RNA regulates repeat-associated non-AUG (RAN) translation initiation in C9orf72 FTD/ALS

Repeat-associated non-AUG (RAN) translation synthesizes protein in the absence of a cognate AUG start codon ^1,2 In frontotemporal dementia and amyotrophic lateral sclerosis, a GGGGCC (G ₄ C ₂ ) repeat expansion in an intron of C9orf72 leads to synthesis of neurotoxic dipeptide-repeat proteins, underscoring the need to understand the mechanism of C9orf72 RAN translation ^1-5 . RNA sequence and structure have been implicated, but how they direct C9orf72 RAN translation, particularly the rate-limiting, multi-step initiation phase, remains unclear ^6-10 . We applied single-molecule biophysics to a reconstituted human translation initiation system and tracked fluorescently labeled ribosomes and initiation factors in real time. We show that RNA G ₄ C ₂ repeats and sequence context alter initiation factor dynamics after ribosomal scanning, generating a kinetic bottleneck in the commitment to initiate at a near-cognate CUG start codon. Our model of C9orf72 RAN translation provides a mechanistic framework for how repeat expansions change underlying translation dynamics and may be broadly relevant to other disorders that involve RAN translation.