CD28 Regulation of Alternative Splicing Changes in Human T cells

项目摘要 选择性剪接包括选择性地包含或排除在成熟mRNA之外的外显子 成绩单。事实上，大多数蛋白质编码基因，约95%，包含一个选择性外显子。先前 研究发现，10- 15%的选择性剪接基因的子集经历异构体丰度的变化， 在原代人CD 4 + T细胞活化后。信号诱导替代的一个充分表征的例子 剪接变化包括酪氨酸磷酸酶受体CD 45，其中增加的酪氨酸磷酸酶受体CD 45的丰度， 较短的RNA同种型与来自未来刺激的增加的激活阈值相关。监管 机制包括信号通路和RNA结合蛋白的作用，影响剪接的变化 已经被我们的实验室和其他实验室鉴定过了然而，该领域缺乏对这一作用的一般了解， 差异性T细胞刺激调节整体剪接变化的机制。 以前的研究调查了信号诱导的选择性剪接变化与T细胞活化， 抗CD 3和抗CD 28刺激物的存在。CD 3是T细胞受体（TCR）的一种成分， 负责识别在感染情况下由抗原呈递细胞呈递的肽。CD28 是增强TCR下游的许多信号传导事件的共刺激受体。我们问如果全球 选择性剪接的变化受到CD 28共刺激的影响， 人CD 4 + T细胞。因此，我从三个人供体获得了原代CD 4 + CD 45 RO- T细胞， 在以下条件下培养细胞：单独的培养基、抗-CD 28抗体、抗-CD 3抗体或抗- CD 3/CD 28结合。我收集RNA进行高通量测序分析，发现了一个 与剪接事件相比，受CD 28共刺激影响的选择性剪接事件 由单独的CD 3刺激调节。这些剪接事件之一包括多外显子跳跃事件， Casapse-9转录本尚未在CD 4 + T细胞中表征。我们假设多外显子 跳跃事件在刺激时调节T细胞存活。在我们的提案中，我们计划阐明 受CD 28共刺激调节的剪接变化以及这些事件的功能后果，例如 Caspase-9中的多外显子跳跃事件以及对T细胞存活的影响。总的来说，这项工作将是一个步骤， 了解全球可变剪接事件是如何调节的，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.