Internal ribosome entry sequences

The plasmid vector pTEV-DHFR was used in this study. Internal ribosome entry sequence originated from tobacco etch virus (TEV) was locatai at upstream of dihydrofolate reductase (DHFR) gene. 

In a special class of internal initiations, the ribosomes bind at a specific internal AUG codon to start translation. The internal AUG may be located as far as 800 bases from the 5 end as in picornaviruses with uncapped, (+)-sense genomic RNAs. According to the model of cap-independent translation or internal ribosome entry (55), the internal RBS or ribosome landing pad is a 5 UTR sequence that forms a stable stem-loop structure. Hepatitis B virus (HBV) and some retroviruses also use an internal AUG codon, rather than ribosomal frameshifting, to express their reverse transcriptase genes (56,57). 

During a secondary screen we validated 6 of the 17 hits for their capacity to selectively reduce luciferase expression translationally driven from the APP 5 UTR but maintain co-expression of GFP with an internally controlled viral Internal Ribosome Entry Site (Dicistronic construct) [7]. For this puipose SY5Y neuroblastoma cells were stably transfected with the dicistronic construct (pJRl). The FDA drugs, dimercaptopropanol, paroxetine, azithromycin, quinoline-gluconate, tamsulosin and atorvastatin each suppressed luciferase reporter mRNA translation via the 146 nt APP 5 UTR sequence [111]. The iron... 

Figure 5, Primary screen far drugs targeted to the APP S untranslated region. In a screen of a library of 1,200 FDA-pre-approved compounds phenserine(ACHEi) and the potent intracellular iron chelator, desferrioxamine, were the validated positive control drugs that suppressed APP mRNA translation through APP 5 UTR sequences. In this transfection based screen several lead APP-5 UTR directed drugs were identified to limit luciferase gene expression driven by the APP 5 UTR. As an internal selectivity control for this screen, downstream dicistronic GFP gene expression (at the translational level by a viral internal ribosome entry site (IRES)) was unresponsive to drug action. Several leads (i,e, dimercaptopropanol) were identified to be chelators as described.

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