Fig. 4: YBX1 maintains an oncogenic protein network in AML at the post-transcriptional level.

A Heatmap derived from hierarchical clustering of proteins that were detected as being differentially abundant in mass-spectrometry based whole proteome analysis. YBX1 was inactivated in MOLM-13 cells using CRISPR-Cas9 mediated knockout (2 different sgRNAs). Lentiviral transductions were performed in quadruplicates and 2 × 10⁶ cells per replicate were FACS-sorted 14 days after transduction for the strongest guide-expressing cells (RFP-high) before cell lysis, trypsin digest and analysis via mass-spectrometry. Proteins significantly up- or down-regulated with both guide RNAs (ANOVA p < 0.05) were considered. B Gene-set enrichment analysis for Gene-ontology terms on proteins that were significantly down-regulated in the whole proteome analysis. Selected terms are annotated. C Gene-set enrichment analysis for Gene-ontology terms on proteins that were significantly up-regulated in the whole proteome analysis. Selected terms are annotated. D Volcano-plot of differentially expressed genes in RNAseq at day 7 after knockout of YBX1 by CRISPR-Cas9 (2 different guide RNAs). Every dot represents a gene with a p value below 0.05. The dots highlighted in blue are genes among those that show a fold change above 1.5. E Two of the top Gene-ontology terms that were found to be lost in GSEA of RNAseq data in MOLM-13 cells with knockout of YBX1. F Depiction of differentially expressed genes from RNAseq in YBX1-deficient MOLM-13 cells that were previously identified as RNA-binding targets of YBX1 [2]. Left: Pie chart showing the proportion of DEGs that are known binding partners of YBX1 (blue) in relation to genes that have not been shown to bind to YBX1 (gray). Right: iCLIP-seq IGV-tracks of selected DEGs, that are established binding partners of YBX1 [2]. G RIP-qPCR of YBX1 from MOLM-13 cells. Left: Western-blot and RNA-tape bands as a quality control of the YBX1-enrichment during immunoprecipitation and RNA integrity post IP. Right: results of RIP-qPCR shown as % of enrichment over input validating the binding of EIF4B, EIF3L, EIF3D and EEF2 to YBX1 in MOLM-13 cells. H Analysis of differential splicing in YBX1-deficient cells (sgRNA1/sgRNA2) compared to vector control. The computational analysis to call alternative splicing events was performed using the “IsoformSwitchAnalyzeR”-package [33].