Signal transduction and gene induction in lymphocytes

淋巴细胞中的信号转导和基因诱导

• 批准号: 8713901
• 负责人: Patrick Hogan
• 金额: $ 43.5万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8713901
• 关键词: Address; Affinity Chromatography; Antigens; Autoimmunity; B-Lymphocytes; Back; Binding; Binding Sites; Biochemical; Biological Process; Brain; Bypass; CSNK1A1 gene; Calcineurin; Calcium; Calmodulin; Cell Nucleus; Cell physiology; Cells; Chimeric Proteins; Complex; Cytoplasm; Cytoskeletal Proteins; DNA; Data; Disease; Exons; Family; Figs - dietary; Functional RNA; Funding; Gene Expression; Gene Targeting; Generations; Genes; Grant; Human; Immune; Immune system; Importins; Inositol; Interferon Type II; Interferons; Interleukin-2; Interphase Cell; Laboratories; Luciferases; Lymphocyte; Mammalian Cell; Mediating; MicroRNAs; Mitogen-Activated Protein Kinases; Modeling; Molecular; Molecular Weight; Mus; Myocardium; NFAT Pathway; Names; Nuclear; Nuclear Import; Nuclear Translocation; Paper; Pathway interactions; Phospholipase C; Phosphotransferases; Plasmids; Play; Positioning Attribute; Production; Protein Dephosphorylation; Proteins; Proteomics; RNA; RNA Interference; RNA Recognition Motif; RNA-Binding Proteins; Reagent; Recruitment Activity; Regulation; Reporter; Rest; Role; Scaffolding Protein; Second Messenger Systems; Signal Pathway; Signal Transduction; Signaling Protein; Stimulus; T-Lymphocyte; Techniques; Time; Transduction Gene; Travel; Work; calcineurin phosphatase; cytokine; follow-up; gene induction; genetic regulatory protein; genome wide association study; genome-wide; in vivo; innovation; member; muscular system; next generation sequencing; novel; nucleocytoplasmic transport; protein complex; receptor; receptor coupling; response; scaffold; second messenger; thymocyte; transcription factor

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.

描述（由申请人提供）：转录因子NFAT在免疫和非免疫细胞中起重要作用。在T细胞中，通过STIM-ORAI途径的钙池操纵的Ca 2+内流触发磷酸酶钙调磷酸酶，其使NFAT脱磷酸并促进其核转位。NFAT激酶CK 1、GSK 3和DYRK对抗钙调磷酸酶的作用，并使NFAT在细胞质中保持磷酸化的非活性状态。在本申请中，我们集中于称为NRON（NFAT的非编码阻遏物）的长基因间非编码RNA（lincRNA）在调节T细胞中的Ca 2 +-钙调神经磷酸酶-NFAT信号传导途径中的作用。NRON先前被鉴定为NFAT的负调节剂，并且显示结合支架蛋白IQGAP 1和核输入蛋白。我们通过显示NRON、NFAT、IQGAP 1和NFAT激酶在细胞质中形成高分子量复合物来扩展这些发现。针对NRON、IQGAP 1或两者的siRNA响应于低水平刺激而增强NFAT活化，并且显著增加Jurkat T细胞产生NFAT依赖性细胞因子白细胞介素-2（IL-2）。同样，携带Iqgap 1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lincRNA NRON、RNA结合蛋白（RBP）和在全基因组RNAi筛选中鉴定的NFAT信号传导的新型调节剂在调节T细胞中Ca 2 +-钙调神经磷酸酶- NFAT途径的活性中的作用。在目标1中，我们将定义含有NFAT，NRON，IQGAP 1，钙调蛋白，NFAT激酶和输入蛋白的支架复合物在静息和活化T细胞中的生物化学特征，询问复合物的组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。我们还将确定NRON的蛋白质伴侣和钙调磷酸酶调节剂RCAN 1的RNA伴侣。在目标2中，我们将通过小鼠中的条件性基因破坏来检查NRON在胸腺细胞和T细胞中的体内作用。小型非编码RNA，特别是微小RNA的功能已得到广泛研究，但长非编码lincRNA在生物过程中的作用才刚刚开始阐明。LincRNA可以将转录因子靶向到它们在DNA中的结合位点，并且还可以作为调节基因表达的RNA-蛋白质复合物的关键组分发挥作用。我们提出的研究应该阐明这些可能性的重要的Ca 2 +-calcineurin-NFAT信号通路在免疫细胞。我们已经开发了大量的创新试剂和技术来研究这一途径，因此具有独特的优势来阐明Ca 2 +-钙调磷酸酶和NRON支架复合物激活NFAT的分子机制。