PROJECT SUMMARY Widespread aberrant splicing is now considered a hallmark of cancer, and many of the resulting transcript isoforms have been functionally implicated in tumorigenesis. However, the underlying regulatory pathways that govern RNA splicing and the extent to which they play a role in cancer progression remain largely unknown. Due to our limited knowledge of splicing regulation, we cannot solely rely on annotated pathways to study changes in transcript isoforms. Moreover, bioinformatic strategies that are often used in discovering transcriptional regulatory pathways often fail to capture the complexities of post-transcriptional regulation. For example, common methodologies for cis-element discovery focus on sequence alone and largely ignore the integral role of the RNA structural code in splicing. To address this challenge, we have developed a computational framework that performs context-aware analysis of alternative splicing events to identify known and novel regulators of RNA processing. Using this approach, we have discovered and partly characterized a previously unknown RNA structural splicing enhancer (SSE) that drives aberrant splicing in highly metastatic breast cancer. We have identified the associated RNA-binding protein that serves as the trans factor that interacts with this novel SSE. We have shown that this pathway drives metastatic progression in xenograft mouse models and that its activity is strongly associated with poor survival in patients. In this proposal, we will build on this previous work to (i) perform a detailed dissection of this non-canonical regulatory pathway of alternative splicing, its underlying molecular machinery, and its governing RNA structural code; (ii) evaluate the contribution of this pathway to breast cancer progression; and (iii) establish the clinical relevance of this pathway and its regulon to cancer.