m⁶A modulates RAN translation from CAG repeat expansion RNA

Nucleotide repeat expansions contribute to the development of a number of neurodegenerative diseases. Recent studies revealed that DNA sequences with CAG and other nucleotide repeat expansions can undergo bidirectional transcription, and the ensuing transcripts could be translated into proteins through repeat-associated non-AUG (RAN) translation; however, not much is known about the precise mechanisms underlying RAN translation. Here, we demonstrated that m⁶A, installed by METTL3 and removed by FTO, promotes RAN translation in all three reading frames from the expanded CAG repeat RNA derived from the human ATXN3 gene, in which repeat expansion contributes to spinocerebellar ataxia type 3 (SCA3). Genetic depletion and pharmacological inhibition of METTL3 result in significantly diminished levels of RAN translation products from all three reading frames, which could be restored by ectopic expression of wild-type METTL3, but not its catalytically inactive mutant. Conversely, genetic ablation of FTO led to augmented RAN translation in all three reading frames. Moreover, one of the RAN translation products, poly(serine) exhibits gel-like aggregates in cells. Together, our study unveiled a crucial role of m⁶A in modulating RAN translation from expanded CAG repeat RNA transcribed from the human ATXN3 gene, and documented new biophysical properties of the poly(serine) RAN translation product.