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

描述(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，从而增加了蛋白质多样性和控制蛋白质表达。几乎所有的哺乳动物多外显子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节机制和功能后果只对一小部分基因进行了很好的表征。免疫系统的细胞尤其被期望利用替代剪接来控制细胞活动，因为这些细胞必须对接收危险信号的环境做出动态反应，并将这些信号转换为效应器功能。具体地说，T细胞通过其T细胞受体被刺激，从而触发导致增殖和细胞因子分泌的信号级联反应。然而，对基因调控T细胞激活的了解主要集中在转录变化上，对选择性剪接对T细胞效应器功能的贡献的研究要少得多。我们实验室的研究试图填补我们对选择性剪接和T细胞生物学理解的这一关键空白。下一代测序最近导致了168个基因的鉴定，这些基因经历了强大的信号诱导的选择性剪接，以响应T细胞信号。对这168个基因中的典型例子进行详细研究，将有助于更深入地理解这些基因的剪接是如何调控的，以及这种选择性剪接是如何影响细胞功能的。这个建议集中在一个基因，MKK7，基于剪接变化的稳健性和与T细胞生物学相关的本体论。MKK7是一种激活JNK信号通路的激酶，而JNK信号通路是T细胞活动所必需的。此外，初步数据表明，MKK7的剪接受到一种不同于先前在信号诱导的选择性剪接中涉及的机制的调控。为了研究MKK7选择性剪接的调控机制，首先将使用标准的微型基因和突变方法来定义足以在信号诱导的MKK7剪接中发生变化的顺式调控元件。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，这些蛋白与MKK7剪接的功能相关性将通过细胞中的击倒来确定。最后，将使用实验室中常用的标准信号和蛋白质结合分析来研究激活诱导选择性剪接导致的MKK7替代蛋白亚型之间的功能差异。综上所述，这项提案中概述的研究将揭示信号诱导T细胞剪接调控的新途径的独特见解，并将提供对如何替代 剪接被用来塑造人类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.