CUG initiation and frameshifting enable production of dipeptide repeat proteins from ALS/FTD C9ORF72 transcripts

Expansion of G₄C₂ repeats in the C9ORF72 gene is the most prevalent inherited form of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded transcripts undergo repeat-associated non-AUG (RAN) translation producing dipeptide repeat proteins from all reading frames. We determined cis-factors and trans-factors influencing translation of the human C9ORF72 transcripts. G₄C₂ translation operates through a 5'-3' cap-dependent scanning mechanism, requiring a CUG codon located upstream of the repeats and an initiator Met-tRNA^Met_i. Production of poly-GA, poly-GP, and poly-GR proteins from the three frames is influenced by mutation of the same CUG start codon supporting a frameshifting mechanism. RAN translation is also regulated by an upstream open reading frame (uORF) present in mis-spliced C9ORF72 transcripts. Inhibitors of the pre-initiation ribosomal complex and RNA antisense oligonucleotides selectively targeting the 5'-flanking G₄C₂ sequence block ribosomal scanning and prevent translation. Finally, we identified an unexpected affinity of expanded transcripts for the ribosomal subunits independently from translation.