RNA pseudoknot domain of tobacco mosaic virus can functionally substitute for a poly(A) tail in plant and animal cells

Abstract

The genomes of many RNA viruses terminate in a tertiary structure similar to the L-conformation of tRNAs and this structure is recognized by many tRNA-specific enzymes such as aminoacyl-tRNA synthetase. Virtually the entire 3′-untranslated region (UTR) of tobacco mosaic virus (TMV) RNA is involved in an extended tertiary structure containing, in addition to a tRNA-like structure, a pseudoknot domain that lies immediately upstream. Although the functions of these structures are not well understood, they are essential to the virus. We demonstrate that the addition of the 204-base TMV 3′-untranslated region to foreign mRNA constructs can increase gene expression up to 100-fold compared to nonadenylated mRNA. The 3′-UTR of TMV was equal to or greater than a polyadenylated tail in enhancing gene expression in electroporated dicot and monocot protoplasts. The TMV 3′-UTR is functionally similar to a polyadenylated tail in that it increases mRNA stability and translation and must be positioned at the 3′ terminus to function efficiently. Similar effects on expression were observed in Chinese hamster ovary cells, demonstrating that the sequence functions in a wide range of eukaryotes. When the extended tertiary structure was dissected, the upstream pseudoknot domain was found to be largely responsible for increasing expression. The inclusion of the tRNA-like structure, however, was important for full regulation.