Extended Data Fig. 7: Adenosine-ending codon frequency correlates with translation defects.
From: Reprogramming neuroblastoma by diet-enhanced polyamine depletion
a) Top down- and upregulated Reactome pathways in the GSEA analysis on the protein level when comparing the diet-drug combination to the control diet (ProArg-free DFMO vs. CD). b) Fraction of codons ending with respective nucleotide across all Reactome pathways (grey), cell cycle related (blue) and neuronal system (red). c) The fraction of A-ending codons is more divergent than the fraction of G-ending codons in cell cycle related (blue) and neuronal system (red) emphasizing A-ending codon content as a differential metric for regulating translation of these two pathways. d) Enrichment analysis based on the A, T, G or C ending codon fraction per gene. Pathways positively enriched (positive p-adj) have higher A, T, G or C ending fractional enrichment whereas negatively enriched pathways have a lower fraction. e) Across Reactome pathways, increasing enrichment of adenosine-ending codons correlates to downregulation on the protein level when comparing combined diet-drug treatment to control. Enrichment was computed using gene set enrichment analysis, where transcripts are ranked by adenosine-ending codon fraction and proteins by fold change on the protein levels. f) Protein intensity across cell cycle, the most downregulated pathway, where the difference between ProArg-free DFMO and CD is evaluated by protein fold-change on the y axis. Fold change distribution on the right side. g) ProArg-free DFMO and CD DFMO highlighting the additive diet effect on the downregulation of cell cycle proteins. Four top-down regulated proteins were identified in both comparisons. Fold change distribution on the right side. h) Protein intensities across the most upregulated pathway, neuronal system, where the difference between ProArg-free DFMO and CD is evaluated by protein fold-change on the y axis. Fold change distribution on the right side. i) ProArg-free DFMO and CD DFMO highlighting the additive diet effect on the upregulation of neuronal system proteins. Common top-upregulated proteins (log2 fold change >1) were identified in both comparisons. Fold change distribution on the right side. j) Relative codon preference comparing the Itgb3bp gene (CENPR protein) to the whole transcriptome. Percentages above the line highlight a preferential use for encoding the amino acid in CENPR by that specific codon type (red is adenosine ending). k) Relative amino acid codon preference comparing the pathway cell cycle to the whole transcriptome. Marks above the line highlight the preferential utilization of defined codons in the cell cycle genes to encode the same amino acid. l) The relative ribosome pausing sum as the sum of ribosome occupancy ratios of ProArg-free DFMO to CD according to the nucleotide at codon position three in Itgb3bp (CENPR protein). Specific dysfunction in decoding of A-ending codons is observed. m) Relative ribosome occupancy across Itgb3bp (CENPR protein) reveals a dysfunction in decoding adenosine-ending codons. Occupancy ratios were calculated between ProArg-free DFMO and CD. Summed by the nucleotide at codon position three in i). n) Percentage of codons with the respective nucleotide at the ending position in the whole transcriptome. o) Relative pausing sum of Cep57 and Kif2c, where the relative ribosome occupancy ratio between ProArg-free DFMO and CD are summed according to the nucleotide at the codon ending position. Hus1 did not show sufficient coverage. p) Correlation of relative pausing sum with protein levels in the top up and down regulated proteins. The relative ribosome occupancy ratio between ProArg-free DFMO and CD are summed according to the nucleotide at the codon ending position and normalized for gene length. For a, d, f, g, h, i and p: Proteomics ProArg-free DFMO n = 6; CD DFMO n = 6; CD n = 5. For l, m, o, and p: Ribo-Seq n = 5 per group; For b-e, the legend for pathways is shared above. Abbreviation: ‘Respiratory electron transport …’, ‘Respiratory electron transport; ATP synthesis by chemiosmotic coupling, and heat production by uncoupling proteins ‘; NES, Normalized Enrichment Score.
