Sequence-specific Capture for Discovering Protein-IncRNA Interactions in Prostate Cancer

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 DESCRIPTION (provided by applicant): Protein-nucleic acid interactions play key roles in both transcriptional and translational regulation, not only in the direct interaction of proteins with DA and mRNA to regulate these processes, but also the interactions among proteins, lncRNAs, and DNAs. Understanding these interactions requires knowledge of the molecules that are involved. lncRNAs interact with proteins to achieve several consequences in gene regulation. First, lncRNAs can act as a "molecular sink" for proteins that may interact with DNA or RNA. Their interaction with the lncRNA thus diverts the protein from binding to its primary target. lncRNAs can also act as guides for proteins, leading them to their DNA targets to either repress or activate transcription. Finally, lncRNAs can act as platforms upon which molecules can congregate to perform a function, such as histone modification, as a team at a specific location and time. Each of these functions requires the interaction of lncRNAs with a diverse set of proteins. Knowledge of the proteins bound to a specific lncRNA will allow for determination of the mechanisms by which that lncRNA affects gene expression. Unraveling this tremendous diversity of interactions demands tools capable of rapid identification and quantification of proteins. In this proposal we focus on the particular problem of identifying the proteins that are bound to specific lncRNA molecules, their relationship to the progression of prostate cancer and their potential as markers of the disease and its progression. We describe an RNA-centric approach to discovery of the lncRNA-binding proteins that is innovative compared with existing technologies in two major ways. First, crosslinking of proteins to lncRNAs is done in vivo under normal cellular conditions. Therefore, proteins and lncRNAs will be folded normally, will be present at their normal cellular concentrations, and will be in their normal cellular locations. Second, sequence-specific capture of the lncRNA is used to extract the RNA of interest along with its crosslinked proteins. This is a universal capture strategy that does not rely on the availability of any other capture reagent (e.g. antibody or aptamer). Following enrichment by sequence-specific capture, mass spectrometry will be utilized to identify and quantify the associated proteins.