Fig. 3: MSI2 expression stimulates the MYC, JUN and HCV IRES activity in Huh7 cells.

A Ratio of IRES-containing genes per 21,000 genes was increased after anti-MSI2 RIP-seq enrichment. B Molecular modeling of MSI2 binding motif on the MYC IRES. The MSI2 binding consensus sequence has complementarity to the CUG initiation codon (non-AUG) of MYC IRES and antagonizes binding to the 3′ end of 18S ribosomal RNA. C Comparison of the MSI2 binding sites (in color) of IRES elements of MYC and JUN mRNAs and HCV RNA. The representative secondary structures are shown. D Schematic representation of the reporter constructs pRMF, pRHCV, pRAF/pRBF and pRF. pRF is the parental bicistronic reporter vector lacking an IRES element. pRMF is the construct containing the MYC-IRES. Other bicistronic constructs contained IRES elements from HCV (pRHCV), APAF1 (pRAF) and BAG1 (pRBF) as indicated. E Dependence of IRES-driven luciferase activity on MSI2 binding motif and response to MSI2 expression. Luciferase activity from various IRES reporter plasmids as indicated were assayed under conditions of MSI2 overexpression (OE) or silencing. pRMF reporter activity showed the greatest dependence on MSI2 for maximal relative luminescence activity compared to other IRES reporters. Little or negligible change of luciferase activity was observed for reporter vectors without IRES (pRF) or with APAF-1 or BAG-1 IRES elements. Complementation of MSI2 binding motif to APAF-1 or BAG-1 IRES restored IRES-driven luciferase activity upon MSI2 expression. F CD133(+) TICs have elevated levels of MYC IRES activity, indicating that MSI2^High cells support corresponding elevated levels of synthesized MYC protein. G EMSA assays were performed with recombinant MSI2 protein or MSI2 protein with deleted RNA-binding domain (ΔRBD) in the presence or absence of wild type or mutant MYC IRES RNA probes. H The MSI2 binding consensus sequence has homology to JUN IRES. I Schematic representation of the reporter constructs pR-JUN-F and pR-VEGFA-F. phpR-JUN-F and phpR-VEGFA-F were constructed by inserting the hairpin sequence (−55 Kcal/mol) upstream of Renilla luciferase coding sequence. These hairpin-containing plasmids were made to inhibit translational initiation by ribosome scanning from the 5′ cap structure and to exclude the impact of leaky SV40 promoter activity on mRNA levels. J Luciferase activities were determined in the presence or absence of MSI2. Data represent mean ± S.D. from three independent experiments. *P < 0.05. K The MSI2 binding consensus sequence has homology to HCV IRES. L Reduction of HCV IRES activity with mutations of HCV IRES MSI2-binding sites. Reduction of HCV IRES activity after mutation of HCV IRES MSI2-binding sites. M (Left panel) Luciferase assays were performed in the presence or absence of mutations in HCV IRES MSI2-binding sites. Synthetic MSI2-binding site-mimetic oligonucleotides were transduced into HCV infected or sub-genomic replicon Huh7 cells. (Right panel) Repression of HCV replication and sub-genomic replicon by antisense oligos to HCV IRES MSI2-binding sites. RT-qPCR results of HCV RNA level in HCV infected or sub-genomic replicon Huh7 cells. CD133(+) TICs have elevated levels of HCV RNAs, indicating that MSI2^High cells supported elevated levels of HCV RNA.