Ribosome profiling identifies mRNAs with reduced translation efficiency upon DAP5 KD in hESCs. (A) Principal component analysis (PCA) of transcript profiles of DAP5-KD (shDAP5) and NT (shNT) hESCs Ribo-seq and total RNA-seq, from four independent experiments. (B) Scatter plot of total mRNA transcript expression in DAP5-KD and NT hESCs, on a log2 scale. Transcripts with decreased abundance in DAP5-KD hESCs (FC < 0.5, FDR < 0.05) are highlighted in red, and transcripts with increased abundance (FC > 2, FDR < 0.05) are highlighted in blue. (C) Pathway analysis by GeneAnalytics of differentially expressed genes identified by RNA-seq of total mRNA upon KD of DAP5 in hESCs, compared to control NT KD. Scores are based on P-values, after correction for multiple comparisons by the false discovery rate (FDR) method; top 10 high quality score pathways are listed. Numbers at right correspond to the number of matched genes out of the total number of genes in the pathway. (D) Comparative analysis of the translation efficiency (TE) in DAP5-KD and NT hESCs. Genes are plotted as a scatter plot according to changes in ribosome occupancy (RP) on the y-axis and mRNA abundance (total mRNA) on the x-axis. The values are shown on a log2 scale. Each dot represents an individual gene (n = 14,511). Genes without significant changes in TE are indicated in gray. Genes with increased or decreased TE (FC > 1.5-fold or <0.67-fold, P-value < 0.001) are highlighted in blue (16 genes) and red (68 genes), respectively. (E) PCA of MS analysis of DAP5 and control NT KD hESCs, from four independent experiments. (F) Volcano plot showing protein abundance as determined by MS analysis of DAP5 KD and NT KD hESCs, expressed as log2(fold-change). Dashed line indicates P < 0.05, all dots above the line showed a significant change upon DAP5 depletion. Proteins with significant decreased abundance that were identified as DAP5 activated translation targets in the ribosome profiling screen are colored in red. (G) Venn diagram showing the overlap between the set of significantly decreased proteins identified by MS, and the set of translationally activated mRNAs that were detected by RP. (H) Western blotting was performed on lysates from shNT and shDAP5 hESCs to detect protein levels of DAP5 and potential DAP5 translation targets, as indicated. Shown are representative blots from one of three (two for MPRIP) experiments. Tubulin was used as a loading control on each of the four blots shown. I. Venn diagram showing overlap between set of mRNAs down-regulated upon KO of Kmt2d in mESCs and KD of DAP5 in hESCs. Note that sequence coverage was much larger in the former experiment; only genes detected in both screens were included. P-values for overlap in G and I were calculated by hypergeometric test.
