Project Summary The genetic landscape of patient samples has illustrated a vast array of potential diagnostic and clinical biomarkers through profiling of chromosomal rearrangements and examining subsequent alterations in RNA abundance. However, genome-wide analyses have also discovered that there are discrepancies between transcript expression levels and corresponding protein abundance, highlighting the limited predictive power of bulk RNA-based prognostic strategies to assist in therapy. Moreover, there are an increasing number of studies depicting thousands of missed and unannotated peptides. These discoveries underscore the importance of understanding RNA regulation in the context of disease to showcase the complete landscape of genetic expression and target the phenotype of malignant cells. Our findings have noted that select mRNAs are translationally increased, independent of their transcriptional regulation, in response to disease progression, and can be studied to create novel therapeutic avenues unique per disease type. Mapping post-transcriptional regulation and the un-annotated proteome, including RNA localization and modifications to assist in translational regulation, however, remains poorly understood. As cells rely on the dynamic response of protein synthesis to quickly respond to environmental cues, this leaves an unexplored area for discovery that is required for cell survival and is promising for innovative therapies. We have identified essential signaling advantages activated to rewire RNA translation during initiation and for the maintenance of aggressive disease states. During these processes, ribosome initiation is altered and increased at non-canonical start sites, which can alter protein variants and produce micropeptides. Our research focuses on mechanistically determining how growth and adaptive signaling cascades interact by: 1) mapping the post-transcriptional aspects of gene expression missing from our current annotated genomes, focusing on alternative ribosome initiation, and 2) investigating the RNA-modification and binding proteins influencing selection and spatial interactions of RNA. This regulatory control rapidly reprograms the cellular phenotype at the level of RNA translation, increasing genetic diversity, and we will be able to characterize the global utilization of nonconventional initiation to study cell state changes modeled during cancer progression. Our multidisciplinary approach is designed to connect unbiased ribosome sequencing with proteomic profiles to identify the complete oncogenic proteome and the features that impose RNA selection. We will identify the trans- factors, cis- domains, and RNA modifications utilized by adaptive signaling to commandeer the translational response between cell states. Our studies will provide unprecedented insight into RNA regulation through focusing on localization and modifications alongside ribosome initiation, and how their interplay alters gene expression. This ...