PROJECT SUMMARY Large non-coding RNAs (lncRNAs) comprise a newly recognized class of thousands of functional RNA molecules that play important roles in a wide variety of cellular pathways. These lncRNAs can have significant effects on gene expression, and are involved in controlling regulatory networks critical for cellular growth and differentiation. Many lncRNAs are important players in human diseases, especially human cancers. An emerging hypothesis is that lncRNAs regulate gene expression and cellular function by binding functional protein complexes to organize and coordinate their activities. However, a main challenge in understanding the mechanisms of lncRNA-mediated regulation of gene expression and cellular function has been a lack of knowledge of their protein interaction partners. To address this research bottleneck, I have developed a method to comprehensively define the proteins that directly interact with a lncRNA, termed RNA Antisense Purification with Mass Spectrometry (RAP-MS). This method has so far been highly successful in identifying direct and specific interaction partners of several cellular RNAs, including Xist lncRNA, 18S ribosomal RNA, U1 small nuclear RNA, and 45S pre-ribosomal RNA. In the proposed work I will use RAP-MS to identify the functional protein interactors of three lncRNAs (Tsix, Neat1, and Malat1) then identify the interacting regions of lncRNAs and proteins and create detailed high resolution structural models of lncRNA and protein complexes. Dissection of the sequence determinants of lncRNA-protein interactions at the level of nucleotide and amino acid interactions will lead to an improved understanding of the mechanisms of action of lncRNAs in controlling gene expression and regulating cellular function at a molecular level. The goals of this study are to identify direct and specific lncRNA-interacting proteins using RAP-MS (Aim 1), determine the functions of lncRNA-interacting proteins (Aim 2), and dissect the molecular structure of lncRNA- protein complexes (Aim 3). Taken together, the results of these experiments will reveal how lncRNAs bind and coordinate proteins into higher-order complexes to achieve their roles in regulating gene expression and cellular function. An understanding of functional lncRNA-protein interactions at the level of amino acid and nucleotide interactions will provide insight into the mechanisms of action of lncRNAs and reveal molecular principles guiding lncRNA-protein assembly. In addition, identification of the underlying principles of lncRNA- protein function at a detailed molecular level will lead to new opportunities to control and manipulate the action of non-coding RNAs in mammalian cells during the initiation and progression of cancer and other disease processes.