FIG. 4.

(A) Effects of removal of upstream cistron on IRES activity of recovered elements. Two micrograms of uncapped monocistronic RNAs consisting of the stem-loop structure followed by either the 50-nt sequences (PS1-4, NS1), no intervening sequence (None), the EMCV IRES variant (EMCV), or its mutant form (emcv) was used in in vitro HeLa translation reactions. Each reaction mixture also contained 500 ng of capped CAT RNA for comparative purposes, and the results of densitometric analysis of bands representing translated proteins, normalized to levels of translation in the PS1 reaction, are shown below. Translation of EGFP directed by NS1, None, and emcv was undetectable by densitometric quantitation. (B) Demonstration of context independence of elements PS3 and PS4. Two micrograms of uncapped monocistronic RNAs, consisting of either the 50-nt sequences (PS1-4, NS1), the multiple cloning site from pDF-N (MCS), the EMCV IRES from pDF-E (EMCV), or the mutant EMCV IRES from pDF-e (emcv) upstream of firefly luciferase (Luc), was used in in vitro HeLa translation reactions. Each reaction mixture also contained 500 ng of capped CAT RNA, and densitometric analysis of translated proteins is shown below. (C) PS3 and PS4 possess context-independent IRES activity in vivo. The 50-nt elements, MCS, or EMCV IRES were placed in a bicistronic plasmid between Renilla luciferase (Rluc) and firefly luciferase (Luc). Twenty-four hours after transfection into HEK293 cells, cells were analyzed for Luc and Rluc activity. The Luc/Rluc ratios, normalized to that found in cells transfected with the MCS-containing plasmid, are shown. The experiment was performed two separate times in triplicate.