Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence

Abstract

Eukaryotic messenger RNAs are translated with unequal efficiencies in vivo and in vitro and the molecular basis of this phenomenon is not understood. As an approach to understanding the role of the 5' untranslated leader sequence in regulating mRNA translational efficiency, chimaeric mRNAs have been generated by joining a heterologous leader to complementary DNA (cDNA) sequences, followed by in vitro transcription using SP6 RNA polymerase and in vitro protein synthesis. We used the untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), a well-translated, highly competitive message, to replace the leader sequence of barley alpha-amylase (B alpha A) and human interleukin 1 beta (IL-1 beta) cDNAs. Deletion of transcribed vector sequences and replacement of the native untranslated region with the AMV RNA 4 leader can result in as much as a 35-fold increase in mRNA translational efficiency; moreover, the translational efficiency of the chimaeric mRNAs containing the AMV RNA 4 leader is at least as great as that of virion RNA 4. The results suggest that the chimaeric AMV-mRNAs have either a higher relative affinity or a diminished requirement for a limiting component(s) of the translational machinery; in addition, it may be feasible, through use of heterologous leader sequences, to increase expression of engineered genes or cDNAs without changing the antigenic or biological properties of the encoded protein.