The Role of Alternative Splicing and Alternative Polyadenylation in Regulating Activated T cell Biology
Genetics and Genomics
Alternative splicing and alternative polyadenylation are now recognized to occur in the vast majority of human genes. Both have the potential to alter the identity of the encoded protein, as well as control protein abundance, protein localization, or association with other factors. T cells provide a unique model to study RNA processing events, such as alternative splicing and polyadenylation, due to the dynamic and robust changes in isoform usage induced upon activation. However, detailed characterization of the regulation of RNA processing and its impact on biological consequences on T cells remain limited. The major goal of this thesis is to further elucidate the regulation of global alternative splicing and polyadenylation changes induced upon activation, and to exemplify their biological consequences on cell survival mechanisms. I created a carefully controlled dataset comprising of human naive CD4+ T cells stimulated with anti-CD28, anti-CD3, and anti-CD3/CD28 for 8 and 48hrs, to further elucidate the signal-specific regulation of alternative splicing and alternative polyadenylation changes upon stimulation. I show that splicing and polyadenylation changes that occur between early (8hrs) and late (48hrs) T cell activation regulate distinct sets of genes that are enriched for differential biological functions and display minimal overlap with genes regulated through differential expression. In addition, a small subset of alternative splicing and polyadenylation events, ~10-30%, are regulated by CD28 costimulation. Through the creation of CRISPR clones and AMO transfection experiments, I excitingly found isoforms of Caspase-9, Bim, and Bax, that increase in abundance upon T cell stimulation and work in an additive manner to promote T cell survival. Finally, preliminary analysis indicates that alternative polyadenylation may regulate T cell survival by controlling cFLIP levels through promoting proximal PAS usage within the 3'UTR tail. In summary, this dissertation demonstrates broad alternative splicing and polyadenylation changes are fine-tuned to specific T cell stimuli and exert biological impact on cell survival signaling.