(a) Left panel: Schematic view of the MT bench technology. A GFP-labeled RBP fused to MBD (Microtubule-Binding domain, yellow) was brought to microtubules in U2OS cells to attract endogenous mRNAs (in red) on the microtubule network (grey). Middle panel: Image of a 96-well plates seeded with U2OS cells. Right panel: Image of a single well processed by HCS imager showing the expression of MDP-GFP-YB-1 in U2OS cells (green). (b) U2OS cells expressing MBD-GFP-YB-1 (bait in green, GFP). mRNAs in red (in situ hybridization, poly(dT) probe). Nuclei in blue (DAPI). The images were obtained with an HCS imager (40 x, water immersed objective operating in confocal mode). (i) DAPI and the red channel (mRNA) were used to detect automatically the nuclei and cytoplasm, respectively. (ii) Using HARMONY “find spots” procedure, elongated spots along the microtubules were detected using the green channel (the bait, RBP). Spots were selected owing to their width-to-length ratio (<0.22) and their enrichment in GFP (YB-1). Scale bar: 20 μm. (c) Left panels: The enrichment of mRNAs in single selected spots (spot/cytoplasm intensity ratio, red channel) and spot bait intensity on microtubules (green channel) show a linear relationship when YB-1 was used as bait. The slope of the regression line reflects the affinity of an RBP for mRNAs. A large number of cells can be analyzed by HCS (>500 cells well with in average 10–50 spots per cell). Slopes from linear regression were measured for each well with a 95% confidence interval. Right panel: SSMD value estimated by measuring the normalized slopes in 48 negative controls (MBD-GFP used as bait) and 48 positive controls (YB-1 was used as bait). The SSMD value is 8.1 for a 96-well plate. Spot data from all wells are shown in Figure 3—figure supplement 2.3a. (d) Bar diagram representing the enrichments of 13 different mRNAs measured by RT-PCR after two different purification procedures, co-sedimentation (MT pellet) and immunoprecipitation (Beads), and for 2 different RBPs, YB-1 and HuR; the purification procedures are illustrated in Figure 3—figure supplement 2.3, data and correlation analysis are provided in Appendix 5—table 5 for 3 RBPs (YB-1, HuR, and FUS). (continued).