Davia Blake (Mentor: Kristen Lynch, PhD)

“Alternative splicing of apoptosis genes promotes T cell survival”

Davia Blake, Caleb Radens and Kristen Lynch

Alternative splicing consists of exons that are selectively either included or excluded from the mature mRNA transcript. Previous studies have demonstrated that ~10-15% of alternatively spliced genes undergo signal-induced changes in isoform abundance in CD3/CD28 activated primary human CD4+ T cells. CD28 enhances various signaling events downstream of the TCR, however the contribution of CD28 signaling with splicing changes has not been investigated. Here we test the hypothesis that CD28 exerts some of its functional impact through the enhancement of splicing changes in T cells. We utilized poly(A) RNA-seq and the MAJIQ alternative splicing algorithm to analyze splicing events of human CD4+ T cells stimulated with CD3, with or without CD28. Consistent with previous studies, we identified approximately 400 and 1,000 significant splicing events induced by CD3/CD28 stimulation at 8 and 48 hours of culture, respectively. Alternative splicing changes induced by CD3 and CD3/CD28 stimuli are highly correlative, however, approximately 33% of alternative splicing changes are further enhanced with additional CD28 costimulation at 8 hours of culture.  This enhancement drops to 12% of alternative splicing events by 48 hours. Through further downstream analysis, we have found that a splicing event within Caspase-9 is regulated by CD28 costimulation and is validated to do so via RT-PCR. The splicing event within Caspase-9 led us to test if other splicing events within death signaling pathway genes are also regulated by splicing changes in activated T cells. After a survey of splicing events within over 10 signaling genes, Caspase-9, Caspase-1, Bim, and Bax are found to be heavily regulated by alternative splicing and display an increased abundance of the shorter isoform upon T cell stimulation. Furthermore, by utilizing the Crispr/Cas9 system in Jurkats, the shorter isoform of Caspase-9, Bim, and Bax promotes Jurkat T cell survival upon apoptosis induction. Therefore, the increased abundance of the shorter isoform of Caspase-9, Bim, and Bax may increase primary CD4+ T cell survival upon T cell activation.