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.
Bibliographical noteFunding Information:
We would like to thank Paul Hagerman (University of California, Davis) for parent FMR1 constructs, Jerry Pelletier (McGill University) for hippuristanol, Nicolas Charlet-Burguerand and Dan Southworth for helpful discussions, and Mike Sutton and Christian Althaus for help with rat primary neuron and use of their confocal microscope. We thank Nathan Blewett and Chase Weidmann for early work on luciferase reporter constructs and Trista Schagat for advice on reporter design. We thank Hank Paulson, Sami Barmada, and Fang He for reviewing the manuscript. We apologize to colleagues whose important work we could not cite due to manuscript size limitations. Funding for this work was provided by the NIH (K08NS069809 to P.K.T., R01NS086810 to P.K.T., F32NS089124 to M.G.K., F31NS090883 to C.M.R., T32GM007315 to K.M.G. and A.E.L.), the Veterans Health Administration (1I01BX001689 and 1I21BX001841 to P.K.T.), and the M-Cubed Initiative (University of Michigan).
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