Fig. 7.

A competition-based approach preferentially mislocalizes APC-dependent RNAs from protrusions. a Schematic of exogenously expressed constructs containing the β-globin coding sequence followed by the indicated 3′UTRs (β-globin UTR (HBB; spaced dashed line); Rab13 UTR (dashed line), Pkp4 UTR (solid line), or deletion fragments of Pkp4 UTR (PkpB, PkpA12, and PkpA12.1)). Representative in situ hybridization images detecting the β-globin RNA, and the ability of each construct to localize at protrusions are shown. Images focus on individual protrusions. Scale bar: 10 μm. Whole-cell images and PDI values are shown in Supplementary Fig. 7a, b. b Cell lines, stably expressing β-globin constructs with the indicated UTRs, were analyzed by in situ hybridization and RNA distributions were assessed through PDI quantitation. (Ddr2, Kank2: APC-dependent RNAs; Arpc3: control, diffuse RNA). N-values of observed cells, from at least 3 independent experiments, are indicated within each bar. Error bars: standard error. p-values: ***<0.001, **<0.01, *<0.05 by analysis of variance with Dunnett’s multiple comparisons test. c Pie chart of non-localized or Ps-enriched RNAs in control HBB cells from 4 replicate experiments. Cutoffs for Ps-enrichment were set at FC > 2 and p-value < 0.05. d Cumulative fraction plot of log2 fold-change differences between control HBB and Pkp4 cUTR-expressing cells, for RNAs non-localized or localized in protrusions of control HBB cells. e For all Ps-localized RNAs of control HBB cells, the log₂ differences in FC values, between HBB control and Pkp4 cUTR-expressing cells, were plotted against the corresponding p-values. Applying the cutoffs marked by the red lines (see Supplementary Data 3 for details), Ps-localized RNAs were distinguished into groups less- or equally-enriched in protrusions upon Pkp4 cUTR expression. f Categories of molecular functions, derived through IPA analysis, significantly represented in RNA groups equally- or less-enriched in protrusions of Pkp4 cUTR-expressing cells. g Results of differential enrichment analysis showing significance of overlap between APC-dependent RNAs and RNAs mislocalized upon Pkp4 cUTR expression