Signal transduction and gene induction in lymphocytes

描述（由申请人提供）：转录因子NFAT在免疫细胞和非免疫细胞中都起重要作用。在T细胞中，储存操作的Ca2+通过STIM-ORAI途径内流触发磷酸酶钙调磷酸酶，其脱磷磷酸化NFAT并促进其核易位。NFAT激酶CK1、GSK3和DYRK对抗钙调磷酸酶的作用，并在细胞质中维持NFAT处于磷酸化、无活性状态。在本应用中，我们重点研究了一种名为NRON （NFAT的非编码抑制因子）的长基因间非编码RNA （lincRNA）在T细胞中调节Ca2+-钙调磷酸酶-NFAT信号通路中的作用。NRON先前被鉴定为NFAT的负调节因子，并与支架蛋白IQGAP1和核输入蛋白结合。我们通过证明NRON、NFAT、IQGAP1和NFAT激酶在细胞质中形成一个高分子量复合物来扩展这些发现。针对NRON、IQGAP1或两者的sirna在低水平刺激下增强了NFAT的激活，并显著增加了Jurkat T细胞中NFAT依赖性细胞因子白介素-2 （IL-2）的产生。同样，携带Iqgap1基因破坏的小鼠T细胞比野生型T细胞产生更多的干扰素γ （IFN¿）。在这里，我们将研究在全基因组RNAi筛选中发现的lincRNA NRON、rna结合蛋白（rbp）和新型NFAT信号调节剂在调节T细胞中Ca2+-钙调磷酸酶- NFAT通路活性中的作用。在Aim 1中，我们将定义静止和活化T细胞中含有NFAT、NRON、IQGAP1、钙调蛋白、NFAT激酶和进口蛋白的支架复合体的生化特征，询问该复合体的成分是否参与在细胞质中招募钙调磷酸酶，以及它们是否在细胞核中具有潜在的功能。我们还将确定NRON的蛋白伴侣和钙调磷酸酶调节因子RCAN1的RNA伴侣。在目的2中，我们将通过小鼠条件性基因破坏来研究NRON在胸腺细胞和T细胞中的体内作用。小的非编码rna，特别是microrna的功能已经被广泛研究，但长链非编码lincrna在生物过程中的作用才刚刚开始被阐明。LincRNAs可以将转录因子靶向到它们在DNA中的结合位点，也可以作为rna -蛋白复合物调节基因表达的关键组分。我们提出的研究应该阐明免疫细胞中重要的Ca2+-钙调磷酸酶- nfat信号通路的这些可能性。我们已经开发了大量的创新试剂和技术来研究这一途径，因此具有独特的定位来阐明Ca2+-钙调磷酸酶和NRON支架复合物激活NFAT的分子机制。

DESCRIPTION (provided by applicant): 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.