A repeated IMP-binding motif controls oskar mRNA translation and anchoring independently of Drosophila melanogaster IMP

Zip code–binding protein 1 (ZBP-1) and its Xenopus laevis homologue, Vg1 RNA and endoplasmic reticulum–associated protein (VERA)/Vg1 RNA-binding protein (RBP), bind repeated motifs in the 3′ untranslated regions (UTRs) of localized mRNAs. Although these motifs are required for RNA localization, the necessity of ZBP-1/VERA remains unresolved. We address the role of ZBP-1/VERA through analysis of the Drosophila melanogaster homologue insulin growth factor II mRNA–binding protein (IMP). Using systematic evolution of ligands by exponential enrichment, we identified the IMP-binding element (IBE) UUUAY, a motif that occurs 13 times in the oskar 3′UTR. IMP colocalizes with oskar mRNA at the oocyte posterior, and this depends on the IBEs. Furthermore, mutation of all, or subsets of, the IBEs prevents oskar mRNA translation and anchoring at the posterior. However, oocytes lacking IMP localize and translate oskar mRNA normally, illustrating that one cannot necessarily infer the function of an RBP from mutations in its binding sites. Thus, the translational activation of oskar mRNA must depend on the binding of another factor to the IBEs, and IMP may serve a different purpose, such as masking IBEs in RNAs where they occur by chance. Our findings establish a parallel requirement for IBEs in the regulation of localized maternal mRNAs in D. melanogaster and X. laevis

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.

Expanding the Diversity of Imaging-Based RNAi Screen Applications Using Cell Spot Microarrays

microarray