Fig 2: Male-biased expression of EIF1A protein in the heart. (A) Signal/noise values for Y-specific peptides in XX (gray) and XY (green) samples. Set refers to the 11-plex experiment (out of three total) in which the peptide was detected. The dotted line shows average signal/noise value in XX samples. (B) Amino-acid sequence of EIF1AX/Y: X- and Y-specific amino acids are superscripted and subscripted, respectively. X-specific (gold), Y-specific (blue), and X–Y shared (purple) peptides detected by mass spectrometry are shown, along with the number of 11-plex experiments in which each peptide was detected. (C) Relative abundance of X and Y protein isoforms in XX (n = 12) and XY (n = 21) heart tissue samples by mass spectrometry. For each X-Y pair, points show the levels of the X isoform (gold) or the total level of the X and Y isoform (purple) in XX samples compared with XY samples, from which the relative proportion of X and Y isoform expression in XY samples can be inferred (dotted white line). P-values by estimated by permutation. (D) Comparison of estimated Y/X expression ratios and sex-biased expression from RNA-seq and mass spectrometry. Asterisks indicate statistical significance in the corresponding analysis. (E) Abundance of EIF1A and GAPDH by western blot in pooled XX and XY protein lysates. (F) Quantification of EIF1A levels in pooled XX and XY samples by western blot; P-value by Welch's t-test.

Fig 3: Y-specific loss of the miR-1 target site led to elevated EIF1AY expression in XY heart and tissue. (A) Each point shows expression level of EIF1AY (blue) or EIF1AX (gold) in a single tissue sample from an XY individual. Lines show median expression level. (B) Alignment of 3′ UTRs of EIF1AY, EIF1AX, and their orthologs; miR-1 target site in pink. Key branch points annotated with estimated divergence times in millions of years ago (mya). Fully conserved sites annotated with an asterisk; sites consistent with a single evolutionary substitution event annotated with a dot. (C) Quantile-normalized expression levels of miR-1 across human tissues. (D) Activity of luciferase reporter fused to 3′ UTR sequences of EIF1AX or EIF1AY with intact (+) or disrupted (−) miR-1 site in HEK293 cells, upon transfection with miR-1 or miR-124. Luciferase activity of each reporter with a disrupted miR-1 site is normalized to activity of corresponding reporter with intact site. P-values from two-sided Welch's t-test. (E,F) Each point shows Log2(Y/X expression ratio) for EIF1AY/EIF1AX orthologs in macaque (E) and chimpanzee (F).

Fig 4: Transcriptional regulation of p21 by EIF1AX is p53‐independent. A, The recruitment of EIF1AX to the p21 promoter in p53+/+ HCT‐116 cells. Soluble chromatin from p53+/+ HCT‐116 cells was immunoprecipitated with anti‐EIF1AX, rabbit normal IgG antibody served as control. qRT‐PCR analysis of the final extracted DNA using the primers specific to the proximal promoter region of the p21 gene. B, The recruitment of EIF1AX to the p21 promoter in p53−/− HCT‐116 cells. Soluble chromatin from p53−/− HCT‐116 cells was immunoprecipitated with anti‐EIF1AX, rabbit normal IgG antibody served as control. The final extracted DNA samples were amplified by qRT‐PCR using primers specific to the proximal promoter region of the p21 gene. C, The effect of EIF1AX on p21 promoter activity in p53+/+ HCT‐116 cells. Luciferase assay of p53+/+ HCT‐116 cells transfected with p21‐Luc reporter construct and 0, 0.1, 0.2 and 0.4 µg/well EIF1AX expression construct. D, The effect of EIF1AX on p21 promoter expression in p53−/− HCT‐116 cells. Luciferase assay of p53−/− HCT‐116 cells transfected with p21‐Luc reporter construct and 0, 0.1, 0.2 and 0.4 µg/well EIF1AX expression construct. E, qRT‐PCR analysis of p21 in EIF1AX knock‐down p53+/+ (left panel) and p53−/− (right panel) HCT‐116 cells. F, Western blotting analysis of p21 in EIF1AX knock‐down p53+/+ (left panel) and p53−/− (right panel) HCT‐116 cells. G, Quantification of proteins in (F)

Fig 5: EIF1AX promotes breast cancer cell proliferation and tumorigenesis. A, The effect of EIF1AX overexpression on cell cycle progression. Cell cycle analysis of control and EIF1AX overexpressed MCF‐7 cells by flow cytometry. The right panel data are presented as the mean ± SD from three independent experiments. The left panel shows representative results. B, The effect of EIF1AX knock‐down on cell cycle progression. Cell cycle analysis of control and EIF1AX knock‐down MCF‐7 cells using flow cytometry. The right panel data are presented as the mean ± SD from three independent experiments. Representative results are shown in the left panel. C and D, Colony formation assay. Represent images of EIF1AX overexpressed and knock‐down MCF‐7 cell colonies (C). Statistics of the colony number of each group (D). Data are presented as the mean ± SD from triplicate experiments. *P < .05, Student's t test. E, The effect of EIF1AX on cell proliferation. Cell proliferation analysis of EIF1AX overexpressed and knock‐down MCF‐7 cells. Data are expressed as the mean ± SD from triplicate experiments. *P < .05; **P < .01, compared with empty vector. # P < .05; ## P < .01, compared with control siRNA, Student's t test. F and G, EIF1AX promotes breast tumorigenesis. Athymic mice were injected with EIF1AX‐overexpressing or EIF1AX shRNA‐expressing MCF‐7 cells. Representative tumour images (F) and the average tumour mass (G). Each point represents the mean ± SD *P < .05, Student's t test