Figure 1.

The FMRpolyG protein itself causes aggregate formation in several cell lines. (A) Schematic representation of the wtHP-99Gly-GFP and mutHP-90Gly-GFP constructs used in this study. Both contain the part of the FMR1 5′UTR that encodes the entire FMRpolyG protein (shown in blue). The wtHP-99Gly-GFP construct gives rise to both a CGG RNA hairpin and the FMRpolyGlycine protein. In mutHP-90Gly-GFP, the CGGs have been substituted with alternative glycine codons to abolish hairpin formation. (B) Confocal images of GFP-positive aggregates in the indicated cell lines, transfected with wtHP-99Gly-GFP or mutHP-90Gly-GFP and analyzed 24–48 h after transfection. (C,D) Quantification of FMRpolyG-GFP aggregates in HEK293 cells 24 h after transfection, presented as number of aggregates per 100 cells (C) or 100 GFP-positive cells (D). Untransfected cells and cells transfected with GFP-C1 were used as negative controls. Small aggregates range from 1 to 3 μm² in size, while large aggregates are those over 3 μm². Calculations are based on three individual experiments, each including >4,500 cells for each plasmid. Error bars represent standard deviation (SD).