Fig. 2. MSI2 promotes MYC protein expression through 5′UTR and 3′UTR-dependent processes.

A Validation of MSI2-RIP-seq data by RT-qPCR. RIP was performed with anti-MSI2 monoclonal antibody and candidate mRNAs were quantitated using RT-qPCR. Expression analysis showed the enrichment for MSI2-target MYC and MIR22HG gene products was augmented in MSI2 overexpressing (OE) Huh7 cells and that was reduced in sh-MSI2-silenced cells. B Diagram of transcription start sites for MYC and corresponding protein products. (Inset) Western blot results showed an increase in longer MYC protein expression in HCC and TICs whereas primary hepatocytes showed relatively low level of longer MYC isoform. C Effect of MSI2 silencing on MYC level. Two different shRNAs against MSI2 were tested as shown. Uncropped film images are shown in separate files. D Basal expression of MYC and MSI2 in primary hepatocytes vs. Huh7 cells (Left panel). Expression level of MSI2 and corresponding effects on MYC. Western Blot results showed an increase in MYC protein expression in MSI2 overexpressing cells whereas a decrease in MYC protein level was seen in MSI2-silenced cells. β-ACTIN was used as loading control (Right panel). E Overexpression and knockdown of MSI2 exerts minimal effect on MYC mRNA level. RT-qPCR was employed for examination of expression levels of MYC mRNA. GAPDH was used for normalization. F MSI2 silencing did not affect protein stability of MYC. Huh7 cells were transduced with scramble shRNA (sh-scr) or shRNA targeting MSI2. Seventy-two hours after transduction, cycloheximide (CHX) was added and cells were harvested at the indicated times. MYC protein levels were detected by immunoblotting (Top panel). Results of a representative experiment (n = 3) are plotted as percentage of starting MYC protein level for half-life determination (Bottom panel). G MSI2 regulates MYC translation through the 5′- and 3′-untranslated regions of the MYC mRNA. Schematic illustration of MYC expression constructs which contain coding sequence only (C) and in addition to coding sequence either the 3′UTR (C3′) or the 5′UTR (5′C) or a combination of both (5′C3′), as indicated (Left panel). Huh7 cells stably expressing sh-scramble or sh-MSI2 were transfected with the indicated MYC constructs (Center top panel). Huh7 cells were co-transfected with vector or MSI2 and the indicated MYC cDNA constructs (Center bottom panel). Cell lysates harvested 48 h after transfection were subjected to SDS-PAGE and immunoblotting analysis with MYC and MSI2 antibodies. The densitogram of immunoblots for MYC and MSI2 protein level were quantified by ImageJ. (Right panel). H A hypothetical model demonstrates that MSI2 binding stimulates IRES-dependent initiation of MYC translation.