Figure 2. Analysis of Single-Mutant Variant Binding to PUM2.
(A) Top: color code for the scaffolds in Figure 1B; the arrow points to affinities for each position 1 sequence variant. Bottom: KD values of PUM2 for single mutants at each position of the UGUAUAUA consensus. Bars indicate weighted means of two replicate measurements and error bars indicate weighted replicate errors. The dashed line indicates the average affinity for the consensus sequence across the four scaffolds, and the consensus residues are circled. Asterisks indicate variants with significant differences between scaffolds (10% FDR).
(B) Scaffold variance before and after accounting for RNA secondary structure and after excluding sequences with predicted structure. The bars indicate standard deviations of the distribution of differences between each measured value (part A) and the scaffold mean for the respective sequence variant; see also Figure S2A and STAR Methods. Dashed lines indicate the standard deviation of measurement error. The experimental standard deviation was higher at 37°C than 25°C because of weaker binding and the absence of an independent duplicate experiment.
(C) Model for RNA structure effects on PUM2 binding. Occluded RNA molecules increase the observed dissociation constant (weaken binding) by stabilizing the unbound state (see also Figure S2B and STAR Methods).
(D) Single-mutant affinities after accounting for structure effects predicted by RNAfold (solid bars; Lorenz et al., 2011); the transparent region indicates the structure correction. Error bars indicate weighted replicate errors. Asterisks indicate variants with significant scaffold differences after accounting for structure effects.
(E) Median effects of each single mutation (residues 1–8) across scaffolds and across 5A/C/U backgrounds at 25°C, after excluding variants with alternative binding registers and after accounting for structure. Error bars indicate 95% CIs of the median. Mutational effects were calculated relative to the weighted mean affinity for the UGUA[A/C/U]AUA consensus across scaffolds. Position 9 specificity was derived as shown in Figure S2F and the mutational effect was calculated relative to the most tightly bound residue (G).
(F) Comparison of single-mutant affinities measured by RNA-MaP (Figure 2E) and by gel shift. 1C, purple; 2A, yellow; 2C, green; 3A, white; 3G, red; 4G, orange; 4U, blue; 5G, wheat; 7C, brown; 7G, magenta; 9A, lime; 9C, cyan; 9U, gray. The gel-shift values are averages and 95% CIs from two to four measurements.
See also Figures S2 and S3.