Figure 5. Differential A-to-I RNA editing in 3’UTR regions of normal HSPCs and BC LSC.

(A and B) Volcano plot showing the A-to-I(G) editome of cell cycle genes in ADAR1 WT-transduced cord blood CD34⁺ cells compared with lentiviral vector controls (n=3) (A) and in CP progenitors (n=7) compared with BC counterparts (n=6) (B). (C) A-to-I RNA editing of MDM2 3’UTR in individual CP (n=7) and BC (n=6) samples. The predicted miRNA binding sites within MDM2 3’UTR using miRcode transcriptome-wide miRNA target prediction tool were shown (Jeggari et al., 2012). (D) Relative miRNA expression determined by miRNA qPCR array of 84 miRNAs in CML CP CD34⁺ cells transduced with backbone or ADAR1 WT (n=3). (E) Relative miRNA expression in normal aged-matched (>55 year old) CD34⁺ cells (n=4) and BC CML CD34⁺ cells (n=3). (F and G) The expression of MDM2/p53 pathway transcripts, MDM2 (F) and TP53 (G) in progenitor population of normal peripheral blood (NPB), CML CP (n=7), and CML BC (n=6) determined by RNA-seq. (H) MDM2 expression in HEK293T cells transduced with ADAR1 WT, ADAR1^E912A alone or in combination with miR-155 (n=3 experiments). (I) Structure of “wt” or “edited” MDM2 3’UTR reporter construct with A-to-(I)G changes introduced at miRNA targeting sites (highlighted in red). The genomic loci of A-to-(I)G changes were also indicated with arrows. (J) The MDM2 3’UTR reporters were transfected into HEK293T cells and then challenged with miR-155 overexpressing lentivirus (n=3 experimental triplicate). The miRNA targeting efficiency was measured as the relative luciferase activity (GLuc/SEAP ratio). (K) MDM2 and LIN28B expression in BC CML CD34⁺ cells with shControl or shADAR1 (n=3). All graphs show mean with SEM and statistical analysis was calculated using the Student’s t-test. *p<0.05, **p<0.005, ***p<0.0005. See also Figure S4 and Table S3.