Signal transduction and gene induction in lymphocytes

描述(申请人提供)：转录因子NFAT在免疫和非免疫细胞中都扮演着重要的角色。在T细胞中，储存操作的钙离子通过STIM-ORAI途径内流触发磷酸酶钙调神经磷酸酶，使NFAT去磷酸化并促进其核转位。NFAT激酶CK1、GSK3和Dyrk对抗钙调神经磷酸酶的作用，并在细胞质中将NFAT维持在磷酸化的非活性状态。在这一应用中，我们重点研究了一种名为NRON(Non-Coding Repressor of NFAT)的长基因间非编码RNA(LincRNA)在调节T细胞钙-钙调神经磷酸酶-NFAT信号通路中的作用。NRON以前被认为是NFAT的负调控因子，并被证明与支架蛋白IQGAP1和核进口蛋白结合。我们通过显示NRON、NFAT、IQGAP1和NFAT激酶在细胞质中形成一个高分子量复合体来扩展这些发现。针对NRON和/或IQGAP1的siRNA可增强NFAT在低水平刺激下的激活，并显著增加Jurkat T细胞产生依赖NFAT的细胞因子白介素2(IL-2)。同样，携带Iqgap1基因突变的小鼠T细胞比野生型T细胞产生更多的干扰素-γ(干扰素�)。在这里，我们将研究全基因组RNAi筛选中发现的lincRNA NRON、RNA结合蛋白(RBPs)和新的NFAT信号调节器在调节T细胞中钙-钙调神经磷酸酶-NFAT途径的活性中的作用。在目标1中，我们将确定含有NFAT、NRON、IQGAP1、钙调素、NFAT激酶和重要蛋白的支架复合体在静息和激活的T细胞中的生化特征，询问该复合体的组成部分是否参与在细胞质中招募钙调神经磷酸酶，以及它们是否在细胞核中具有潜在的功能。我们还将为NRON确定蛋白质伙伴，为钙调神经磷酸酶调节因子RCAN1确定RNA伙伴。在目标2中，我们将通过条件性基因破坏在小鼠体内检测NRON在胸腺细胞和T细胞中的作用。小的非编码RNA，特别是小RNA的功能已经被广泛研究，但长的非编码连接RNA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DNA中的结合部位，也可以作为RNA-蛋白质复合体的关键成分来调节基因的表达。我们提出的研究应该阐明免疫细胞中重要的钙-钙调神经磷酸酶-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，从而独特地定位于阐明钙-钙调神经磷酸酶和NRON支架复合体激活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.