Figure 6. Reduced cellular translation due to loss of MYSM1 preferentially decreases translation efficiency of iron transport, storage and metabolism genes, resulting in overall decrease of protein level.

(A) Real time PCR analysis of iron transport, storage and metabolism gene expression of sorted CD34⁺CD45RA⁻CD90⁺CD133⁺ cells. (B) Western blot analysis of key iron handling and ferroptosis genes in indicated fractions of culture CD34⁺ HSPCs edited by AAVS1 control or MYSM1. (C) Volcano plot of MYSM1 KO vs. AAVS1 differentially translated genes, highlighting genes directly relevant to cellular iron transport, storage and metabolism, and ferroptosis. (D) Top enriched signatures in differentially translated genes in MYSM1 KO cells. (E) Gene set enrichment analysis (GSEA) showing ferroptosis protective gene translation is significantly impaired in MYSM1 KO cells. (F) Representative translation efficiency change of ferroptosis genes in MYSM1 KO cells. (G) Motif discovery analysis identified genes with specific 5’ UTR motif pattern tend to be less or more efficiently translated due to MYSM1 loss. (H) Percentage of 5’UTRs of genes of different groups in downregulated or upregulated motifs discovered in (G). (I) Western blot analysis of the translation of motif modified mRNA of MYSM1 deficient CD34⁺CD45RA⁻CD90⁺ cells. FTH1, SLC40A1 and ALAS 5’UTRs were engineered by placing three repeats of first two motifs identified in up side shown in (G) and HSPB1 and RELB were engineered by placing three repeats of first two motifs identified in down side shown in (G).