Figure 3.

Knockdown of METTL3 in prostate cancer alters gene expression and translation. A, Generation of LNCaP cell lines with two doxycycline-inducible shRNAs targeting METTL3, or a nontargeting shRNA targeting GFP. Cells were treated with doxycycline for 96 hours followed by Western blot analysis for METTL3. B, Significantly (adjusted P value < 0.05) differentially expressed genes with METTL3 knockdown in the two inducible shRNA lines (n = 3). Common genes between the two shRNA lines are highlighted with a black border. C, Six transcripts are significantly downregulated and seven transcripts are significantly upregulated with METTL3 knockdown in both shRNA lines, but not the nontargeting line (n = 3). D, Correlation (Pearson r = 0.6306, P < 1E-15) between the average mRNA (n = 6) and ribosome footprint (n = 4) from the METTL3 shRNA lines without doxycycline. Outliers with extreme high (red) and low (blue) TE (Ribo/RNA) were calculated using ROUT analysis (Q = 1%). E, Riborex analysis of Ribo-seq data identifies genes with changes in TE independent of changes in mRNA expression. Shown is the fold change with doxycycline treatment in both METTL3 shRNAs combined as determined by DESeq2 (RNA-seq, n = 12; Ribo-seq, n = 10). F, Gene set enrichment analysis of genes ranked by the fold change in TE with METTL3 knockdown as determined by Riborex.