Extended Data Fig. 9: Disorder and charge analyses of proteins created by frameshifts in candidate proteins.

(a, c) Disorder analysis of HMGB1, HMGB3, FOXC1, FOXF1, MYOD1, RAX, RUNX1, CALR, FOXL2, PHOX2B, SOX2 and SQSTM1 wild type and frameshift mutant sequences using the PONDR algorithm. The PONDR scores for the wild type sequences are plotted with grey dashed line, the PONDR scores for the mutant sequences are plotted in red. The positions of the DNA binding domains (DBD) are highlighted with black bars and frameshift position is highlighted with red arrow. (b, d) Charge plots of wild type and mutant sequences. Note the increased positive charge in C-terminus of frameshift variants. Isoelectric points (pI) for the protein sequence following the frameshift position in wild type and mutant sequences are shown beside the charge plots. (e) Quantification of nucleolar enrichment of the indicated proteins in the FIB1-RFP co-expression experiments. Median is shown as a line within the boxplot, which spans from 25^th to 75^th percentiles. Whiskers depict a 1.5x interquartile range. *** P <10⁻³, **** P < 10⁻⁴ from two-tailed Welch’s t-test, n = number of nuclei examined per condition. (f) Representative images of U2OS cells co-expressing RFP-Fibrillarin and EGFP-tagged DVL1 proteins. Scale bar = 10 µm. (g) Relative fluorescence intensity of bleached EGFP-tagged DVL1 in U2OS cells before and after photobleaching with identical laser settings. Line: mean, lighter shade: ± SD. (h) Representative images of hiPSCs co-expressing RFP-Fibrillarin and EGFP-tagged DVL1 proteins. Note the nucleolar signal in the cells expressing Mutant EGFP-DVL1. Scale bar = 10 µm. (i) Quantification of nucleolar enrichment of DVL1 in the FIB1-RFP co-expression experiments in hiPSCs. Median is shown as a line within the boxplot, which spans from 25^th to 75^th percentiles. Whiskers depict a 1.5x interquartile range, **** P < 10⁻⁴, two-tailed Welch’s t-test, n = number of nuclei examined per condition.