CD28 Regulation of Alternative Splicing Changes in Human T cells

项目摘要 选择性剪接由外显子组成，这些外显子选择性地包括或排除在成熟的mRNA中 文字记录。事实上，大多数编码蛋白质的基因，大约95%，都含有一个可供选择的外显子。上一首 研究发现，10%-15%的选择性剪接基因的子集经历了异构体丰度的变化 当原代人类CD4+T细胞被激活时。信号诱导替代的一个很好的特征例子 剪接的变化包括酪氨酸磷酸酶受体CD45，其中增加的丰度 较短的RNA异构体与来自未来刺激的激活阈值增加相关。监管 影响剪接变化的机制包括信号通路和RNA结合蛋白的作用 已经被我们的实验室和其他人所描述。然而，该领域缺乏对这一角色的一般知识， 差异T细胞刺激调节整体剪接变化的机制。 以前的研究研究了信号诱导的选择性剪接变化与T细胞在 存在抗CD3和抗CD28刺激物。CD3是T细胞受体(TCR)的一个组成部分，它是 负责在感染的情况下识别抗原提呈细胞提呈的多肽。CD28 是一种共刺激受体，能增强TCR下游的许多信号事件。我们问，如果全球 选择性剪接变化受原发灶中CD28共刺激的存在的不同影响 人的CD4+T细胞。因此，我从三名人类捐赠者那里获得了原代CD4+CD45RO-T细胞，并 在以下条件下培养细胞：单独培养液、抗CD28抗体、抗CD3抗体或抗CD3抗体。 CD3/CD28共同表达。我收集了用于高通量测序分析的RNA，并发现了 与剪接事件相比，受存在CD28共刺激影响的选择性剪接事件 仅受CD3刺激的调节。这些剪接事件之一包括多外显子跳过事件 尚未在CD4+T细胞中鉴定的Casapse-9转录本。我们假设多外显子 跳跃事件是调节T细胞在刺激下的存活。在我们的提案中，我们计划阐明机制 由CD28共刺激调控的剪接变化以及此类事件的功能后果，例如 Caspase-9多外显子跳跃事件及其对T细胞存活的影响总的来说，这项工作将是迈出的一步 斯通了解全球选择性剪接事件是如何调控的，以及对细胞功能的影响。

Project Summary Alternative splicing consists of exons that are selectively included or excluded from the mature mRNA transcript. In fact, most protein encoding genes, approximately 95%, contain an alternative exon. Previous studies recognized that a subset of alternative spliced genes, 10-15%, undergo changes in isoform abundance upon activation of primary human CD4+ T cells. A well-characterized example of signal-induced alternative splicing changes includes a tyrosine phosphatase receptor, CD45, where the increased abundance of the shorter RNA isoform is correlated with an increased activation threshold from future stimuli. Regulatory mechanisms including the role of signaling pathways and RNA binding proteins influencing splicing changes have been characterized by our lab and others. However, the field lacks general knowledge of the role and mechanism of differential T cell stimuli to regulate global splicing changes. Previous studies investigated signal-induced alternative splicing changes with T cells activated in the presence of anti-CD3 and anti-CD28 stimuli. CD3 is a component of the T cell receptor (TCR), which is responsible for recognizing peptides presented by antigen presenting cells in the context of an infection. CD28 is a costimulatory receptor that enhances many signaling events downstream of the TCR. We ask if global alternative splicing changes are differentially influenced by the presence of CD28 costimulation in primary human CD4+ T cells. Therefore, I acquired primary CD4+ CD45RO- T cells from three human donors and cultured the cells in the following conditions: media alone, anti-CD28 antibody, anti-CD3 antibody, or anti- CD3/CD28 together. I harvested RNA for high-throughput sequencing analysis and discovered a subset of alternative splicing events that are influenced by the presence CD28 costimulation compared to splicing events regulated by CD3 stimulation alone. One of these splicing events includes a multi-exon skipping event within Casapse-9 transcript that has not been characterized in CD4+ T cells. We hypothesize that the multi-exon skipping event is regulating T cell survival upon stimulation. In our proposal, we plan to elucidate mechanisms of splicing changes regulated by CD28 costimulation and the functional consequences of such events, such as the multi-exon skipping event in Caspase-9 and impact on T cell survival. Overall, this work will be the stepping stone into knowing how global alternative splicing events are regulated and the impact on cellular functions.