Extended Data Fig. 8: Nuclear YBX1 regulates Mknk1 mRNA splicing in JAK2VF cells.
From: Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms
a, Experimental design of RNA sequencing and data analysis. b, Bars represent number of retained intron events significantly upregulated in YBX1-depleted cells with two-tailed test P value < 0.05 (1,064 RI events), then filtered for P value < 0.01 and ΔPSI 0.1, reducing the number to 472 highly significant RI events. c, Network map displaying enrichment of gene sets in the 472 highly significant RI events. Each node represents significantly enriched gene sets. Clusters of functionally related gene sets are circled, and labels are highlighted. Two-sided hypergeometric test, P value correction–Bonferroni step down. d, Visualization of spliced Mknk1 mRNA product after in vitro splicing assay. Nuclear extracts with and without YBX1 knockdown and with and without ATP were incubated for 2 h with biotin-labelled Mknk1 pre-mRNA (exon 12–exon 13). mRNA was isolated and reverse transcribed, PCR was performed using the primers at the indicated arrow, and products were agarose gel resolved and visualized using gel-red stain. Representative images from n = 3 biological experiments. e, f, Flow cytometric analysis of MKNK1 protein expression rescue experiment in CRISPR–Cas9-induced YBX1 knockout mouse JAK2VF cells upon nonsense-mediated decay (NMD) inhibition. Representative flow cytometry histogram and violin plot showing quantification of MKNK1–DyLight 649 mean fluorescence intensity (MFI) upon e, compound C treatment (NMD inhibitor (dorsomorphin), termed CC, 10 μM, for 24 h), and f, VG-1 treatment (5 μM and 10 μM, for 20 h) (n = 3, two-sided t-test; mean ± s.d.). g, Genomic track profile of human MKNK1, ARAF and BRAF loci in HEL cells from YBX1 ChIP sequence data set (n = 2). h, Genomic track profile of mouse Mknk1, Araf and Braf loci in Ba/F3 JAK2VF cells from YBX1 ChIP sequence data set. n = 3 biological experiments. Genomic track profile of human and mouse Prkcb shown as positive control from the YBX1 ChIP-seq data set compared to IgG controls. i, Western blot showing the regulation of MKNK1 protein abundance in YBX1 phosphomutants expressing Ba/F3 JAK2VF cells as indicated. n = 3 independent experiments.
