PROJECT SUMMARY/ABSTRACT More than 15 years after the completion of the Human Genome Project, our understanding of the “annotated” genome is still incomplete. Alternative ribosome initiation sites that are not part of the “annotated genome” encode novel open reading frames (ORFs) that are either variants of annotated proteins, distinct ORFs upstream or downstream of annotated proteins, or even ORFs on long non-coding RNAs. These novel ORFs are emerging as a translational control mechanism to rapidly reprogram specific genes and protein synthesis networks, especially during stress and cell-state transformations such as tumorigenesis. Furthermore, these new ORFs encode putative peptides that remain uncharacterized. Thus, understanding the translational control mechanisms, as well as the regulatory functions of the encoded peptides, could reveal fundamental biology and targets for therapeutics. Existing studies of alternative initiation or alternative ORF-encoded peptides have taken an ad hoc approach, owing to a lack of tools to profile them at genome-wide scale. Our goal is to develop and apply deep sequencing and high-throughput, CRISPR-based methods to map the functional roles of these nonconventional translation transcriptome-wide, using tumorigenesis as a model system. Functional genomic approaches are uniquely poised to address the deficit in our understanding of functional alternative ORFs. In this proposal we will aim to characterize the global utilization of nonconventional initiation sites during tumorigenesis using ribosome profiling and RNA-seq at various time points (Aim 1). This will define novel ORFs that are actively translated, and how the translation of these ORFs are regulated to promote expression of oncogenic genes and peptides. Then, we will use CRISPR screens to identify ORFs necessary for tumor growth, and define the functions of the novel peptides by characterizing localization, physical interactions, and genetic interactions (Aim 2). Finally, we will mechanistically interrogate alternative start site usage to investigate how translation is tuned during cell-state changes (Aim 3). Overall, the results from the proposal will address long- standing questions about translational control, and reveal the regulatory roles of novel proteins. The combination of mentored support, skills, and data obtained in the K99 phase will provide Dr. Chen a springboard to achieving independence as an investigator in the R00 phase and beyond. The results of our studies will provide new insights into fundamental aspects of translational control, and will define new paradigms relevant to biology and disease.