Reconstitution of C9orf72 (GGGGCC) repeat-associated non-AUG translation with purified components.
Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is the most common genetic cause underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonical process known as repeat-associated non-AUG (RAN) translation. In order to ascertain the essential components required for RAN translation, we successfully recapitulated G4C2-RAN translation using an in vitro reconstituted translation system comprising human factors, namely the human PURE system. Our findings conclusively demonstrate that the presence of fundamental translation factors is sufficient to mediate the elongation from the G4C2 repeat. Additionally, we observed ribosomal frameshifting from the poly Gly-Ala dipeptide frame to other frames during the elongation process. Furthermore, the initiation mechanism proceeded in a 5 cap-dependent manner, independent of eIF2A or eIF2D. In contrast to cell lysate-mediated RAN translation, where longer G4C2 repeats enhanced translation, we discovered that the expansion of the G4C2 repeats inhibited translation elongation using the human PURE system. These results suggest that the repeat RNA itself functions as a repressor of RAN translation. Taken together, our utilization of a reconstituted RAN translation system employing minimal factors represents a distinctive and potent approach for elucidating the intricacies underlying RAN translation mechanism. Significance statementEvery cellular protein undergoes synthesis through a process known as translation. While the fundamental aspects of translation have been established, recent advancements have unveiled various noncanonical translation pathways, including the translation originating from "noncoding" RNAs. Within this context, certain neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), are linked to the translation of noncoding RNAs, referred to as repeat-associated non-AUG (RAN) translation, the underlying mechanism of which remains controversial. To dissect the complicated nature of RAN translation, this study employs a reconstituted cell-free translation system comprised of human translation factors. By reconstituting RAN translation utilizing a minimal set of factors, this bottom-up approach not only facilitates the elucidation of its mechanism but also offers a distinctive avenue for pharmaceutical development.