Fig. 5: Impact of effector interactions on RNA binding by Unkempt.
From: Regulation by the RNA-binding protein Unkempt at its effector interface
A, B Altered RNA sequence recognition by UNK mutants. A Heatmaps illustrate positional frequencies of the 64 possible trimers within UNK-binding sites between 15 nts upstream and downstream of the binding-site maxima for UNKWT, UNKdPAM2, and UNK3M. Plots above the heatmaps profile the mean enrichment of different sets of trimers, considering the upstream UAG trimer (brown), the downstream U/A-rich trimers (orange), and all other trimers (gray; see “Methods” section). B Overlays of the mean enrichment profiles for each set of trimers shown in (A). C Density scatterplots comparing crosslink events per peak pairwise among the combined iCLIP replicates (n = 4) for UNKWT, UNKdPAM2, and UNK3M. D Normalized UNKWT, UNKdPAM2, and UNK3M iCLIP coverage tracks for three UNK target transcripts showing mutant-specific reduced binding strength at UAG-containing binding sites and the emergence of UAG-less satellite peaks. Arrowheads indicate positions of all UAG motifs; positions of UAG motifs immediately upstream of UNKWT binding sites are highlighted by dashed vertical lines. Arrows point to UAG-less satellite peaks. E Transcriptome-wide occurrence of the UAG trimer within 15 nts upstream of WT-specific (WT-3M and WT-dPAM2) or mutant-specific peaks (3M–WT and dPAM2-WT). Data are presented as mean ± SD. Statistical significance for the indicated comparisons considering four iCLIP replicates per condition was determined using Student’s two-tailed t-test. F Proportional metatranscript analysis of iCLIP data showing the positional frequency of crosslink events for UNKWT, UNKdPAM2, and UNK3M on different segments of mRNA. Data points represent normalized crosslink events summarized over every percent of a given mRNA segment. Gray data points show total counts of the UAG trimer. Note the 5′-shift of RNA binding into coding sequences (CDS) by UNK3M and especially by UNKdPAM2 despite the relative depletion of UAG in this mRNA segment. See “Methods” section for determination of statistical significance for pairwise comparisons in (B) and (F).
