Fig. 6

METTL16 preferentially localizes to the granular component (GC) of the nucleolus and facilitates rRNA processing and ribosome biogenesis. A Representative SIM images of FBL (green) and METTL members (red; including METTL3, M3; METTL14, M14; METTL16, M16) in the nucleus of Huh7 cells. FBL, nucleolar marker; DAPI, nuclear marker. B, C Pearson’s correlation analysis between distributions of FBL and the three METTL members (B) or between SC35 and the three METTL members in Huh7 cells (C) (n = 10; mean ± SD). SC35, nuclear speckle marker. D, E Bubble plots showing the METTL16-interacting (D) and METTL3-interacting (E) proteins identified by BioID assay. The size of each dot represents the P value of probability of binding of METTL16 or METTL3 with each protein. The proteins within the rectangle specifically interact with METTL16-BirA* (D) or METTL3-BirA* (E); while the proteins within the oval interact with both METTL16 and METTL3-BirA*. BirA*, BirA R118G variant. F GO enrichment analysis of the specific METTL16-interacting proteins. BP, biological process; CC, cellular component. G Representative confocal images showing the nucleolus in normal (CL48) and cancer (PCL/PRF/5) cells. H Statistical results of nucleolar numbers in normal cells and cancer cells (n > 40). I, J Representative confocal images (I) and the statistical results (J) showing the effects of METTL16 KO and rescued expression on nucleolar numbers in Huh7 cells (n > 50). K Representative confocal images showing the subnucleolar localization of METTL16 in Huh7 cells. FBL, DFC marker; NPM1, GC marker. L Pearson’s correlation analysis between METTL16 and FBL or NPM1 in Huh7 cells (n = 10; mean ± SD). M Simplified schematic of rRNA processing and the probes we used for Northern blotting and qPCR. N The effects of METTL16 KO and rescued expression on pre-rRNA levels in Huh7 cells as determined by qPCR (n = 3; mean ± SD). P1 was used to detect 47S pre-rRNA; while P2 was used to detect 47S, 45S, and 30S pre-rRNAs. O Representative images showing the effects of METTL16 KO and rescued expression on pre-rRNA levels as determined by Northern blotting in HepG2 cells. P Representative Co-IP images showing the direct interaction between METTL16 and DDX47, DDX49, or BOP1 in HepG2 cells. Q Representative confocal images showing the colocalization of METTL16 with DDX47, DDX49, or BOP1 in the nucleolus of Huh7 cells. Statistical analyses: unpaired t-test (B, C, H, J, L, N); ns, not significant; **P < 0.01, ***P < 0.001