Studying the role of TERRA in telomere protection and length regulation

研究TERRA在端粒保护和长度调节中的作用

• 批准号: 10605982
• 负责人: Bersabel Wondimagegnhu
• 金额: $ 4.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605982
• 关键词: Apoptosis; Binding; Biochemical; Biological; Biological Assay; Cell Aging; Cell physiology; Cells; Cellular biology; Chromosomes; Complex; DNA; DNA-Directed RNA Polymerase; Detection; Elongation by Telomerase; Exhibits; G-Quartets; Genetic Transcription; Heterogeneity; Hybrids; Individual; Invaded; Kinetics; Length; Malignant Neoplasms; Measurement; Measures; Mediating; Methods; Microscope; Modeling; Molecular; Molecular Conformation; Movement; Nucleoproteins; Pancreatic ribonuclease; Pathway interactions; Proteins; Protocols documentation; RNA; RNA Binding; RNA annealing; Rad51 recombinase; Regulation; Ribonucleases; Role; Sister Chromatid; Somatic Cell; Structure; System; Tandem Repeat Sequences; Telomerase; Telomere Maintenance; Telomere Pathway; Transcript; Untranslated RNA; Up-Regulation; Visualization; alternative treatment; cancer cell; experience; helicase; homologous recombination; insight; overexpression; oxidative damage; prediction algorithm; protein complex; recruit; ribonuclease H1; single molecule; single-molecule FRET; telomere; telomere loss; ultra high resolution

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.

项目总结 端粒是一种特殊的DNA-蛋白质复合体，它保护线性染色体的末端。哺乳动物 端粒由高度保守的双链(TTAGGG)n串联重复序列组成，以a结尾 与端粒特异蛋白质结合的3‘单链(TTAGGG)n序列，称为保护素 很复杂。在健康的体细胞中，端粒的进行性丢失会导致细胞衰老。85%的癌症患者 然而，端粒是由端粒酶拉长的，而剩下的15%的癌症患者使用的是 端粒延长(ALT)。ALT途径导致含有端粒重复序列的RNA过表达 (Terra)，一种从端粒转录而来的长的非编码RNA。Terra能够通过以下方式进行反式退火 侵入端粒双链形成R-环(DNA-RNA杂交)结构。始终如一地，积累 R环是ALT癌症的特征之一。此外，最近的一项研究表明，R环的形成是 被RAD51重组酶促进，但被RNaseH1拮抗。尽管有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。这里, 我建议阐明Terra重新招募和侵入端粒的分子机制 保护素蛋白的背景和Terra调节细胞端粒长度的程度。目标1将 探查Terra诱导的R-环的形成和Shelterin蛋白的作用。我使用Single的初步结果 分子(Sm)FRET表明Terra反式作用于端粒DNA，并出人意料地产生R-环 表现出由TRF2稳定的动态运动。我还使用了sm-co-ococation分析来测量 Terra对端粒DNA的反式退火效率。我的化验结果显示， 表示亚端粒DNA的非Terra序列，以及G-四链的存在 (G4)端粒悬垂显著增强了Terra与端粒双链的结合。目标2将阐明 RAD51和Shelterin蛋白对Terra募集和入侵的机制。我向大家展示我的 利用sm-共定位实验的初步结果表明RAD51-Terra复合体入侵端粒 双工比单独使用Terra更高效。目标3将测量端粒长度作为Terra水平的函数 依赖ALT和端粒酶的癌细胞。此外，该目标还将研究TRF2、POT1、Rad51、 RNaseH1在Terra中对细胞端粒的调节。完成这些目标将提供一种分子 支持Terra在端粒保护和长度调节中作用的机制，从而有助于发展 TERRA表达上调的ALT阳性癌症的治疗。