Fig 1: Validation of the genotype and hypusination status of the eif5a2-K50R mouse model. (A) Mice were genotyped to confirm the eif5a2-K50R mutation using mouse tail DNA preps. The O208/O209 primer pair was used for PCR resulting in a 503 bp product, which was digested using PflFI. eif5a2-K50R mutant PCR product is cut into two bands, while the wild-type product is not digested. (B) Protein lysates from brain tissue of the same mice were used to detect hypusinated eif5a2 levels. Brain tissue was used because of its high endogenous eif5a2 expression. As expected, eif5a2K50R/K50R mice lack hypusinated eif5a2. (C) Quantification of eif5a2 levels relative to ß-actin in (B). * indicates a statistically significant difference in eif5a2 protein levels in wild-type and eif5a2-K50R mouse brain lysates (P=0.0194). WT, wild-type.
Fig 2: eif5a1-eif5a2 mRNA expression in mouse tissue. The eif5a1 and eif5a2 mRNA tissue expression patterns were analyzed using the mouse ENCODE database that profiles mouse embryonic and adult tissues using RNA sequencing through the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). mRNA levels show that eif5a1 is almost universally expressed in all tissues investigated, whereas eif5a2 shows a much more restricted expression pattern, at around 30-50 times lower levels. Absolute mRNA levels are expressed as reads per kilobase per million (RKPM).
Fig 3: eIF5A1-eIF5A2 gene aberrations in human cancer. An overview of eIF5A1 and eIF5A2 gene aberrations in human cancer was obtained using the cBioPortal website for Cancer Genomics (https://www.cbioportal.org/). Quantitative analysis of eIF5A1/2 genetic abnormalities using the 32 TCGA Pan Cancer studies, with 10,967 samples representing most major human cancer types. Analyses was performed using website default settings.
Fig 4: Metabolomics of primary mouse fibroblasts harboring eif5a2-K50R mutation reveal broad metabolic changes. (A) A heatmap of significantly differentially abundant metabolites between wild-type and eif5a2K50R/K50R-derived fibroblasts. (B) A volcano plot reveals that 47 metabolites are differentially abundant, with 41 metabolites showing increased levels (FDR<0.05) in K50R cells. Within these significantly elevated metabolites, interesting patterns emerged with glutathione metabolism (C), and vitamins and cofactors (D). Data are expressed as mean±s.e. GSH, glutathione; NAD, nicotinamide adenine dinucleotide; FAD, flavin adenine dinucleotide; CoA, coenzyme A. WT, wild-type.
Fig 5: eIF5A1-eIF5A2 mRNA expression in human cancer. An overview of eIF5A1/2 mRNA expression in human cancer, using the ICGC/TCGA dataset (ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium) with 2922 samples representing 33 major human cancer types. Analyses were performed in cBioPortal as in Fig. 5.
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