Fig. 5

MATR3 overexpression mitigates DPR production from G4C2₆₀ transcripts in an RNA-dependent manner. a Representative confocal images of HEK293T cells co-transfected with G4C2₆₀-Dendra2 RAN translation reporter plasmid and either FLAG (empty vector), FLAG-MATR3 or FLAG-MATR3-ΔRRM2 plasmids. G4C2₆₀ expression resulted in formation of G4C2 RNA foci (red) and, further, the G4C2₆₀ transcripts also undergo RAN translation in a subset of cells to produce dipeptide repeats (DPRs), indicated by Dendra2 signal (green) that is in frame with polyGR (white arrows). b Quantification of the percentage of cells that produce GR-Dendra2 (green) RAN product showed that overexpression of FLAG-MATR3 (gray) significantly reduced RAN translation of G4C2₆₀ transcript to GR-Dendra2 RAN product. Overexpression of FLAG-MATR3-ΔRRM2 did not impact GR-Dendra2 production in these cells (One-way ANOVA) n = 15-18 per group. c Quantification of G4C2₆₀-Dendra2 mRNA levels in cells expressing FLAG (empty vector) or FLAG-MATR3 showed no significant difference (One-way ANOVA) n = 5 per group. d Schematic of previously-published codon-optimized approach to produce dipeptide protein repeats by canonical translation of non-repeat RNA (i.e. not carrying GGGGCC repeats). e Representative images of Drosophila eyes and f quantification of external eye degeneration from flies expressing codon-optimized DPRs: GR36, PR36, GA36 and GP36. Flies expressing PR36 and GR36 developed significant external eye degeneration that is more severe is GR36 compared to PR36. Flies expressing either GA36 or GP36 did not show any external eye degeneration. Expression of MATR3 in these flies did not modify the phenotypes (Kruskal–Wallis test) n ≥ 50 per genotype. Error bars indicate S.E.M. **p value < 0.01