The intracellular localisation of metallothionein isoform mRNA : role of the 3' untranslated region and the cytoskeleton

• Main Author: Mahon, P.
• Published: University of Aberdeen 1997
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.593020

For the purpose of creating a novel cell culture model to study the physiological significance of MT mRNA localisation and also to initially investigate the possibility of whether any other region of the MT-1 mRNA (apart from the 3' UTR) is involved in intracellular targeting. CHO cells (which do not express MT genes) were transfected with constructs consisting of the wild-type MT-1 cDNA as well as the 5' UTR and coding region of the MT-1 cDNA linked to the glutathione peroxidase (GSH-Px) 3' UTR (which is not thought to contain a localisation signal). <I>In situ</I> hybridisation using a digoxigenin-labelled riboprobe revealed that the native MT-1 transcript was localised to the perinuclear region of the cytoplasm in the transfected CHO cells whereas the hybrid MT1-GSH-Px transcripts were distributed evenly throughout the cytoplasm in a random manner. Clones expressing similar levels of MT-1 protein were then established from these cell lines for the purpose of functional studies. These studies suggest that the MT-1 mRNA is localised in rat hepatoma cells and is associated with the cytoskeleton whilst in the process of translation, with the MT-II mRNA showing a weaker association; suggesting compartmentalisation of these mRNA isoforms. Furthermore, the observed ability of the MT-I 3' UTR to target a reporter sequence to the cytoskeleton and to the perinuclear cytoplasm as well as the loss of localisation observed upon replacement of the MT-I 3' UTR with that of the GSH-Px 3' UTR suggests that the 3' UTR of the MT-1 transcript contains a <I>cis</I>-acting localisation signal which is responsible for the observed intracellular targeting and cytoskeleton association of this transcript.