Mechanisms of signal-responsive alternative splicing in T-cell activation

T 细胞激活中信号响应选择性剪接的机制

• 批准号: 8400614
• 负责人: Nicole Martinez
• 金额: $ 4.22万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8400614
• 关键词: Address; Affinity Chromatography; Alternative Splicing; Antigens; Binding; Biological Assay; Biology; Cell Line; Cell Proliferation; Cell physiology; Cells; Cellular biology; Complex; Cytotoxin; DNA Sequence Rearrangement; Data; Environment; Event; Exons; Future; Gene Expression; Gene Expression Regulation; Genes; Goals; Human; Immune; Immune response; Immune system; Immunobiology; Investigation; Laboratories; Lead; MAPK8 gene; Mediating; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mutagenesis; Ontology; Pathogenesis; Pattern; Phosphotransferases; Play; Process; Proliferating; Protein Isoforms; Proteins; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Regulatory Element; Role; Shapes; Signal Pathway; Signal Transduction; Signaling Protein; Spliced Genes; T cell regulation; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Testing; Tissues; Trans-Activators; Transcriptional Regulation; antigen challenge; base; crosslink; cytokine; experience; extracellular; human disease; immunoregulation; insight; intercellular communication; knock-down; next generation sequencing; novel; pathogen; programs; protein expression; protein function; public health relevance; response; therapeutic development; tool; transcriptome sequencing

DESCRIPTION (provided by applicant): Alternative splicing provides cells with the ability to generate distinct proteins from the same gene, thus increasing protein diversity and controlling protein expression. Almost all multi-exon mammalian genes undergo alternative splicing in a tissue specific or signal-induced manner, however the regulatory mechanisms and functional consequences of such splicing changes have only been well-characterized for a small fraction of genes. Cells of the immune system are particularly expected to utilize alternative splicing to control cellular activity, as these cells must respond dynamically to their environment receiving danger signals and converting these into effector functions. Specifically, T-cells are stimulated through their T-cell receptors to trigger signaling cascades that lead to proliferation and cytokines secretion. However, the understanding of gene regulation upon T-cell activation has focused on transcriptional changes, with much less investigation of the contribution of alternative splicing to T-cell effector functions. Studies in our laboratory seek t fill this critical gap in our understanding of both alternative splicing and T-cell biolgy. Next-generation sequencing has recently led to the identification of 168 genes that undergo robust signal-induced alternative splicing in response to T-cell signaling. Detailed investigation of prototypical examples from among these 168 genes will lead to a deeper understanding of how splicing of these genes is regulated, and how such alternative splicing impacts the function of the cell. This proposal is focused on one gene, MKK7, based on the robustness of splicing change and ontology relevant to T-cell biology. MKK7 is a kinase that activates the JNK-signaling pathway that is essential for T-cell activity. Moreover, preliminary data suggests that splicing of MKK7 is regulated by a mechanism distinct from those previously implicated in signal-induced alternative splicing. To investigate the regulatory mechanisms governing alternative splicing of MKK7, standard minigene and mutagenesis approaches will first be used to define the cis-regulatory elements that are sufficient to confer signal-induced changes in MKK7 splicing. RNA-affinity purification and UV crosslinking will then be used to identify RNA-binding proteins that associate with these cis-regulatory elements, and the functional relevance of such proteins to MKK7 splicing will be determined by knock-down in cells. Finally, the functional distinction between the alternate protein isoforms of MKK7 that result from activation-induced alternative splicing will be investigated using standard signaling and protein association assays that are in common use in the laboratory. Taken together, the studies outlined in this proposal will reveal unique insight into a novel pathway of signal- induced splicing regulation in T-cells and will provide further understanding as to how alternative splicing is utilized to shape the functional response of the human immune system.

描述（由申请人提供）：替代剪接使细胞具有与同一基因产生不同蛋白质的能力，从而增加了蛋白质的多样性和控制蛋白质的表达。 几乎所有多种多样的哺乳动物基因都以组织特异性或信号诱导的方式进行替代剪接，但是，这种剪接变化的调节机制和功能后果仅适用于一小部分基因的特征。预期免疫系统的细胞会利用替代剪接来控制细胞活性，因为这些细胞必须动态响应其环境，从而接收危险信号并将其转换为效应子功能。 具体而言，T细胞通过其T细胞受体刺激，以触发导致增殖和细胞因子分泌的信号传导级联反应。然而，对基因调节在T细胞激活时的理解集中在转录变化上，对替代剪接对T细胞效应子功能的贡献的研究很少。我们在实验室中寻求T的研究填补了我们对替代剪接和T细胞生物的理解。 下一代测序最近导致鉴定了168个基因，这些基因响应T细胞信号传导而经历了强大的信号诱导的替代剪接。在这168个基因中，对原型示例的详细研究将使人们更深入地了解这些基因的剪接如何受到调节，以及这种替代剪接如何影响细胞功能。该提议的重点是基于与T细胞生物学相关的剪接变化和本体的稳健性，该建议是MKK7。 MKK7是一种激活JNK信号途径的激酶，这对于T细胞活性至关重要。 此外，初步数据表明，MKK7的剪接受到与以前有关的替代剪接涉及的机制不同的机制。为了调查有关MKK7替代剪接的调节机制，将首先使用标准的微型基因和诱变方法来定义顺式调节元件，这些元件足以赋予信号诱导的MKK7剪接变化。然后，将使用RNA亲和力纯化和紫外线交联来识别与这些顺式调节元件相关的RNA结合蛋白，并且此类蛋白与MKK7拼接的功能相关性将通过细胞中的敲除确定。最后，将使用在实验室中常用的标准信号传导和蛋白质关联测定法研究，将研究由激活诱导的替代剪接引起的MKK7的替代蛋白质同工型之间的功能区别。 综上所述，该提案中概述的研究将揭示对T细胞中信号诱导的剪接调控途径的独特见解，并将提供有关如何替代的进一步理解 剪接用于塑造人免疫系统的功能响应。