CD28 Regulation of Alternative Splicing Changes in Human T cells

点击翻译按钮获取中文摘要

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.