Project Summary/Abstract Cells and organisms, from simple to complex, carry the same genetic DNA sequence organized into genes. Multicellular eukaryotes transcribe and process genes into RNA isoforms through a process called alternative splicing. Alternative splicing is developmentally and cell-type specifically regulated. It is foundational to how higher organisms’ genomes are decoded. Yet, critical and fundamental questions regarding its regulation and the function of its output remain unanswered. For example, circRNA being a ubiquitous product of alternative splicing was only discovered in 2012, and its regulation and function remains enigmatic. circRNAs’ discovery revealed a larger critical knowledge gap in the field for “what, how and why” genes are alternatively spliced. What RNA splice variants are expressed, how splicing is regulated and which spliced RNAs have essential functions? Answering these questions is critical for predicting which of myriad genetic variants cause disease and why they do so. Answers will also enable a new generation of digital nucleic acid biomarkers and diagnostics for disease, drug targets for correcting dysregulated splicing and identification of pathogenic protein- or non-coding products (respectively) as well as fundamental basic scientific insight into evolution and function of eukaryotic genomes. The proposed research will couple novel statistical analyses of -omics data by taking an unbiased approach and including biological features that are understudied or un-annotated. Predictions will be coupled with incisive experimental validation to reveal new principles of how RNAs, including circRNAs, are spliced and how they function. This research potentiates significant new discoveries in why alternative splicing exists and how this understanding can be used for precision medicine.