American Society of Hematology

Figure 4.

YBX1 binds m6A-tagged RNA mediated by IGF2BPs. (A) The distribution of YBX1-binding peaks within different gene regions identified by FLAG-YBX1 RIP-seq in HEK293T cells. (B) Metagene profiles showing higher overlap of YBX1-binding sites and m6A modifications across the mRNA transcriptome. GSE29714 data were used for MeRIP-seq analysis. (C) Experimental scheme for RNA pull-down assay. Biotin-labeled single-strand RNA ss-m6A and biotin-labeled ss-A were used. (D) Western blot shows that IFG2BPs and YBX1 protein were selectively pulled down from HEK293T nuclear extract. (E) RNA pull-down assay showing the pull-down of purified GST-IGF2BP1/3 proteins, but not GST-YBX1 by biotin-labeled ss-m6A. (F) Coimmunoprecipitation and western blot showing the binding of YBX1 with IGF2BP1/3 in HEK293T cells. (G) Schematic structures showing the organization of YBX1 protein and YBX1 variants used in this study. Green box, the ala (A) and pro (P) enrich domain (A/P-rich domain); yellow box, an evolutionarily conserved CSD; pink and white separated boxes showing long C-terminal domain (CTD) with 4 arginine-rich motifs (ARMs) (pink boxes) and 4 acid motifs (AcidMs) (white boxes). (H-I) Coimmunoprecipitation and western blot showing the binding of FLAG-tagged YBX1 and its variants with hemagglutinin antigen (HA)-tagged IGF2BP1 in HEK293T cells (H) and of FLAG-CSD with endogenous IGF2BP1/3 in HEK293T cells (I). (J) RNA pull down with ss-m6A or ss-A in YBX1 KD or control HEK293T cells. Western blotting showing the similar pull-down efficiency of IGF2BP1/3 between YBX1 KD and control group. (K) RNA pull down with ss-m6A or ss-A in IGF2BPs KD or control HEK293T cells. Western blots showing the reduced pull-down amounts of IGF2BP1/3 and YBX1 between IGF2BP1/3 KD and control group. (L) Venn diagram showing the percentages of shared high-confidence targets among YBX1 and IGF2BPs. IGF2BPs iCLIP-seq data from GSE90686 were compared with YBX1 RIP-seq data. (M) Top consensus sequences of YBX1-binding sites, IGF2BP-binding sites, and the m6A motif detected by motif analysis. (N) YBX1 RIP-qPCR showing binding of FLAG-YBX1 to the representative targets (MYC, BCL2, TK1, MARCKSL1) in HEK293T cells. (O) Reducing mRNA half-life of the representative targets (MYC, BCL2, TK1, MARCKSL1) by deleting YBX1 in HEK293T cells. (P) Relative Fluc activity of MYC and BCL2 3′ UTR reporters with wild-type (CRD-WT) or mutated (CRD-mut) CRD in HEK293T cells with overexpressed YBX1. (Q) Relative luciferase activity (Rluc) ofCRD-WT orCRD-mut in IGF2BP1 KDor controlHEK293T cellswith expression of YBX1. (R) Relative luciferase activity ofCRD-WT or CRD-mut in YBX1 KD or control HEK293T cells with expression of IGF2BP1. RNA pull-down assays and western blotting show 1 representative of 3 independent experiments (D-K). Panels N and P-R show 1 representative of at least 3 independent experiments. Two-tailed Student t test: **P < .01; ***P < .001.; IP, immunoprecipittion; mut, mutant; shCon, shControl; Vec, vector.

YBX1 binds m⁶A-tagged RNA mediated by IGF2BPs. (A) The distribution of YBX1-binding peaks within different gene regions identified by FLAG-YBX1 RIP-seq in HEK293T cells. (B) Metagene profiles showing higher overlap of YBX1-binding sites and m⁶A modifications across the mRNA transcriptome. GSE29714 data were used for MeRIP-seq analysis. (C) Experimental scheme for RNA pull-down assay. Biotin-labeled single-strand RNA ss-m⁶A and biotin-labeled ss-A were used. (D) Western blot shows that IFG2BPs and YBX1 protein were selectively pulled down from HEK293T nuclear extract. (E) RNA pull-down assay showing the pull-down of purified GST-IGF2BP1/3 proteins, but not GST-YBX1 by biotin-labeled ss-m⁶A. (F) Coimmunoprecipitation and western blot showing the binding of YBX1 with IGF2BP1/3 in HEK293T cells. (G) Schematic structures showing the organization of YBX1 protein and YBX1 variants used in this study. Green box, the ala (A) and pro (P) enrich domain (A/P-rich domain); yellow box, an evolutionarily conserved CSD; pink and white separated boxes showing long C-terminal domain (CTD) with 4 arginine-rich motifs (ARMs) (pink boxes) and 4 acid motifs (AcidMs) (white boxes). (H-I) Coimmunoprecipitation and western blot showing the binding of FLAG-tagged YBX1 and its variants with hemagglutinin antigen (HA)-tagged IGF2BP1 in HEK293T cells (H) and of FLAG-CSD with endogenous IGF2BP1/3 in HEK293T cells (I). (J) RNA pull down with ss-m⁶A or ss-A in YBX1 KD or control HEK293T cells. Western blotting showing the similar pull-down efficiency of IGF2BP1/3 between YBX1 KD and control group. (K) RNA pull down with ss-m⁶A or ss-A in IGF2BPs KD or control HEK293T cells. Western blots showing the reduced pull-down amounts of IGF2BP1/3 and YBX1 between IGF2BP1/3 KD and control group. (L) Venn diagram showing the percentages of shared high-confidence targets among YBX1 and IGF2BPs. IGF2BPs iCLIP-seq data from GSE90686 were compared with YBX1 RIP-seq data. (M) Top consensus sequences of YBX1-binding sites, IGF2BP-binding sites, and the m⁶A motif detected by motif analysis. (N) YBX1 RIP-qPCR showing binding of FLAG-YBX1 to the representative targets (MYC, BCL2, TK1, MARCKSL1) in HEK293T cells. (O) Reducing mRNA half-life of the representative targets (MYC, BCL2, TK1, MARCKSL1) by deleting YBX1 in HEK293T cells. (P) Relative Fluc activity of MYC and BCL2 3′ UTR reporters with wild-type (CRD-WT) or mutated (CRD-mut) CRD in HEK293T cells with overexpressed YBX1. (Q) Relative luciferase activity (Rluc) ofCRD-WT orCRD-mut in IGF2BP1 KDor controlHEK293T cellswith expression of YBX1. (R) Relative luciferase activity ofCRD-WT or CRD-mut in YBX1 KD or control HEK293T cells with expression of IGF2BP1. RNA pull-down assays and western blotting show 1 representative of 3 independent experiments (D-K). Panels N and P-R show 1 representative of at least 3 independent experiments. Two-tailed Student t test: **P < .01; ***P < .001.; IP, immunoprecipittion; mut, mutant; shCon, shControl; Vec, vector.

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