Upstream non-AUG initiation and ribosomal -1 frameshifting in ATXN8OS CTA/CTG repeat-associated translation

Microsatellite repeat expansions contribute to the pathogenesis of many neurodegenerative disorders. In spinocerebellar ataxia type 8 (SCA8), abnormal expansion of CTA/CTG repeats in the 3' untranslated region of the ATXN8OS (ATXN8 Opposite Strand) gene has been implicated in disease pathology. Although the occurrence of repeat-associated non-AUG (RAN) translation from the ATXN8 transcript has been reported, whether and how RAN translation occurs from the ATXN8OS transcript has remained unexplored. Here, using a cell-free translation system and cultured cells, we showed that ATXN8OS undergoes robust AUG-independent translation in a repeat length-dependent manner. Mechanistic analyses revealed that translation of the poly L (0) frame initiates at a non-AUG codon located upstream of the repeats. Moreover, using live-cell imaging at a single messenger RNA level, we directly visualized ribosomal -1 frameshifting from the poly L (0) frame to the poly T-poly A (+2) frame during translation elongation. We further showed that ATXN8OS translation was enhanced upon activation of the integrated stress response. Together, these findings establish both the occurrence and the molecular mechanisms of ATXN8OS translation from expanded CTA/CTG repeats and provide insights into the pathogenic processes underlying SCA8.