Translational enhancer function of an RNA trafficking sequence

First, this thesis describes the use of fluorescence quenching and dequenching to analyze molecular interactions of RNA in vitro and in vivo. RNA degradation causes changes in intra-molecular interactions resulting in fluorescence dequenching manifested as a time dependent increase in fluorescence quantum yield that can be measured spectroscopically in vitro or microscopically in vivo. Intermolecular interactions between RNA and other macromolecular ligands cause fluorescence superquenching manifested as a time dependent decrease in quantum yield that can also be measured microscopically in living cells. This assay provides a powerful system for analysis of dynamic and kinetic aspects of both RNA degradation (dequenching) and RNA trafficking (superquenching) in living cells.^ Second, I developed an in vivo translation assay using green fluorescent protein (GFP) as a reporter, to follow, in real time, the processes of translation and transport for a particular localized mRNA within a single living cell. My studies demonstrate that the RTS, which was originally identified as an RNA trafficking sequence in MBP mRNA, functions as an enhancer of cap-dependent translation in vivo and in vitro. This represents the first specific translational enhancer to be identified in a mammalian system. The translational enhancer function of the RTS is position, copy number and cell type independent and requires hnRNP A2, which binds to the RTS, as a trans-acting factor. It is proposed that RTS/hnRNP A2 cis/trans determinants are required for both transport and translational regulation of a family of different mRNAs.