CD28 Regulation of Alternative Splicing Changes in Human T cells

CD28 对人类 T 细胞选择性剪接变化的调节

• 批准号: 10252785
• 负责人: Davia Blake
• 金额: $ 3.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10252785
• 关键词: Alternative Splicing; Antibodies; Antigen-Presenting Cells; Apoptotic; CASP9 gene; CD28 gene; CD3 Antigens; CD4 Positive T Lymphocytes; Cell Death; Cell Survival; Cell physiology; Cells; Computer software; Cultured Cells; Data; Event; Exhibits; Exons; Future; Gene Expression; Genes; Genetic; Goals; Harvest; High-Throughput Nucleotide Sequencing; Human; Immune system; Infection; Knowledge; Messenger RNA; Monoclonal Antibody HuM291; PTPRC gene; Peptides; Phenotype; Physiological; Poly A; Process; Production; Protein Isoforms; Protein Tyrosine Phosphatase; Proteins; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Signal Pathway; Signal Transduction; Spliced Genes; Stimulus; Suggestion; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Testing; Transcript; Work; cytokine; exon skipping; novel; pathogen; receptor; response; tool; transcriptome sequencing

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.

项目概要 选择性剪接由选择性包含或排除成熟 mRNA 的外显子组成 成绩单。事实上，大多数蛋白质编码基因（大约 95%）都含有替代外显子。以前的 研究认识到选择性剪接基因的一个子集（10-15%）会发生异构体丰度的变化 原代人 CD4+ T 细胞激活后。信号诱导替代方案的一个典型例子 剪接变化包括酪氨酸磷酸酶受体 CD45，其中 较短的 RNA 亚型与未来刺激的激活阈值增加相关。监管 机制，包括信号通路和 RNA 结合蛋白影响剪接变化的作用 我们的实验室和其他实验室已经对其进行了表征。然而，该领域缺乏对其角色和作用的一般了解。 差异T细胞刺激调节整体剪接变化的机制。 先前的研究调查了信号诱导的选择性剪接变化，其中 T 细胞被激活 抗CD3和抗CD28刺激的存在。 CD3 是 T 细胞受体 (TCR) 的组成部分， 负责识别感染情况下抗原呈递细胞呈递的肽。 CD28 是一种共刺激受体，可增强 TCR 下游的许多信号转导事件。我们询问是否全球 原代细胞中 CD28 共刺激的存在对可变剪接变化有不同的影响 人类 CD4+ T 细胞。因此，我从三个人类捐赠者那里获得了原代 CD4+ CD45RO- T 细胞， 在以下条件下培养细胞：单独培养基、抗CD28抗体、抗CD3抗体或抗- CD3/CD28 一起。我收集了 RNA 进行高通量测序分析，并发现了一个子集 与剪接事件相比，受 CD28 共刺激影响的选择性剪接事件 仅受 CD3 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 CD4+ T 细胞中进行表征。我们假设多外显子 跳跃事件调节 T 细胞在刺激后的存活。在我们的提案中，我们计划阐明机制 CD28 共刺激调节的剪接变化以及此类事件的功能后果，例如 Caspase-9 中的多外显子跳跃事件及其对 T 细胞存活的影响。总体而言，这项工作将成为 深入了解全局选择性剪接事件是如何调节的以及对细胞功能的影响。