Fine-tuning levels of heterologous gene expression in plants by orthogonal variation of the untranslated regions of a nonreplicating transient expression system

A transient expression system based on a deleted version of Cowpea mosaic virus (CPMV) RNA-2, termed CPMV-HT, in which the sequence to be expressed is positioned between a modified 5 ' UTR and the 3 ' UTR has been successfully used for the plant-based expression of a wide range of proteins, including heteromultimeric complexes. While previous work has demonstrated that alterations to the sequence of the 5 ' UTR can dramatically influence expression levels, the role of the 3 ' UTR in enhancing expression has not been determined. In this work, we have examined the effect of different mutations in the 3 ' UTR of CPMV RNA-2 on expression levels using the reporter protein GFP encoded by the expression vector, pEAQexpress-HT-GFP. The results showed that the presence of a 3 ' UTR in the CPMV-HT system is important for achieving maximal expression levels. Removal of the entire 3 ' UTR reduced expression to approximately 30% of that obtained in its presence. It was found that the Y-shaped secondary structure formed by nucleotides 125-165 of the 3 ' UTR plays a key role in its function; mutations that disrupt this Y-shaped structure have an effect equivalent to the deletion of the entire 3 ' UTR. Our results suggest that the Y-shaped secondary structure acts by enhancing mRNA accumulation rather than by having a direct effect on RNA translation. The work described in this paper shows that the 5 ' and 3 ' UTRs in CPMV-HT act orthogonally and that mutations introduced into them allow fine modulation of protein expression levels.