The transcription factor NFAT plays essential roles in both immune and non-immune cells. In T cells, store- operated Ca2+ influx through the STIM-ORAI pathway triggers the phosphatase calcineurin, which dephos- phorylates NFAT and promotes its nuclear translocation. The NFAT kinases CK1, GSK3, and DYRK counter the action of calcineurin and maintain NFAT in a phosphorylated, inactive state in the cytoplasm. In this application, we focus on the role of a long intergenic non-coding RNA (lincRNA) named NRON (non- coding repressor of NFAT) in regulation of the Ca2+-calcineurin-NFAT signalling pathway in T cells. NRON was previously identified as a negative regulator of NFAT, and shown to bind the scaffold protein IQGAP1 and nuclear import proteins. We extended these findings by showing that NRON, NFAT, IQGAP1 and NFAT kinases form a high-molecular-weight complex in the cell cytoplasm. siRNAs against NRON, IQGAP1 or both enhanced NFAT activation in response to low-level stimulation, and dramatically increased production of the NFAT-dependent cytokine Interleukin-2 (IL-2) by Jurkat T cells. Likewise, mouse T cells bearing a disruption of the Iqgap1 gene produced considerably more interferon-gamma (IFN) than wild type T cells. Here we will investigate the role of the lincRNA NRON, RNA-binding proteins (RBPs), and novel modulators of NFAT signalling identified in a genome-wide RNAi screen, in regulating the activity of the Ca2+-calcineurin- NFAT pathway in T cells. In Aim 1, we will define the biochemical features of the scaffold complex containing NFAT, NRON, IQGAP1, calmodulin, NFAT kinases and importins in resting and activated T cells, asking if components of the complex are involved in recruiting calcineurin in the cytoplasm and if they have a potential function in the nucleus. We will also identify protein partners for NRON and RNA partners for the calcineurin regulator RCAN1. In Aim 2, we will examine the in vivo role of NRON in thymocytes and T cells by conditional gene disruption in mice. The functions of small non-coding RNAs, particularly microRNAs, have been extensively studied, but the roles of long noncoding lincRNAs in biological processes are just beginning to be elucidated. LincRNAs can target transcription factors to their binding sites in DNA, and can also function as critical components of RNA-protein complexes that regulate gene expression. Our proposed studies should illuminate these possibilities for the important Ca2+-calcineurin-NFAT signaling pathway in immune cells. We have developed a large number of innovative reagents and techniques to study this pathway, and thus are uniquely positioned to elucidate the molecular mechanisms underlying NFAT activation by Ca2+-calcineurin and the NRON scaffold complex. PUBLIC HEALTH RELEVANCE: Calcium is a universal second messenger that is used by all cells, including those of the brain, heart, muscle and immune system, to control a huge diversity of cellular functions. When calcium enters cells, a protein known as NFAT turns on genes important for the proper functioning of the cell. In the previous project period, we showed that NFAT existed in a large complex that contains both RNA and other proteins. Here we will use cutting-edge approaches, including proteomics and next-generation sequencing, to investigate how NFAT travels to the nucleus in response to calcium entry. We expect to identify new regulatory proteins that will provide novel therapies for autoimmunity and other important diseases.