Studying the role of TERRA in telomere protection and length regulation

项目摘要 端粒是一种特殊的DNA-蛋白质复合物，保护线性染色体的末端。哺乳动物 端粒是由高度保守的串联重复序列组成的，以（TTAGGG）n结尾， 3'单链（TTAGGG）n序列，与称为shelterin的端粒特异性蛋白质结合 复杂.在健康的体细胞中，端粒的逐渐丢失导致细胞衰老。85%的癌症 然而，端粒被端粒酶延长，而其余15%的癌症使用替代疗法。 端粒延长（ALT）。ALT途径导致含端粒重复序列的RNA过表达 （TERRA），一种从端粒转录的长的非编码RNA。TERRA能够进行反式退火， 侵入端粒双链体以形成R环（DNA-RNA杂合）结构。因此，积累 R环是ALT癌症的标志之一。此外，最近的一项研究表明，R环的形成是 Rad 51重组酶促进，但它被RNase H1拮抗。尽管越来越多的证据表明 由于TERRA在端粒结构和功能中的关键作用，我们对TERRA的分子理解是有限的。在这里， 我建议阐明TERRA募集和侵入端粒的分子机制， shelterin蛋白的背景和TERRA调节细胞端粒长度的程度。目标1将 探针TERRA诱导的R环形成和shelterin蛋白的作用。我的初步结果使用单一 分子（sm）FRET显示TERRA反式退火到端粒DNA上，出乎意料的是，产生的R环 表现出由TRF 2稳定的动态运动。我还采用了sm-共定位分析来测量 TERRA与端粒DNA反式退火的效率。我的化验结果显示 非TERRA序列，代表亚端粒DNA，以及G-四链体的存在 (G4)端粒突出端显著增强TERRA与端粒双链体的结合。目标2将阐明 RAD 51和shelterin蛋白对TERRA募集和侵袭的潜在机制。给你我最 使用sm-共定位分析的初步结果显示，RAD 51-TERRA复合物侵入端粒 比单独使用TERRA更有效。目标3将测量端粒长度作为TERRA水平的函数， ALT和端粒酶依赖性癌细胞。此外，本研究还将研究TRF 2、POT 1、Rad 51、 RNA酶H1在TERRA调节细胞端粒中的作用。这些目标的完成将提供一个分子 在端粒保护和长度调节中支持TERRA功能的机制， 治疗ALT阳性癌症，其中TERRA上调。

PROJECT SUMMARY Telomeres are specialized DNA-protein complex that protect the ends of linear chromosomes. Mammalian telomeres are composed of highly conserved tandem repeat sequences of duplexed (TTAGGG)n, ending with a 3’ single stranded (TTAGGG)n sequence that is bound to telomere specific proteins known as the shelterin complex. In healthy somatic cells, progressive loss of telomeres results in cellular senescence. In 85% of cancer cells, however telomeres are elongated by telomerase, while the remaining 15% of cancers use alternative lengthening of telomeres (ALT). ALT pathway leads to overexpression of Telomeric repeat containing RNA (TERRA), a long noncoding RNA that is transcribed from telomeres. TERRA is capable of trans annealing by invading the telomeric duplex to form an R-loop (DNA-RNA hybrid) structure. Consistently, the accumulation of R-loops is one of the hallmarks of ALT cancer. In addition, a recent study showed that R-loop formation is promoted by Rad51 recombinase, but it is antagonized by RNase H1. Despite the mounting evidence of TERRA’s critical role in telomere structure and function, our molecular understanding of TERRA is limited. Here, I propose to elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin proteins and the extent to which TERRA regulates telomere length in cells. Aim 1 will probe TERRA-induced R-loop formation and the role of shelterin proteins. My preliminary results using single molecule (sm) FRET show that TERRA trans anneals to telomeric DNA, and unexpectedly, the resulting R-loop exhibits dynamic movement that is stabilized by TRF2. I also employed an sm-colocalization assay to measure the efficiency for trans annealing of TERRA to telomeric DNA. My results from this assay show that the presence of non-TERRA sequence, which represents the subtelomeric DNA, as well as the presence of G-quadruplex (G4) telomeric overhang significantly enhances TERRA binding to telomeric duplex. Aim 2 will elucidate the mechanism underlying TERRA recruitment and invasion by Rad51 and shelterin proteins. I present my preliminary results using an sm-colocalization assay which show that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Aim 3 will measure telomere length as a function of TERRA levels in ALT- and telomerase dependent cancer cells. Additionally, this aim will study the role of TRF2, POT1, Rad51, RNaseH1 in TERRA regulation of telomeres in cells. Completion of these aims will provide a molecular mechanism underpinning TERRA function in telomere protection and length regulation and thereby help develop treatment for ALT positive cancers in which TERRA is upregulated.