SRA IncRNA Recognition by the SHARP Epigenetic Regulatory Protein Studied by NMR

通过 NMR 研究 SHARP 表观遗传调节蛋白对 SRA IncRNA 的识别

• 批准号: 9558660
• 负责人: THOMAS C LEEPER
• 金额: $ 12.84万
• 依托单位: KENNESAW STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9558660
• 关键词: SRA; IncRNA; Recognition; SHARP; Epigenetic

Project Summary - SRA lncRNA recognition by the SHARP epigenetic regulatory protein studied by NMR Thomas Leeper, Kennesaw State University. The aim of the proposed research is to use NMR to determine the structures of ribonucleoproteins (RNPs) derived from SHARP, an epigenetic regulatory protein, and the steroid receptor activator epigenetic regulator RNA (SRA1). This RNA is part of a co-activator network for androgen receptor (AR) that modulates chromatin and enhances AR mediated oncogenic expression. AR response stimulates prostate cancer cell hyper- proliferation. Thus understanding the structures and biochemical details of AR regulator factors is important to understand this type of cancer. The SRA1 RNA molecule is a member of the emerging class of long-non- coding RNAs (lncRNA) that is a well established component of chromatin remodeling complexes. SHARP is also associated with the cancer related Wnt and Notch signaling pathways. These structures will give us some of the first glimpses of lncRNA ribonucleoprotein particle structures and suggest regions where structure-based drug discovery efforts should focus their attention if therapies are to be developed targeting lncRNAs. This project spans several disciplines but is most appropriately described as structural biology with the explicit goal of providing structures from this RNP scaffolding complex. The specific aims are: Aim 1: Determine the NMR structures of the RRM domains of human SHARP. a. Express, purify, and NMR screen RRM domains derived from a fragment of SHARP. b. Determine the NMR structures of the well-folded SHARP RRMs. Aim 2: Delineation of the human SRA lncRNA/SHARP interaction. a. Assay RNP formation by immobilized protein RNA pull-down assays to find RNA-domain boundaries. b. Chemical probe experiments to determine if RNA truncations have fold-parity with the full RNA. c. Footprinting studies monitored by SHAPE and nuclease/DMS protection. Aim 3: Determine the structure of human SRA1 RNP complexes. a. NMR monitored binding experiments to assess viability for structure determination. b. RNP complex NMR structures of portions of SRA1 RNA (i.e. STR7) bound to SHARP RRM domains.

项目总结-通过NMR研究SHARP表观遗传调节蛋白对SRA lncRNA的识别 托马斯利珀，肯尼索州立大学。 本研究的目的是利用核磁共振技术来确定核糖核蛋白的结构 来源于SHARP，一种表观遗传调节蛋白，和类固醇受体激活剂表观遗传调节因子 RNA（SRA1）。这种RNA是调节染色质的雄激素受体（AR）共激活网络的一部分 并增强AR介导的致癌表达。AR反应刺激前列腺癌细胞超- 增殖因此，了解AR调节因子的结构和生物化学细节对于 了解这种癌症。SRA 1 RNA分子是一种新兴的长非短链RNA分子， 编码RNA（lncRNA），其是染色质重塑复合物的公认组分。夏普是 也与癌症相关的Wnt和Notch信号通路有关。这些结构会给我们一些 lncRNA核糖核蛋白颗粒结构的第一次一瞥，并建议基于结构的区域 如果要开发靶向lncRNA的治疗方法，药物发现工作应该集中注意力。这 一个项目跨越几个学科，但最恰当的描述是结构生物学与明确的目标 从这个RNP支架复合体中提供结构。 具体目标是： 目的1：确定人SHARP的RRM结构域的NMR结构。 a.表达、纯化和NMR筛选源自SHARP片段的RRM结构域。 B.确定良好折叠的SHARP RRM的NMR结构。 目的2：描述人SRA lncRNA/SHARP相互作用。 a.通过固定化蛋白质RNA下拉测定法测定RNP形成，以发现RNA结构域边界。 B.化学探针实验，以确定RNA截短是否与完整RNA具有倍数奇偶性。 C.通过SHAPE和核酸酶/DMS保护监测足迹研究。 目的3：确定人SRA 1 RNP复合物的结构。 a. NMR监测结合实验以评估结构测定的可行性。 B.与SHARP RRM结构域结合的SRA 1 RNA（即STR 7）部分的RNP复合物NMR结构。