Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location

Abstract

The internal ribosomal entry site (IRES) from encephalomyocarditis virus (EMCV) is a popular RNA element used widely in experimental and pharmaceutical applications to express proteins in eukaryotic cells or cell-free extracts. Inclusion of the wild-type element in monocistronic or bicistronic messenger RNAs (mRNAs) confers a high level of cap-independent translation activity to appropriately configured cistrons. The history of this element and the experimental consequences of sequence derivations inherent to commercial IRES vectors are less well known. Compared head-to-head with dual-luciferase reporter constructs, a native EMCV IRES in a bicistronic configuration directed 8- to 10-fold more protein than a simi larly configured pIRES vector. It also produced nearly twice as much protein as pCITE®-1, an early monocistronic iteration, harboring a suboptimal A7 sequence in a crucial structural motif. The results indicate that investigators should be aware of and carefully report the sequence of their IRES in any comparative study. The preferred IRES (viral bases 273–845) and the minimum IRES (viral bases 400–836) for optimum activity are illustrated.