CGG Repeat-Associated Non-AUG Translation Utilizes a Cap-Dependent Scanning Mechanism of Initiation to Produce Toxic Proteins

Repeat-associated non-AUG (RAN) translation produces toxic polypeptides from nucleotide repeat expansions in the absence of an AUG start codon and contributes to neurodegenerative disorders such as ALS and fragile X-associated tremor/ataxia syndrome. How RAN translation occurs is unknown. Here we define the critical sequence and initiation factors that mediate CGG repeat RAN translation in the 5' leader of fragile X mRNA, FMR1. Our results reveal that CGG RAN translation is 30%-40% as efficient as AUG-initiated translation, is m⁷G cap and eIF4E dependent, requires the eIF4A helicase, and is strongly influenced by repeat length. However, it displays a dichotomous requirement for initiation site selection between reading frames, with initiation in the +1 frame, but not the +2 frame, occurring at near-cognate start codons upstream of the repeat. These data support a model in which RAN translation at CGG repeats uses cap-dependent ribosomal scanning, yet bypasses normal requirements for start codon selection. AUG-independent translation of expanded nucleotide repeats produces toxic neuronal proteins. Kearse et al. show that RAN translation of CGG repeats initiates similarly to canonical translation, requiring an m⁷G cap and 40S ribosomal scanning. However, initiation codon selection differs across repeat reading frames and with repeat expansion.