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. 2019 Nov 20;8:e48718. doi: 10.7554/eLife.48718

Figure 2. Receptor-ribosome coupling is mRNA dependent and DCC and Nrp1 bind to specific RBPs and mRNAs.

(A) Relative 18S and 28S ribosomal RNA abundance after control (IgG) pulldown or receptors pulldowns with or without EDTA or RNase A/T1 treatments (two-way ANOVA with Bonferroni’s multiple comparisons test; three biological replicates; Bars indicate mean, error bars indicate standard deviation; ***p<0.0001). (B) Western blot analysis and quantification of ribosomal proteins after DCC and (C) Nrp1 pulldowns. (two-way ANOVA with Bonferroni’s multiple comparisons test; three biological replicates; Bars indicate mean, error bars indicate standard deviation; **p<0.01; ***p<0.0001). (D) Hierarchically-clustered heatmap of detected RBPs after DCC and Nrp1 pulldown. LFQ intensities are plotted for each IP-MS replicate. (E) Mander’s overlap coefficients analysed using dual immunohistochemistry of DCC and Staufen1 or hnRNPA2B1 in axonal growth cones (unpaired two-tailed t-test; three biological replicates; individual data points are shown, error bars indicate SEM; p=0.03913). (F) Mander’s overlap coefficients analysed using dual immunohistochemistry Nrp1 and Staufen1 or hnRNPA2B1 in axonal growth cones (unpaired two-tailed t-test; three biological replicates; individual data points are shown, error bars indicate SEM; p=0.00161). (G) Volcano plot showing differential expression analysis for DCC and Nrp1 pulldowns. (H) Enrichment analysis plot of known RBP targets of Staufen1 and hnRNPA2B1 detected in RNA-sequencing data after DCC and Nrp1 pulldown (individual data points are shown, error bars indicate standard deviation, Mann-Whitney test, Wilcoxon rank sum test DCC versus Nrp1; p=0.001511).

Figure 2—source data 1. Spreadsheet containing all Manders Overlap Coefficient values for each axonal growth cone in Figure 2E and F.
Figure 2—source data 2. Spreadsheet containing RNA-sequencing analysis of DCC and Nrp1 bound mRNAs and GO analysis of high abundant (FPKM >100) detected mRNAs for DCC and Nrp1.

Figure 2.

Figure 2—figure supplement 1. Polysome fractionation analysis, RNase sensitivity of Nrp1-Staufen1 interaction and additional RNA-seq analyses.

Figure 2—figure supplement 1.

(A) Control and (B) EDTA treated polysome fractions and Western blot showing the distribution of DCC and Nrp1 across fractions. (C) Relative quantification of DCC and Nrp1 protein levels in ribosome-free and ribosomal fractions for control and EDTA-treated samples (DCC control n = 2, DCC EDTA n = 2, Nrp1 control n = 2, Nrp1 EDTA n = 1; Bars indicate mean, errors bars indicate standard deviation). (D) UV absorbance profiles after sucrose density gradient fractionation for control and RNAseA/T1 treated lysates. (E) Western blot analysis and quantification of Staufen1 after Nrp1 pulldowns. (paired t-test; three biological replicates; bars indicate mean, error bars indicate standard deviation; p=0.0136). (F) Bioanalyzer gel analysis of RNA. (G) Distance matrix showing a high correlation between replicates and a distinct signature between samples. (H) Gene ontology enrichment plot of mRNAs after DCC or (I) Nrp1 pulldowns.