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

项目总结 癌细胞通过激活端粒维持机制确保复制性永生。 大约15%的癌症通过同源定向修复(Hdr)无限期延长其端粒。 驱动机制，称为交替延长的端粒(ALT)。通过ALT延长端粒 其机制依赖于两条非典型的HDR途径：(1)依赖RAD51-HOP2的同源 重组和(2)RAD52通过增殖细胞核抗原-Rfc1-Pold复制体介导端粒从头合成。我们的 实验室先前已经证明，RAD51AP1是一种辅助的HR蛋白，是ALT-HDR的重要介质 小路。RAD51AP1的缺失会导致ALT阳性(ALT+)癌细胞的端粒缩短。然而， RAD51AP1通过ALT-HDR介导的端粒延长的确切机制尚不清楚。 有趣的是，我们的初步数据显示，RAD51AP1的破坏导致端粒重复减少- 含有lincRNA(Terra)的。此外，我们的初步数据确定了一个ALT特异性RAD51AP1相互作用体 含有多种参与HDR偶联转录沉默的蛋白质。Terra的失调已经被 与端粒HDR不足有关。因此，了解Terra参与的过程和关键蛋白 ALT癌症内的调解将为ALT疗法的发展提供必要的信息。我 假设RAD51AP1在ALT端粒稳定Terra RNA-DNA杂交体，并作为 ALT-HDR的关键介体的集合。 我将通过两个目的来研究RAD51AP1在调节Terra在ALT中的作用。目标1将 描述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，验证了RNA-DNA杂交体的稳定性。 这一目标将建立RAD51AP1与Terra相互作用的新特征。目标2将定义 RAD51AP1和Terra功能相互作用在ALT-HDR中的作用拟议中的实验 将通过可视化RAD51AP1对Terra的上位效应来确定RAD51AP1调节Terra的顺序机制 RAD51AP1相互作用组中Terra、RAD51AP1和RNA调节蛋白的缺失。 此外，RAD51AP1调节Terra的作用机制将通过 RAD51AP1及调节RAD51AP1相互作用的RNA缺失对转录影响的观察 ALT细胞中偶联的HDR机制。这一目的将阐明RAD51AP1在交叉口的功能 DNA-RNA介导ALT的HDR。 完成这些目标将提供对ALT内部基本监管机制的洞察 将推动ALT疗法发展的途径和潜在的蛋白质靶点。

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.