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: 10543406
• 负责人: Davia Blake
• 金额: $ 1.01万
• 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 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543406
• 关键词: Alternative Splicing; Antibodies; Antigen Presentation; 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; Exclusion; 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; Process; Production; Proliferating; Protein Isoforms; Protein Tyrosine Phosphatase; Proteins; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Signal Induction; Signal Pathway; Signal Transduction; Spliced Genes; Stimulus; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Testing; Titrations; 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.

项目摘要 替代剪接由选择性包括或排除在成熟mRNA中的外显子组成 成绩单。实际上，大多数编码基因的蛋白质（约95％）都包含替代外显子。以前的 研究认识到，替代剪接基因的子集（10-15％）发生了同工型丰度的变化 激活原代人CD4+ T细胞后。信号诱导的替代方案的典型示例 剪接变化包括酪氨酸磷酸酶受体CD45，其中增加的丰度 较短的RNA同工型与未来刺激的激活阈值相关。监管 机制，包括信号通路的作用和影响剪接变化的RNA结合蛋白 以我们的实验室和其他人为特征。但是，该领域缺乏对角色和 差分T细胞刺激的机制调节全局剪接变化。 先前的研究研究了信号引起的替代剪接变化，随着T细胞的激活 抗CD3和抗CD28刺激的存在。 CD3是T细胞受体（TCR）的组成部分， 负责在感染的背景下识别由抗原呈递细胞呈现的肽。 CD28 是一种共同刺激受体，可增强TCR下游的许多信号事件。我们问全球是否 替代剪接变化受主要的CD28共刺激存在差异的影响 人CD4+ T细胞。因此，我从三个人类捐赠者那里获得了主要的CD4+ CD45ro-T细胞， 在以下情况下培养细胞：单独培养基，抗CD28抗体，抗CD3抗体或抗体 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，受到存在CD28共刺激影响的替代剪接事件 仅由CD3刺激调节。这些拼接事件之一包括在 Casapse-9在CD4+ T细胞中尚未表征的转录本。我们假设多述 跳过事件是调节刺激后的T细胞存活。在我们的建议中，我们计划阐明机制 由CD28共刺激调节的剪接变化和此类事件的功能后果，例如 caspase-9中的多外观跳过事件以及对T细胞存活的影响。总的来说，这项工作将是阶梯 知道如何调节全球替代剪接事件以及对细胞功能的影响。