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

描述(申请人提供)：拟议研究的目的是使用核磁共振来确定源自表观遗传调节蛋白SHARP和类固醇受体激活剂表观遗传调节RNA(SRA1)的核糖核蛋白(RNPs)的结构。这种RNA是雄激素受体(AR)共激活网络的一部分，它调节染色质并增强AR介导的致癌表达。AR反应刺激前列腺癌细胞过度增殖。因此，了解AR调节因子的结构和生化细节对于了解这种类型的癌症非常重要。SRA1RNA分子是一类新兴的长非编码RNA(LncRNA)的成员，它是染色质重塑复合体中公认的成分。夏普还与癌症相关的Wnt和Notch信号通路有关。这些结构将让我们第一次瞥见lncRNA核糖核蛋白颗粒结构，并建议如果要开发针对lncRNA的治疗方法，基于结构的药物发现工作应该关注的区域。这个项目跨越了几个学科，但最恰当的描述是结构生物学，明确的目标是提供来自这个RNP脚手架复合体的结构。具体目标是：目标1：确定人夏普RRM结构域的核磁共振结构。A.表达、纯化和核磁共振筛选来自Sharp片段的RRM结构域。B.确定折叠良好的锐利RRMS的核磁共振结构。目的2：描述人类SRA lncRNA/Sharp相互作用。A.通过固定蛋白RNA下拉试验检测RNP的形成，以寻找RNA结构域边界。B.化学探针实验，以确定RNA截断是否与完整RNA具有折叠奇偶性。C.由SHAPE和核酸酶/DMS保护监测的足迹研究。目的3：确定人SRA1 RNP复合体的结构。A.核磁共振监测结合实验，以评估结构确定的可行性。B.与尖锐的RRM结构域结合的部分SRA1 RNA(即STR7)的RNP复合核磁共振结构。

DESCRIPTION (provided by applicant): 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.