Distinct Cellular Effects of Myotonic Dystrophy type 2 RAN Tetrapeptides in Drosophila melanogaster

Myotonic dystrophy type 2 (DM2) is an autosomal dominant, multisystemic disorder caused by the expansion of CCTG repeats in the first intron of the CNBP gene. Repeat-associated non-AUG (RAN) translation of the expanded CCTG RNA may produce two tetrapeptide repeat proteins (TPRs), poly-QAGR and poly-PACL, whose roles in DM2 pathogenesis remain poorly understood. To investigate their individual contributions, we expressed codon-optimized QAGR and PACL peptides with ATG start codon in Drosophila melanogaster. Expression of QAGR and PACL TPRs significantly compromised fly viability and lifespan, induces eye degeneration and locomotor defects. We found that QAGR accumulated in the nucleolus, disrupted nucleolar integrity, and compromised rRNA processing. Moreover, QAGR expression interfered with autophagy, leading to the accumulation of Atg8a- and Ref(2)P-positive aggregates. Genetic interaction studies showed that overexpression of Atg8a or Ref(2)P mitigated QAGR-induced eye-toxicity, while knockdown of autophagy genes exacerbated it. Conversely, PACL repeats promoted stress granule formation, as indicated by their colocalization with TIAR in human cells and their epistatic interaction with the Drosophila orthologue Rox8. Notably, cytoplasmic PACL aggregates were observed in myoblasts of DM2 patient. Together these findings demonstrate that QAGR and PACL peptides exert distinct toxic effects, impairing nucleolar function and autophagy, or altering stress granule dynamics, respectively. Both mechanisms likely converge to drive DM2 pathogenesis and represent promising therapeutic targets.