To date, two different lengths of 5′ UTR (384 nts OTX015 order in accession number NM_001123[25-MAR-2011] and 187 nts in accession numbers NM_001123[31-OCT-2010] and HSU_50196) have been deposited in GeneBank. Because the 384 nts form has been considered to be

a unique species in testis tissue, we performed 5′ RACE analysis to determine the length of the 5′ UTR of ADK mRNA in ORL8 or OR6 cells. Sequence analysis was carried out using more than 20 cDNA clones obtained from each cell line. Consequently, we obtained 319 and 125 nts as the major 5′ UTR species in ORL8 and OR6 cells, respectively. We confirmed these results by RT-PCR analysis using four different primer sets for the 5′ UTR (Fig. 3A). The amount of 384 nts species in ORL8 cells was estimated to be less than one thirtieth the amount of the 319-nts species (Fig. 3A). These results indicate that the length of 5′ UTR in ORL8 cells is longer than that in OR6 cells. From these results, we considered the possibility that the length of the 5′ UTR is associated with the protein level of ADK. To test this possibility, we first compared the expression levels of ADK in various human hepatoma cell lines and human immortalized hepatocyte lines. Low expression level of ADK was observed in HT17 and Hep3B cells as well as OR6 cells, although the other cell lines, including ORL8, HuH-6, MK0683 in vitro HepG2, HLE, and PH5CH8 cells,

showed high expression level of ADK (Fig. 3B and Supporting Fig. 6). We next performed quantitative RT-PCR analysis on the 5′ UTR using total RNAs from OR6, ORL8, HT17, and PH5CH8 cells. Consequently, we found that the 319 nts

species of the 5′ UTR was abundant in PH5CH8 cells, but not in HT17 cells (Fig. 3C), Venetoclax chemical structure indicating good correlation between the amount of 319 nts species and the amount of ADK protein (Fig. 3B,C). These results suggest that the 319 nts species of 5′ UTR is involved in the high protein level of ADK. From the results of 5′ UTR analysis, we assumed that the 319 nts species of the 5′ UTR possesses IRES activity because it is GC rich (72%) and highly structured (estimated ΔG = −110.7 kcal/mol), and because it contains an upstream ORF for 70 amino acids. To test this assumption, we used a bicistronic dual luciferase reporter assay system for the detection of IRES activity (Fig. 4A). As a positive control, we constructed a pGL4-based reporter plasmid containing HCV IRES (377 nts; 341 nts in the 5′ UTR plus the first 36 nts in the Core-encoding region). We next replaced the HCV IRES structure in this plasmid with several different lengths (forward or reverse direction) of the 5′ UTR derived from ORL8 cells. ORL8c cells were transfected with these plasmids, and at 48 hours after transfection, dual luciferase assays were performed. Consequently, we found that the forward 319 nts, but not the forward 125 nts, of 5′ UTR clearly showed IRES activity at the same level as HCV IRES (Fig. 4B).