Abstract
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-tRNAMeti. 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.
Highlights
Expansion of G4C2 repeats in the C9ORF72 gene is the most prevalent inherited form of amyotrophic lateral sclerosis and frontotemporal dementia
To identify cisfactors and trans-factors influencing the translation of G4C2 repeats in the context of the C9ORF72 gene, we used a construct containing 66 repeats that was shown to undergo repeat-associated non-AUG (RAN) translation in all three frames when expressed in cultured cells and in the mouse central nervous system[20,36]
A cell-free translation assay based on rabbit reticulocyte lysates (RRL) was developed to monitor RAN translation efficiency from C9ORF72 transcripts
Summary
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. G4C2 translation operates through a 5′–3′ cap-dependent scanning mechanism, requiring a CUG codon located upstream of the repeats and an initiator Met-tRNAMeti. 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 regulated by an upstream open reading frame (uORF) present in mis-spliced C9ORF72 transcripts. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating neurodegenerative disorders with a considerable clinical and pathological overlap, which is further substantiated by the discovery of. Expansion of a G4C2 hexanucleotide repeat in the first intron of the C9ORF72 gene is identified in.
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