Fig 1: A subset of RBPs localizes to neurites. a Neurite-localized RBPs. Proteomic data from Fig. 1e were overlaid with available databases of mRNA-bound proteins48–51. Neurite-localized RBPs are highlighted in green (protein neurite/soma log2FC > 1, P-values < 0.05; see also Supplementary Data 2), the rest of RBPs are shown in blue. b Western blot validation for selected neurite-enriched RBPs. Histone H3 and TUBB3 were used as soma-enriched markers. c Manual annotation of neurite-enriched RBPs (protein neurite/soma log2FC > 1 and P-values < 0.05) for RNA-related functions. Number of proteins in a given category is indicated on the pie chart. Some RBPs were annotated to multiple GO categories. See also Supplementary Data 7. d Motifs found in mRNAs localized to neurites and locally translated. Motif discovery was done with MEME56 and enrichment calculations with MAST57. Fisher’s exact test was used to assess statistical significance of the association and its enrichment (odds ratio). Alignment of known RBP target sites70 (not restricted to neurite-localized RBPs identified in Fig. 6a) was performed using Tomtom60; only best hits are shown
Fig 2: Alternative polyadenylation sites usage and resulting alternative 3'UTRs affect mRNA localization to neurites. (A) Scheme of neurite/soma separation and 3' mRNA-seq. mESC-derived neurons are grown on a microporous membrane so that neurites extend on the lower side of the membrane to enable separation of the soma and neurites. RNA isolated from subcellular compartments is subjected to 3' mRNA-seq, which is based on oligodT priming and enables amplification and sequencing of polyadenylated mRNA 3' ends. The resulting data are analyzed for the ALE and APA isoforms. (B) Scatterplot illustrating differential localization patterns for transcripts with ALE 3'UTR isoforms. 3'UTR isoforms are designated as A and B on the condition that A is equally or more represented in neurites than B (see the scheme below the plot). Enrichment in neurites for isoform A (X) is plotted against the same enrichment for isoform B (Y). Dots falling on diagonal correspond to genes with similar localization patterns of alternative 3'UTR isoforms. For the rest of the genes, transcripts with alternative 3'UTRs show differential localization between neurites and soma. Coloring indicates local point density. (C) Plot illustrating differential localization patterns for transcripts with APA 3'UTR isoforms. Data are presented as in (B). (D) Distribution of short and long APA isoforms between neurites and soma. The top panel shows percentages of short (]-) and long (]—) APA isoforms, which are enriched in neurites, enriched in soma, or else equally distributed. The lower panel shows localization patterns of short and long APA isoforms deriving from the same gene. Neurites: enriched in neurites >2-fold; equal: <2-fold change between neurites and soma; soma: enriched in soma >2-fold. (E) qRT-PCR for selected differentially localized alternative 3'UTRs. Error bars represent SD for two (neurites) to three (soma) biological replicates. Gapdh (reference RNA), Thyn1, rRNA were used as unlocalized controls, Rbfox3, Slc18a2, Tubb3 as soma-localized, Mapkap2, Kif1c as neurite-localized controls. ENSEMBL identifiers of the isoforms: Cdc42E6 ENSMUST00000030417.9; Cdc42E7 ENSMUST00000051477.12; Kif1b somatic ENSMUST00000060537.12; Kif1b neuritic ENSMUST00000030806.5; Map4 somatic ENSMUST00000169851.7; Map4 neuritic ENSMUST00000035055.14. For a reference, we also show neurite/soma enrichment based on 3' mRNA-seq (purple bars) and RNA-seq data (blue bars, not isoform-specific) next to the qRT-PCR data (red bars). In case of APA isoforms, the qRT-PCR data are shown for a long isoform and both isoforms combined (all), as short APA isoforms cannot be distinguished from long APA isoforms by qPCR.
Fig 3: Segmentation analysis of meningeal innervation. (A) A representative image of meninges stained for ß-tubulin III (green), segmented for morphological analyses. Blue lines represent axons, and yellow filled circles represent endpoints (i.e., CGRP-releasing trigeminal fiber terminal endings). (B) No differences in the total innervation length were observed between the two genotypes (p = 0.73 by unpaired Student’s t-test; n = 10/experimental group). (C) Similarly, the number of trigeminal fiber terminal endings was comparable in WT and K14 meninges (p = 0.26). (D–F) Analysis of the region-specific meningeal innervation patterns. No genotype differences were observed in the percentage of Tubb3 + area (p = 0.22, WT n = 8; K14 n = 10) (D), the intensity of Tubb3 fluorescence signal (p = 0.75) (E), and in the Gaussian distribution of nerves (p = 0.31). Data are presented as mean ± SEM.
Fig 4: Expression of neuronal proteins in hiPSC-derived neurons. (A) Representative confocal images of hiPSC-derived neurons at 30–45 DIV showing expression of the neuronal markers TUBB3 and MAP2 and the astrocyte marker Glial Fibrillary Acidic Protein (GFAP). Scale bar 7.5 µm. (B) Synaptic proteins SV2A (green) and HOMER (red) co-localized at 30–45 DIV. Scale bar 7.5 µm. The rectangle frames are the regions of interest shown below at higher magnification.
Fig 5: Single-cell RNA-seq of iALF and iALFiP neurons at differentiation day 2. (A) Experimental outline. iALF and iALFiP cells were mixed with Tubb3:GFP-expressing iA cells for comparison. (B) UMAP visualization of iALF and iALFiP clustering. Clusters are shown in colors and numbered from 1 to 8. (C–G) Projection of specific genes in panel (B). (C) Transgene expression clearly separates iA (Tubb3:GFP+) from iALF and iALFiP. (D) The transgene expression levels corelate with neural differentiation states suggesting a differentiation cline across cells. (E,F) Both lines contain cells expressing serotoninergic genes but iALFiP represses Phox2b. (G) Projection of representative Cluster 4 (Figure 2D) on the single cell clusters. (C–G) Green = expression in iALF + Tubb3:GFP, Orange = iALFP + Tubb3:GFP.
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