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

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）无限期延长其端粒 端粒替代延长（ALT）。通过ALT延长端粒 该机制依赖于两个非经典HDR途径：（1）RAD 51-HOP 2依赖性同源 （2）RAD 52介导的端粒通过PCNA-RFC 1-Pod复制体从头合成。我们 实验室先前已经表明，辅助HR蛋白RAD 51 AP 1是ALT-HDR 途径。RAD 51 AP 1的耗尽导致ALT阳性（ALT+）癌细胞的端粒缩短。然而，在这方面， RAD 51 AP 1介导的端粒通过ALT-HDR延长的确切机制仍然未知。 有趣的是，我们的初步数据显示，破坏RAD 51 AP 1导致端粒重复序列减少， 含有lincRNA（TERRA）。此外，我们的初步数据确定了ALT特异性RAD 51 AP 1相互作用组， 含有参与HDR偶联转录沉默的多种蛋白质。TERRA的失调 与端粒HDR不足有关因此，了解TERRA的过程和关键蛋白质 ALT癌症中的介导将为ALT治疗剂的开发提供重要信息。我 假设RAD 51 AP 1在ALT端粒处稳定TERRA RNA-DNA杂合体，并作为 ALT-HDR关键介质的组装。 本研究将从两个方面探讨RAD 51 AP 1在ALT中对TERRA的调节作用。目标1将 RAD 51 AP 1与TERRA之间的相互作用。拟议的实验将确定 RAD 51 AP 1的RNA相互作用区及其对端粒TERRA的调控作用 通过一系列免疫共沉淀和免疫荧光实验研究RNA-DNA杂交体的稳定性。 这一目标将建立一个新的特点，RAD 51 AP 1与TERRA的相互作用。目标2将定义 RAD 51 AP 1和TERRA在ALT-HDR期间的功能相互作用。拟议的实验 将通过观察RAD 51 AP 1对TERRA的上位效应来确定RAD 51 AP 1调控的顺序机制。 TERRA、RAD 51 AP 1和来自RAD 51 AP 1相互作用组的RNA调节蛋白的耗竭。 此外，RAD 51 AP 1调节TERRA的作用机制将通过以下方式来解释： 观察RAD 51 AP 1的缺失和调节RAD 51 AP 1相互作用物的RNA如何影响转录 ALT细胞中的HDR机制。这一目标将阐明RAD 51 AP 1在以下交叉点的功能： DNA-RNA介导的ALT HDR。 这些目标的完成将使我们深入了解ALT中的基本监管机制 这将促进ALT疗法的发展。

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.