Fig. 4

METTL16-eIF3a/b interactions are required for translation and proliferation promotion in HCC. A Polysome profiles of HepG2 cells as determined by sucrose density-gradient ultracentrifugation (top). The localization of eIF3a, eIF3b, METTL16, and RPL7 proteins were validated by Western blotting (bottom). B Representative Co-IP images showing the direct interaction between METTL16 and eIF members in HepG2 and Huh7 cells. C In situ detection of METTL16–eIF3a and METTL16–eIF3b interactions through PLA in HepG2 cells. D Representative polysome profiles of Huh7 cells upon METTL16 KO. Data are representative of 3 independent experiments. E Representative Western blotting images of SUnSET assays used to quantify the amount of nascent [puromycin (Puro)-labeled] peptides in Huh7 and HepG2 cells with METTL16 KO and rescued expression. F Schematic describing our in-house CRISPR screening with two HepG2 Cas9 single clones and protein–protein interaction (PPI) models. G Principal component analysis (PCA) of CRISPR screening data from 2 groups of HepG2 Cas9 single clones on day 0 and day 30. H Normalized CRISPR score (NCS) of each sgRNA construct (dot) and smoothed score (line) of the METTL16-tiling survival screen in HepG2 Cas9 single clones. Top, Peptide homology alignment of METTL16 across different species. I The PPI model between eIF3a and METTL16-CRISPR gene tiling scan. Left, PPI modeled structure (model 3, M3); Middle, Visualization of METTL16 surface area within 4 amino acids (4A) from the predicted eIF3a binding models; Right, CRISPR gene tiling scan plotting of METTL16 from the predicted eIF3a binding models. J The amino acids on the METTL16 predicted to be within 4A distance to eIF3a. K The PPI model between eIF3b and METTL16-CRISPR gene tiling scan. Left, PPI modeled structure (model 3, M3); Middle, Visualization of METTL16 surface area within 4A from the predicted eIF3b binding models; Right, CRISPR gene tiling scan plotting of METTL16 from the predicted eIF3b binding models. L The amino acids on the METTL16 predicted to be within 4A distance to eIF3b. Regions 1–6 (R1-R6) were derived from high-density CRISPR gene tiling scans of METTL16. M Rescue effect of regions 1–6 mutated METTL16s on METTL16 KO-induced cell proliferation suppression in Huh7 cells (n = 5; mean ± SEM). N Representative Western blotting images of SUnSET assays in Huh7 and HepG2 cells with METTL16 KO and rescued expression. O, P Representative Co-IP images showing the direct interaction between eIF3a (O) or eIF3b (P) and METTL16 with R1, R2 or R4 mutations in HepG2 cells. Statistical analyses: unpaired t-test (M); ***P < 0.001