Fig 2: YBX1 knockout leads to limited changes in the translatome and the transcriptome. (A) Scatterplot of changes in ribosome footprint counts (Ribo-Seq) plotted against RNA abundance (RNA-Seq) in HEK293T?YB-1 compared to HEK293T. Genes with significantly changed RNA abundance (multiple-testing corrected P-values < 0.05) are highlighted in red (upregulated) or blue (downregulated). (B) Volcano plot of ribosome occupancy changes in HEK293T?YB-1 versus HEK293T; YBX3 is highlighted in green. Genes with non-corrected P-value < 0.01 are labeled. (C) Gene set enrichment analysis (GSEA) of changes in RNA abundance (blue line) or ribosome occupancy (red line). Genes were sorted by signed P-value from left to right according to the changes in RNA abundance (?RNA, blue line) or ribosome occupancy (?RO, red line) in HEK293T?YB-1 versus HEK293T. Vertical bars indicate the location of genes from a particular gene group in the sorted list.

Fig 3: YB-1 controls YB-3 synthesis. (A) The lysates of HEK293T and HEK293T?YB-1 cells were subjected to centrifugation through 15-45% linear sucrose gradient for 1 h at 45,000 rpm in an SW-60 rotor. Profiles of optical density at A254 are shown (upper and middle panels). YB-3 mRNA was detected by qRT-PCR in each fraction of the polysome gradient (lower panel). (B) Changes in the amount of YB-1 in HEK293T cells upon knockout or plasmid-induced expression of YB-1 detected by Western blotting; Fibrillarin is given as a control. (?) Changes in the amount of YB-3 mRNA in HEK293T cells with knockout or overexpression of YB-1 detected by qRT-PCR. YB-3 mRNA amounts were normalized to those of HEK293T cells transfected by the empty vector. Two-tailed Student’s t-test was used to estimate the statistical significance versus HEK293T cells transfected by the empty vector. ***p < 0.001, *p < 0.05. (D) 0.15 pmol of capped and polyadenylated reporter Firefly luciferase mRNAs with indicated UTRs were translated in HEK293T cell-free translation extract in the presence of increasing concentrations of recombinant YB-1 (0, 0.07, 0.14, 0.28, 0.42, and 0.7 µM). After 1h incubation at 30°C, Fluc activity was measured. The values were normalized to the Fluc activity without the addition of YB-1. Values are the means of at least three independent replicates. Error bars show 2 standard deviations.

Fig 4: Akt phosphorylates S209 of YB-1 in vitro. (a) Schematic representation of predicted Akt phosphorylated sites in the YB-1 molecule (ScanSite Z-scores are indicated in blue). Positions of CSD (cold shock domain), NLS (nuclear localization signal), and CRS (cytoplasmic retention site) are shown as colored boxes. Vertebrate sequence alignments of particular segments of the YB-1 protein in the vicinity of phosphorylation sites are shown. Phosphorylation sites and Akt kinase consensus motifs are highlighted in red. (b) In vitro kinase assay for wild-type (WT) and mutant YB-1 proteins. The assay was performed by incubating 1 µg of YB-1 with 0.25 µg of activated Akt kinase in the assay buffer containing [?-32P]-ATP. The reaction products were analyzed by SDS-PAGE (Coomassie staining, bottom panel) and autoradiography (upper panel). (c) Quantification of in vitro kinase assay. The YB-1 phosphorylation level was normalized to that of the WT and corrected for the corresponding protein amount determined by Coomassie staining. The mean ± SD of four independent experiments is shown. #: non-significant, *** p < 0.001, one-sample t-test.

Fig 5: YB-1 is phosphorylated at S102 but is not translocated to the nucleus upon serum stimulation. HeLa (left panel), NIH3T3 (middle panel), and PC3 (right panel) cultivated in normal, serum starvation (24 h w/o FBS, fetal bovine serum), or serum stimulation (24 h without FBS + 2 h 20% FBS) conditions. The cells were (a) lysed and analyzed by Western blot using indicated antibodies, or (b) analyzed by immunofluorescence (IF) microscopy using antibodies against YB-1. Nuclei were visualized by DAPI staining.