Fig. 2: IMP1 binding to neuronal mRNAs correlates with both a rise in the mRNA level and, independently, an increased recruitment of IMP1 to a subset of sites.

a IMP1 iCLIP crosslinks (counts per millions) and RNAseq data (sequencing reads) are mapped onto genomic coordinates in NPCs and neurons for 4 exemplar target mRNAs. iCLIP replicates are shown in blue for neurons and in green for NPCs, while the merged signal is in red. 3’ untranslated region (3ʹUTR) and last exon are marked by a blue box and separated by an arrow, while line arrows indicate other intronic boundaries. In many of the RNA targets, the increase of IMP1 recruitment is not correlated with mRNA level. b Volcano plot of IMP1-bound peaks normalized by gene expression changes in NPCs vs neurons. Peaks with significantly higher signal in NPCs vs neurons, and vice versa are shown respectively in pink and orange. P values were calculated using a two-sided likelihood-ratio test (LRT). A threshold of 1 <Log2 Fold Change < −1 and adjusted p value < 0.05 was used to determine significance. For iCLIP, n = 3 independent iPSC lines for each condition (biological replicates); for NPCs, n = 2 technical replicates from 2 independent iPSC lines + 3 technical replicates from 1 independent iPSC line (biological replicates); for RNAseq n = 3 independent iPSC lines for each condition (biological replicates). The vertical dashed lines denote log2 FCs < −1 or >1, while the horizontal dashed line marks an adjusted p value < 0.05. c Relative expression of IMP1 over GAPDH at NPC and neuronal stages by RT-qPCR (RNA level, left) and WB (protein abundance, right). Data presented as boxplots, where the centre line is the median, limits are the interquartile range and whiskers correspond to 1.5 times the interquartile range. Outliers are not displayed for clarity. For the qPCR experiment n = 4 data points were obtained from independent iPSC lines (biological replicates), while for the WB experiment n = 6 data points (6 replicates, including 4 biological replicates (independent iPSC lines) in 2 experimental blocks). Values are normalized by the relative expression in the NPCs of the corresponding clones. P values were calculated using a two-sided Mann–Whitney U test and reported on the plot. d Left—representative image of βIII-tubulin (top) and skeletonization (bottom) used for the branching analysis. Approximated scale bar is 20 μm. Middle and right—number of triple and quadruple branches in neuronal processes normalized against branch length, in IMP1 siRNA (IMP1 KD) vs non-targeting siRNA control (CTRL). Data presented as boxplots where the centre line is the median, limits are the interquartile range and whiskers correspond to 1.5 times the interquartile range. Outliers are not displayed, for clarity. Data points represent different fields of view, n = 3 independent iPSC lines (biological replicates) in 3 independent experiments. P values were calculated using two-sided Mann–Whitney U test and reported on the plot. Source data are provided as a Source Data file.