Fig. 2: Two-color translation assay revealed translation frameshift frequencies between DPR frames of GGGGCC repeats.

a Schematic of the dual-color frameshift constructs. 24× HA-tag were placed before 70× (GGGGCC) repeats in frame with DPR1. The translation was initiated by a canonical AUG start codon before HA and terminated with a stop codon after GGGGCC repeats. 24× SunTag lacking AUG start codon was placed after 70× (GGGGCC) repeats in the frame of DPR2. 24× MBS was used for RNA visualization and 12× PBS was used to tether mRNA on the cell membrane for long-term tracking. b Expected protein products without or with translation frameshift. c Frameshift reporters used in this study. d Time-lapse images illustrating translation frameshift events for GR-to-GA reporter. Red (RNA): stdMCP-RFP; magenta (GR): HA-Fb-HaloTag; green (GA): SunTag-scFv-sfGFP. See also Supplementary Movie 5. e Fluorescence intensity traces of both HA-tag and SunTag on a single mRNA showing frameshift events (Corresponding to (d)). f Combined TLS tracks in two translation channels for GR-to-GA and GA-to-GR frameshift reporters. Magenta: HA-Fb-HaloTag indicating the translation of AUG-HA-DPR1; green: SunTag-scFv-sfGFP indicating the translation of DPR2-SunTag. g Percentage of translating mRNAs undergoing normal translation (only in HA-DPR1 frame, magenta), frameshift (both frames, orange), or RAN translation (only DPR2-SunTag frame, green). The number of mRNAs under specific state was divided by the total number of tracks to calculate the percentage: GR-to-GA (5 cells, 464 translation events); GA-to-GR (6 cells, 275 of translation events); GA-to-GP (6 cells, 359 of translation events).