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

描述（由申请人提供）：选择性剪接为细胞提供了从相同基因产生不同蛋白质的能力，从而增加蛋白质多样性并控制蛋白质表达。 几乎所有的多外显子哺乳动物基因都以组织特异性或信号诱导的方式进行选择性剪接，然而这种剪接变化的调控机制和功能后果仅对一小部分基因进行了充分表征。特别期望免疫系统的细胞利用选择性剪接来控制细胞活性，因为这些细胞必须动态地响应于它们的环境，接收危险信号并将这些信号转化为效应子功能。 具体地，T细胞通过其T细胞受体被刺激以触发导致增殖和细胞因子分泌的信号级联。然而，对T细胞激活后基因调节的理解主要集中在转录变化上，而对选择性剪接对T细胞效应器功能的贡献的研究却少得多。我们实验室的研究试图填补我们对选择性剪接和T细胞生物学的理解中的这一关键空白。 新一代测序技术最近鉴定了168个基因，这些基因在响应T细胞信号传导时经历了强大的信号诱导的选择性剪接。对这168个基因中典型例子的详细研究将有助于更深入地了解这些基因的剪接是如何调节的，以及这种选择性剪接如何影响细胞的功能。该提议基于剪接变化的鲁棒性和与T细胞生物学相关的本体论而集中于一个基因MKK7。MKK7是一种激活JNK信号通路的激酶，JNK信号通路对T细胞活性至关重要。 此外，初步的数据表明，剪接的MKK7的调节机制不同于那些以前牵连在信号诱导的选择性剪接。为了研究管理MKK7的选择性剪接的调节机制，将首先使用标准小基因和诱变方法来定义足以赋予MKK7剪接中的信号诱导的变化的顺式调节元件。然后将使用RNA亲和纯化和UV交联来鉴定与这些顺式调节元件相关的RNA结合蛋白，并且将通过在细胞中敲低来确定这些蛋白与MKK 7剪接的功能相关性。最后，使用实验室中常用的标准信号传导和蛋白质缔合测定法，研究活化诱导的选择性剪接导致的MKK7的替代蛋白质亚型之间的功能差异。 总之，本提案中概述的研究将揭示对T细胞中信号诱导的剪接调节的新途径的独特见解，并将进一步了解替代性剪接调节是如何发生的。 剪接被用于塑造人免疫系统的功能反应。

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.