title: Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation.
abstract: In this paper we demonstrate that RNA sequences present upstream and downstream of a reporter gene coding region play an important role in determining the amount of protein produced from an mRNA. A translational enhancer, omega, derived from tobacco mosaic virus, when present at the 5'-end of beta-glucuronidase mRNA increased the efficiency of translation 16-fold to 18-fold in electroporated tobacco or carrot protoplasts, and threefold to 11-fold in maize or rice protoplasts. The presence of omega did not alter the half-life of the mRNA in vivo. We also demonstrate for the first time that a minimum polyadenylated tail length of 25 adenylate residues is sufficient to substantially increase the expression and half-life of the reporter mRNA in plants. When in vitro-produced mRNAs were synthesized such that extra sequence was added to the 3'-end of the poly(A) tail, however, the final level of expression was decreased up to 80%. Omega, the translational enhancer, and a poly(A) tail function independently of each other; their combined effect on translation, when both are present in an mRNA, is the multiplication of their individual effects. Histochemical analysis for the presence of beta-glucuronidase in tobacco established that virtually all viable cells receive mRNA during electroporation. Video image analysis of tobacco protoplasts electroporated with luciferase mRNA demonstrated that there is a wide range in the level of expression of this marker. Carrier RNA, when present during electroporation, had only a modest effect on increasing mRNA uptake. Reporter mRNA expression in electroporated protoplasts was directly proportional to the input mRNA up to at least 30 micrograms/ml.
authors.affiliation: Department of Biological Sciences, Stanford University, California 94305-5020.
authors.forename: D R
authors.initials: DR
authors.lastname: Gallie
authors.forename: W J
authors.initials: WJ
authors.lastname: Lucas
authors.forename: V
authors.initials: V
authors.lastname: Walbot
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D005966:Glucuronidase / Q000235:genetics; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D010784:Photomicrography; D010944:Plants / Q000235:genetics* / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D011523:Protoplasts / Q000378:metabolism*; D012315:RNA Caps; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: D012315:RNA Caps; D012333:RNA, Messenger; D011061:Poly A; D005966:Glucuronidase