Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs

In addition to the cap-dependent mechanism, eukaryotic initiation of transl ation can occur by a cap-independent mechanism which directs ribosomes to d efined start codons enabled by internal ribosome entry site (IRES) elements . IRES elements from poliovirus and encephalomyocarditis virus are often us ed to construct bi- or oligocistronic expression vectors to co-express vari ous genes from one mRNA. We found that while cap-dependent translation init iation from bicistronic mRNAs remains comparable to monocistronic expressio n, internal initiation mediated by these viral IRESs is often very ineffici ent. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cyt okines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bic istronic setting is crucial for IRES-dependent translation of the second ci stron. A systematic examination of expression of reporter cistrons from bic istronic mRNAs with respect to position was carried out. Using the dual luc iferase assay system we show that the composition of reading frames on a bi cistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the n ature of the IRES element influence translation strength the dominant deter minant is the nature and the arrangement of cistrons on the mRNA.