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.
The structures of RNA-protein complexes, SHARP and the SRA RNA, involved in mediating epigenetic change and androgen receptor co-activation will be determined using nuclear magnetic resonance spectroscopy. This system of ribonucleoproteins co-activators is a constituent of the prostate cancer cell androgen response network. These structures will improve our understanding of how RNA recognition and ribonucleoprotein assembly relates to long non-coding RNA function in steroid stimulated cancer cell proliferation.
