Fig 2: co-IP of proteins with anti-eIF3g antibody in the nuclear fraction of Bcap37/Tet-on-eIF3g cells. The nuclear protein (200 µg) was incubated with the 5 µg anti-eIF3g antibody and 50 µl protein A/G PLUS-Agarose. Bands that were clearly present and with clear edges for excision in the co-IP sample but absent in the control sample (bands 1, 2, 3 and 4) were selected for protein characterization by mass spectrometry analysis. co-IP, co-immunoprecipitation; eIF3g, eukaryotic translation initiation factor 3 subunit g.

Fig 3: Nuclear localization of eIF3g. (A) Results of fractionation of the-Bcap37/Tet-on-eIF3g cells. GAPDH was used as the cytoplasmic marker and Histone H4 was used as the nuclear marker. The proteins samples were subjected to 12% SDS-PAGE, transferred onto a polyvinylidene fluo-ride membrane and probed by specific antibodies. (B) Immunofluorescent staining was performed. Confocal microscope images for eIF3g are shown (magnification, ×100). eIF3g, eukaryotic translation initiation factor 3 subunit g; W, whole cell lysate; C, cytoplasmic fraction; N, nuclear fraction.

Fig 4: eIF3g interacts with hnRNP U, HSZFP36 and β-actin. (A) Nuclear extracts of Bcap37/Tet-on-eIF3g cells were subjected to immunoprecipitation with anti-eIF3g (5 µg), anti-hnRNP U (5 µg), anti-HSZFP36 (5 µg) and anti-β-actin (5 µg) antibodies, respectively. Precipitated proteins were resolved by 10% SDS-PAGE and analyzed by western blotting with anti-eIF3g antibody. Arrowheads indicate eIF3g. (B) Anti-eIF3g antibody-immunoprecipitated proteins were analyzed by western blotting with anti-hnRNP U antibody. Arrowheads indicate hnRNP U. (C) Anti-eIF3g (5 µg) and anti-hnRNP U (5 µg) antibody-immunoprecipitated proteins were analyzed by western blotting with anti-HSZFP36 antibody. Arrowheads indicate HSZFP36. (D) Anti-eIF3g (5 µg) and anti-β-actin (5 µg) antibody-immunoprecipitated proteins were analyzed by western blotting with anti-β-actin antibody. Arrowheads indicate β-actin. (E) Results of western blotting from the in vitro crosslinking. The nuclear extract from Bcap37/Tet-on-eIF3g cells was crosslinked by DSS, subject ed to 8% SDS-PAGE and electrotransferred onto a polyvinylidene fluoride membrane, followed by incubation with antibodies specific for eIF3g, hnRNP U, HSZFP36 and β-actin. Arrowheads indicate larger complex bands; arrows indicate smaller complex bands. (F) GST-pulldown results. GST or GST-eIF3g proteins were incubated with nuclear lysates of Bcap37 cells and glutathione beads; bound proteins were resolved by 10% SDS-PAGE and detected by western blotting. hnRNP U, HSZFP36 and β-actin were pulled down by GST-eIF3g, with GST protein and GSH beads alone used as negative controls. Arrowheads indicate eIF3g in the Bcap37 cells; arrows indicate the GST-eIF3g protein band. eIF3g, eukaryotic translation initiation factor 3 subunit g; hnRNP U, heterogeneous nuclear ribonucleoprotein U; GST, glutathione S-transferase; GSH, glutathione.

Fig 5: Analysis of Quick irCLIP of eIF3 to RNAs in undifferentiated and differentiated neural progenitor cells.A) Generation of NPCs from hPSCs by embryoid body (EB) and neural rosette selections. B) Western blots of neural markers Pax6 and Sox1 and pluripotent marker Oct4 from hPSCs and hPSC-derived NPCs (NPCs corresponding to passages 1, 2 and 7 are shown). C) Western blots of puromycin-treated (15 min) NPCs. Total protein stain is shown as loading control. Differentiated NPCs were treated with forebrain neuron differentiation medium for the indicated time points. Undifferentiated NPCs were treated with NPC medium for the indicated time points. D) Schematic of the NPC treatment performed in the Quick-irCLIP, APA-Seq, Ribosome profiling, and mRNA-Seq experiments. Treatment consisted of change of media and incubation at 37 °C for 2 hrs. Differentiation media was used for differentiated (Diff.) NPCs and Basal media for undifferentiated (Undiff.) NPCs. E) Immunoprecipitation samples of assembled eIF3 complexes from NPCs. 5% inputs (In), flowthroughs (Ft), and eluates (Elu) are shown. Western blot for subunit EIF3G is not shown because we could not identify an effective antibody for its detection. EIF3J, not shown, is usually dissociated from the eIF3 complex upon immunoprecipitation. However, we detected EIF3G by mass spectrometry in the IPs (Table S1). F) Infrared (IR) image of IR dye-labeled, eIF3 UV-crosslinked RNA transcripts from Diff. and Undiff. NPCs. Regions marked with red boxes, which correspond to subunits EIF3A through EIF3D, were excised from the blot. G) Biological Function enrichment determined using the STRING database for the undifferentiated NPC biological replicates of the EIF3A/B/C/D Quick-irCLIP libraries, using the top 500 mRNA hits from the crosslinking analysis. H) Categories of RNAs crosslinked to eIF3 in undifferentiated and differentiated NPCs. I) mRNA regions that crosslink to eIF3 in undifferentiated and differentiated NPCs. J) Crosslinking of eIF3 across the transcripts of VIM and NES mRNAs in differentiated and undifferentiated NPCs. Read coverage is provided in Counts Per Million (CPM).