Fig. 2

Downregulation of METTL3 disrupts mRNA capping in select genes. (A) Schema of TeloPrime capped mRNA cDNA synthesis and DNAseq analysis. (B) Volcano plot on differentially expressed genes sh-/shMETTL3 A549. Grey points represent no significance (p. value > 0.01 and |Log2FC|< 0.58), green points represent |Log2FC|> 0.58, blue points represent p-value < 0.01, and red points represent significant genes (|Log2FC|> 0.58 and a p-value < 0.01). (C) Gene overlap between top mRNAs or cytoplasmic capped mRNAs and differentially expressed genes. Venn diagrams are scaled with list size. *** represents overlapping Fisher exact test p. value < 0.001. (D) Dot plot of the top 15 pathways identified by IPA for the downregulated capping genes. (E) Representative western blot analysis of protein expression of METTL3, RNGTT, P-RPS6, RPS6 and Ponceau staining as loading control done in triplicate. Original blots are presented in Supplementary Fig. 4. Band intensities were normalized to ponceau stain, and scr- was set to 1. (F) CAP-IP on selected genes in PC9 cells analyzed by RT-qPCR. Data were first normalized to input and then normalized to housekeeping control ACTIN and represented as fold change with scr enrichment set as 1. Error bars represent S.E.M. in N = 7. Ns represents non-significant two-tailed student t-test of scr vs siMETTL3. * Represents two-tailed student’s t-test of scr vs siMETTL3 p. value < 0.05, ** represents p. value < 0.01.