限制，部分原因是无法操纵内源性TERRA 转录水平。为了克服这一挑战，我们设计了一个定期 间隔短回文重复序列（CRISPR）系统允许我们操纵基因 从内源TERRA启动子表达。因此，本提案的目标是 进一步确定TERRA在调节端粒稳定性中的功能， 了解TERRA功能缺陷如何导致癌症的发展。

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.