Investigating how splicing factor homeostasis shapes transcriptomes in pluripotency and differentiation - PROJECT SUMMARY Splicing factors (SFs) are RNA-binding proteins that regulate alternative splicing (AS), enabling a single gene to produce a variety of mRNA transcripts and corresponding proteins. AS plays an integral role in development, cancer, and aging, and many SFs are essential for embryonic development. Therefore, SF levels must be tightly controlled to maintain proper gene expression, which can be achieved through the AS of poison exons (PEs) within their own transcripts. PEs within SF transcripts, or SF-PEs, introduce premature termination codons, triggering nonsense-mediated decay (NMD) to reduce SF protein levels, a process known as AS- NMD. Conversely, PE skipping increases SF abundance. Prior studies highlight SF-PEs as critical for cancer cell survival, but their role in non-cancerous cells remains unclear. The goal of this proposal is to determine how SF-PEs maintain SF homeostasis to modulate transcriptomes that sustain pluripotency and differentiation. Our preliminary data suggest that PEs in Srsf3 and Tra2b, two SFs linked to cancer and developmental disease, are essential for pluripotent stem cell survival and embryonic viability. However, the morphological, functional, and transcriptomic effects of PE knockout remain unclear, as does the broader role of SF-PEs in pluripotent stem cell survival. We hypothesize that SF PEs fine-tune pluripotency by buffering SF gene expression and modulating AS of target genes critical for maintaining pluripotent cell viability. Aim 1 will utilize an in vivo reverse genetics approach and long-read RNA sequencing (LR-seq) to characterize how Srsf3- and Tra2b-PEs shape mouse embryonic development. Aim 2 will investigate SF AS-NMD dynamics in vitro using a high-throughput CRISPR-based exon deletion screen to identify SF-PEs essential for iPSC viability. Conditional knockout iPSC models will be engineered to assess effects of SF-PE knockout on transcriptomes using LR-seq, SF target binding using eCLIP, and differentiation phenotypes using functional assays. Successful completion of these Aims will elucidate how SF-PEs modulate transcriptomes, safeguard cell pluripotency, and drive differentiation. This Fellowship will provide me essential training in RNA splicing, stem cell biology, genomics, and scientific communication—critical for my future career as a physician-scientist translating basic research into clinical applications.
