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

描述（由申请人提供）：拟议研究的目的是使用NMR来确定源自SHARP的核糖核蛋白（RNP）的结构，SHARP是一种表观遗传调节蛋白，类固醇受体激活剂表观遗传调节RNA（SRA 1）。该RNA是雄激素受体（AR）共激活剂网络的一部分，其调节染色质并增强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物化学细节对于了解这种类型的癌症非常重要。SRA 1 RNA分子是新出现的一类长非编码RNA（lncRNA）的成员，其是染色质重塑复合物的公认组分。SHARP还与癌症相关的Wnt和Notch信号通路相关。这些结构将给我们一些lncRNA核糖核蛋白颗粒结构的初步一瞥，并建议如果要开发针对lncRNA的疗法，基于结构的药物发现工作应该集中注意力的区域。这个项目跨越了几个学科，但最恰当的描述是结构生物学，其明确的目标是从RNP支架复合体中提供结构。具体目的是：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。

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.