Fig 1.
(A) Schematic workflow of mRNA-seq, RNC-seq and Ribo-seq of the same batch of cultured cells. (B) Contribution of translation initiation efficiency and elongation velocity to the ribosome density.
Fig 2.
Two mutually exclusive and polarized translation control modes.
(A) The TR and EVI of genes in HeLa cells. The green ellipse indicates the 95% confidence ellipse. Genes outside of this ellipse were clustered in two major clusters (red and blue dots) based on their Euclidean distances. The grey region denotes the region with high TR (genes with top 1% TR) and low EVI (genes with lowest 1% EVI), simultaneously. Rs = Spearman R. (B) The TR and EVI of genes of HBE, A549 and H1299 lung cell lines, respectively. (C) Plot matrix of mutual correlation of the TR (lower half triangle) and EVI (upper half triangle) among the four analyzed cell lines. (D) The heatmap of the Spearman R of the panel (C). All of the P-values are less than 10−38. (E,F) Cluster analysis of the TR (E) and EVI (F) of the four cell lines. The lung-derived cell lines are indicated by a green bar.
Fig 3.
The relevance of low EVI genes to transient translational pausing and protein folding.
(A, B) The RFP coverage of a representative gene with low EVI (A) and high TR (B) in HeLa cells, respectively, are shown with color bars. The highest RFP peaks are indicated by stars. The full length mRNAs are shown in thin lines, while their CDS regions are marked with thick lines. For more examples in all four analyzed cell lines, please refer to S6 Fig. (C) The percentage distribution of the highest RFP peak of each gene relative to the start AUG codon. The highest RFP peaks more than 400 nucleotides are counted in the bin of 400. The P-values of Kolmogorov-Smirnov test between the low EVI genes and high TR genes as well as all genes in each cell line are indicated. The grey dashed lines mark the 240nt position. (D) Correlation between CDS length and EVI in the four cell lines. (E, F) The distribution of genes in HeLa cells according to their protein degradation rate constant (kdeg). Fractions of the genes with low EVI (E) and high TR (F) were indicated in red and blue bars, respectively. For comparison, the fraction distributions of total genes are shown in grey bars as background.
Table 1.
Fraction of positively charged amino acids (lysine, arginine and histidine) in low EVI genes and high TR genes.
Fig 4.
Codon preference of the slow-translating genes.
(A) Codon preference of low EVI genes in the four cell lines based on Preference Score analysis. For details, please refer to Materials and Methods section. Favored and disfavored codons are shown in red and grey bars, respectively. (B) Mutual correlation of PSLow-EVI of codons among all four analyzed cell lines. (C) Percentage of variance explained in the principle component analysis (PCA) on RSCU. (D) Principle component (PC) based clustering analysis. The PCs with the summed explained variance greater than 80% were taken for clustering analyses based on the standardized Euclidean distances. For each cell line, four groups of genes were analyzed. They were low EVI genes, high TR genes, all genes and a random subset of genes that were inside the 95% confidence ellipse (Fig 2A and 2B). The clusters with multiple gene groups are indicated by grey ellipses.
Fig 5.
Codon preference of the high TR genes.
(A) Preference Score of high TR genes (PSHigh-TR) of the four cell lines. (B) Correlation of the PSHigh-TR and PSLow-EVI.
Fig 6.
Relative EVI analysis and cancer relevance.
(A, B) Correlation of the relative changes of TR and EVI comparing cancer with normal cells. A549/HBE (A) and H1299/HBE (B) results were shown, respectively; the genes with relative EVIs of greater than ±10 folds were indicated by blue dots. (C,D) Comparative proteomics analyses on the low-EVI genes comparing A549 cells with HBE cells. Relative abundance distributions of total proteins (C) and the unfolded proteins (D) are shown. (E, F) Top canonical pathway analysis of low relative EVIs by IPA. Low relative EVI genes (<10 folds) in A549/HBE (E) and H1299/HBE (F) comparisons were subjected to IPA analysis. The top canonical pathways with P < 0.001 (Fisher’s exact test provided by IPA indicated by the threshold line in orange) regulated by of these genes were shown respectively. The complete gene lists for IPA are included in the S2 Table with HGNC gene names.