A novel role for RAD51AP1 in regulation of TERRA lincRNA during ALT-mediated telomere extension

RAD51AP1 在 ALT 介导的端粒延伸过程中调节 TERRA lincRNA 的新作用

• 批准号: 10465037
• 负责人: Nicole Kaminski
• 金额: $ 2.04万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10465037
• 关键词: Advanced Development; Adverse effects; Affinity; Binding; Cell Cycle; Cell Death; Cell Line; Cells; Characteristics; Co-Immunoprecipitations; Coupled; DNA; DNA Binding; DNA metabolism; DNA replication fork; Data; Development; Disease; Double Strand Break Repair; Ensure; Exhibits; Gene Silencing; Genetic Transcription; Goals; Hybrids; Immunofluorescence Immunologic; Immunoprecipitation; Impairment; In Vitro; Knowledge; Lead; Link; Malignant Neoplasms; Measurement; Mediating; Mediation; Mediator of activation protein; Metabolism; Pathway interactions; Play; Process; Property; Proteins; Proteomics; RAD52 gene; RNA; RNA metabolism; Regulation; Role; SLC19A1 gene; Series; Single-Stranded DNA; Structure; Telomerase; Telomere Maintenance; Telomere Pathway; Telomere Shortening; Therapeutic; Transcription-Coupled Repair; Untranslated RNA; Work; cancer cell; ds-DNA; experimental study; homologous recombination; improved; insight; link protein; mutant; novel; optical imaging; repaired; telomere; therapeutic development; therapeutic target

PROJECT SUMARY Cancer cells ensure replicative immortality through activation of telomere maintenance mechanisms. Approximately 15% of cancers indefinitely lengthen their telomeres through a homology directed repair (HDR) driven mechanism, termed Alternative Lengthening of Telomeres (ALT). Telomere lengthening via the ALT mechanism is reliant upon two non-canonical HDR pathways: (1) RAD51-HOP2 dependent homologous recombination and (2) RAD52 mediated de novo telomere synthesis via the PCNA-RFC1-Pold replisome. Our lab has previously shown that RAD51AP1, an accessory HR protein, is an essential mediator of both ALT-HDR pathways. Depletion of RAD51AP1 results in telomere shortening of ALT positive (ALT+) cancer cells. However, the exact mechanism behind RAD51AP1 mediated telomere lengthening via ALT-HDR remains unknown. Interestingly, our preliminary data show that disruption of RAD51AP1 results in a decrease of telomere repeat- containing lincRNA (TERRA). Additionally, our preliminary data identify an ALT specific RAD51AP1 interactome containing multiple proteins involved in HDR-coupled transcriptional silencing. Dysregulation of TERRA has been linked to insufficient telomere HDR. Therefore, understanding the processes and key proteins involved in TERRA mediation within ALT cancers will provide essential information toward the development of ALT therapeutics. I hypothesize that RAD51AP1 stabilizes TERRA RNA-DNA hybrids at ALT telomeres and serves as a hub for the assembly of the critical mediators of ALT-HDR. I will investigate the role of RAD51AP1 in the regulation of TERRA in ALT through two aims. Aim 1 will characterize the interaction between RAD51AP1 and TERRA. The proposed experiments will identify the RNA interacting region of RAD51AP1 and determine the regulatory effect of RAD51AP1 on telomeric TERRA RNA-DNA hybrid stability through a series of co-immunoprecipitation and immunofluorescence experiments. This aim will establish a novel characteristic of RAD51AP1 in its interaction with TERRA. Aim 2 will define the role of functional interplay between RAD51AP1 and TERRA during ALT-HDR. The proposed experiments will identify the sequential mechanism of RAD51AP1 regulation of TERRA by visualizing the epistatic effects of depletion of TERRA, RAD51AP1, and the RNA regulating proteins from the RAD51AP1 interactome. Additionally, the mechanism of action in which RAD51AP1 regulates TERRA will be deciphered through observation of how the depletion of RAD51AP1 and RNA regulating RAD51AP1-interactors effect transcription coupled HDR mechanisms in ALT cells. This aim will elucidate the function of RAD51AP1 at the intersection of DNA-RNA mediated HDR in ALT. Completion of these aims will provide insight into essential regulatory mechanisms within the ALT pathway and potential protein targets that will advance the development of ALT therapies.

项目概要 癌细胞通过激活端粒维持机制来确保复制永生。 大约 15% 的癌症通过同源定向修复 (HDR) 无限期地延长其端粒 驱动机制，称为端粒替代延长（ALT）。通过 ALT 延长端粒 该机制依赖于两种非规范 HDR 途径：(1) RAD51-HOP2 依赖性同源 (2) RAD52 通过 PCNA-RFC1-Pold 复制体介导端粒从头合成。我们的 实验室此前已表明，RAD51AP1（一种辅助 HR 蛋白）是 ALT-HDR 的重要调节因子 途径。 RAD51AP1 的耗竭会导致 ALT 阳性 (ALT+) 癌细胞的端粒缩短。然而， RAD51AP1 通过 ALT-HDR 介导端粒延长的确切机制仍不清楚。 有趣的是，我们的初步数据表明 RAD51AP1 的破坏会导致端粒重复的减少 含有 lincRNA (TERRA)。此外，我们的初步数据确定了 ALT 特异性 RAD51AP1 相互作用组 含有多种参与 HDR 偶联转录沉默的蛋白质。 TERRA 的失调已经 与端粒 HDR 不足有关。因此，了解 TERRA 涉及的过程和关键蛋白质 ALT 癌症中的调节将为 ALT 疗法的开发提供重要信息。我 假设 RAD51AP1 稳定 ALT 端粒上的 TERRA RNA-DNA 杂交体，并充当 ALT-HDR 关键介质的组装。 我将通过两个目标来研究 RAD51AP1 在 ALT 中 TERRA 的调节中的作用。目标1将 表征 RAD51AP1 和 TERRA 之间的相互作用。拟议的实验将确定 RAD51AP1的RNA相互作用区并确定RAD51AP1对端粒TERRA的调节作用 通过一系列免疫共沉淀和免疫荧光实验确定 RNA-DNA 杂合体的稳定性。 这一目标将建立 RAD51AP1 在与 TERRA 相互作用中的新特征。目标 2 将定义 RAD51AP1 和 TERRA 在 ALT-HDR 过程中功能相互作用的作用。拟议的实验 将通过可视化上位效应来确定 RAD51AP1 对 TERRA 调节的顺序机制 去除 TERRA、RAD51AP1 和 RAD51AP1 相互作用组中的 RNA 调节蛋白。 此外，RAD51AP1 调节 TERRA 的作用机制将通过 观察 RAD51AP1 和 RNA 调节 RAD51AP1 相互作用子的耗竭如何影响转录 ALT 细胞中的耦合 HDR 机制。该目标将阐明 RAD51AP1 在交叉点的功能 DNA-RNA 介导的 ALT 中的 HDR。 完成这些目标将有助于深入了解 ALT 内的基本监管机制 途径和潜在的蛋白质靶点将推动 ALT 疗法的发展。