A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation
Key findings
Defined an internal ribosome entry site (IRES) within the EMCV 5' nontranslated region by mapping a critical segment (nt 260–484) that drives efficient, cap-independent translation in vitro. Using synthetic mono- and dicistronic mRNAs, the authors showed this RNA fragment enables ribosomes to initiate at an internal site, producing downstream protein independent of 5' cap status and excluding leaky scanning or read-through as explanations.
Experimental evidence came from complementary systems—micrococcal nuclease-treated HeLa cell extracts and rabbit reticulocyte lysates—demonstrating consistent activity across extracts. Truncation and mapping localized the function to the 260–484 region of the EMCV 5' UTR, and bicistronic constructs with this segment produced downstream translation even when upstream initiation was intact, directly supporting an internal-entry mechanism.
This work established the IRES concept for picornavirus RNAs and provided the experimental foundation for bicistronic expression systems. The EMCV-derived IRES became a widely used tool for co-expression constructs and for studies of cap-independent translation, catalyzing widespread adoption of IRES elements in molecular biology and gene-expression technologies.