Figure 4: Association of NCBP3 and NCBP2 with NCBP1 and proteins involved in RNA processing.
From: mRNA export through an additional cap-binding complex consisting of NCBP1 and NCBP3
(a) GFP-tagged NCBP3, NCBP2 or control protein (ctrl) stably expressed in HeLa cells were precipitated and associated proteins were analysed by LC–MS/MS. Volcano plots show the average degrees of enrichment by NCBP3 or NCBP2 over ctrl (ratio of LFQ protein intensities; x axis) and P values (t-test; y axis) for each protein. Bait proteins (red letters), proteins of the exon junction complex (EJC, turquoise dots) or the TREX complex (purple dots), CBC (magenta dots), NELF (olive dots), importin (green dots) and NCBP3, SRRT and PHAX (red dots) are highlighted. Significantly enriched proteins are separated from the background by a hyperbolic curve. Four independent affinity purifications were performed for each bait. (b) Schematic illustration of AP–MS analysis in a focusing on proteins involved in RNA biogenesis. (c) Western blot analysis of representative GFP-precipitated proteins identified by AP–MS analysis. (d) Binding of NCBP1 to NCBP3 in E. coli. Western blot after His-precipitation using RNAse-treated lysates from bacteria co-expressing recombinant SII-tagged NCBP1 and full-length MBP/His-tagged NCBP3 or MBP/His as control. (e) NCBP2 and NCBP3 are required for NCBP1 association to m7GTP. Lysates of HeLa cells treated twice with siRNA against NCBP2, NCBP3 or both were used for m7GTP-affinity purification (m7GTP-AP), followed by western blot analysis. The m7GTP-binding protein EIF4E served as control.
