Expansion of a hexanucleotide GGGGCC repeat within the first intron of the C9orf72 gene is the primary monogenic cause for both Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). Despite its intronic location, expanded GGGGCC repeat is retained in the mature mRNA, supporting a non-canonical translation initiation mechanism known as repeat- associated non-AUG (RAN) translation. RAN translation of C9orf72 mRNA generates toxic dipeptide repeat (DPR) proteins. These DPR proteins aggregate in the brains of affected individuals and have been shown to induce neurodegeneration in model systems. Despite the medical and biological importance of RAN translation, a fundamental question remains unanswered: how does RAN translation initiate? To address this question, this proposal will use cryo-electron microscopy along with biochemical and genetic approaches to determine how expanded GGGGCC repeats physically interact with the ribosome to induce this spurious start site selection in ways that generate DPR proteins. Simultaneously, we will determine the role of protein factors that may bind to the translation initiation complex in neurons and promote RAN translation of C9orf72 mRNA. The innovative approach outlined in this proposal will allow us to address some fundamental questions about mRNA translation initiation in the native environment, specifically identifying protein factors that bind transiently to the ribosomal complex with low affinity and their role in the regulation of RAN translation. Moreover, this proposal will provide a novel tool for studying various neurodegenerative diseases and neuronal functions associated with aberrant mRNA translation. Taken together, our work will provide insights into the molecular mechanism underlying RAN translation initiation while providing insights into the contribution of this aberrant translation to ALS and FTD.