Fig. 5
METTL16 enhances translation-associated pathways and eIF3a mRNA translation efficiency to promote CSC self-renewal. A MA plots displaying the decreased- and increased- expression genes in Huh7 cells upon METTL16 KO. The dashed vertical lines represent Log2(fold change) = 1 or − 1. The significantly increased- or decreased- expression genes are shown in red and blue, respectively (P < 0.01); the grey dots indicate P ≥ 0.01. B Volcano plots showing the enriched gene signatures in Huh7 upon METTL16 depletion. Here, we highlight the top decreased and increased pathways upon METTL16 KO according to GSEA. C GSEA showing of top down-regulated gene signatures in Huh7 upon METTL16 depletion. D Venn diagram showing the overlap between METTL16-, eIF3a-and eIF3b bound transcripts in HepG2 cells. E Venn diagram showing the overlap between the transcripts with decreased translation efficiency (TE) upon METTL16 KO cells and the transcripts directly bound by METTL16-eIF3a- eIF3b. F Pie charts showing the distribution of 957 transcripts with or without significant mRNA level change upon METTL16 KO. G The Gene Oncology Molecular Function (GO MF) enrichment analysis of the 466 transcripts without significant changes at mRNA levels upon METTL16 KO. H Integrative genome viewer (IGV) browser tracks showing M16 (METTL16), eIF3a, and eIF3b binding peaks on eIF3a mRNA. The up three were conducted in HepG2 cells, while the bottom one was conducted in HEK293T cells. I METTL16 CLIP-qPCR analysis showing the interaction between METTL16 protein and eIF3a mRNA in HepG2 cells (n = 3; mean ± SEM). J Western blotting showing the expression levels of eIFs in HepG2 cells upon METTL16 KO and rescue expression. K Ribo-qPCR showing the translation efficiency of eIF3a mRNA in HepG2 cells upon METTL16 KO. L, M Representative images (L) and the statistical results (M) of liver CSC frequency as determined by in vitro LDA in HepG2 cells upon METTL16 KO and rescue expression of METTL16 or eIF3a. N The bar plots showing the colony number in HepG2 cells upon METTL16 KO and rescue expression of METTL16 or eIF3a (n = 6; mean ± SD). O The rescue effect of eIF3a on the cell proliferation of HepG2 cells upon METTL16 KO (n = 3; mean ± SD). Statistical analyses: un-paired t-test (I, K, N); ELDA software (M); Two-way ANOVA (O). *P < 0.05, **P < 0.01, ***P < 0.001