Dissecting the mechanism of NOP56 GGCCUG repeat-associated non-AUG translation using cell-free translation systems

The repeat expansion in the human genome contributes to neurodegenerative disorders such as spinocerebellar ataxia (SCA) and amyotrophic lateral sclerosis. Transcripts with repeat expansions undergo noncanonical translation called repeat-associated non-AUG (RAN) translation. The NOP56 gene, implicated in SCA36, contains a GGCCTG repeat in its first intron. In tissues of patients with SCA36, poly (Gly-Pro) and poly (Pro-Arg) peptides, likely produced through NOP56 RAN translation in (NOP56-RAN), have been detected. However, the detailed mechanism underlying NOP56-RAN remains unclear. To address this, we used cell-free translation systems to investigate the mechanism of NOP56-RAN and identified the following features. (i) Translation occurs in all reading frames of the sense strand of NOP56 intron 1. (ii) Translation is initiated in a 5' cap-dependent manner from near-cognate start codons upstream of the GGCCUG repeat in each frame. (iii) Longer GGCCUG repeats enhance NOP56-RAN. (iv) A frameshift occurs within the GGCCUG repeat. These findings provide insights into the similarities between NOP56-RAN and other types of RAN translation.