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

• Published in: Molecular cell 2016-04, Vol.62 (2), p.314-322
• Main Authors: Kearse, Michael G., Green, Katelyn M., Krans, Amy, Rodriguez, Caitlin M., Linsalata, Alexander E., Goldstrohm, Aaron C., Todd, Peter K.
• Format: Article
• Language: English
• Subjects: Eukaryotic Initiation Factor-4E - genetics; Eukaryotic Initiation Factor-4E - metabolism; Fragile X Mental Retardation Protein - biosynthesis; Fragile X Mental Retardation Protein - genetics; Fragile X Syndrome - diagnosis; Fragile X Syndrome - genetics; Fragile X Syndrome - pathology; Frameshifting, Ribosomal; Genes, Reporter; Genetic Predisposition to Disease; HeLa Cells; Humans; Nerve Degeneration; Neurons - metabolism; Neurons - pathology; Open Reading Frames; Phenotype; Protein Biosynthesis; Ribosomes - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Transcription Initiation Site; Transfection; Trinucleotide Repeat Expansion; Trinucleotide Repeats
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2016.02.034

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. [Display omitted] •Expanded repeats stimulate CGG repeat-associated non-AUG (RAN) translation•CGG RAN translation shares initiation requirements with canonical initiation•RAN translation displays differential start codon selection across reading frames•Sequence context and repeat codon influence CGG RAN translation dynamics 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.