ABSTRACT
Subcellular localization of mRNAs is a key mechanism for the synthesis of proteins close to their site of function. The mRNA encoding MT-1 (metallothionein-1) is localized in the perinuclear cytoplasm, where it is associated with cytoskeletal-bound polysomes. This localization relies on sequences present in the 3'-UTR (3'-untranslated region). The present study aims to characterize the cis-acting localization element(s) within the 3'-UTR. Using transfected cells expressing tagged MT-1 differing in their 3'-UTRs (deleted or mutated), the section(s) of this region required for directing MT-1 transcripts to the perinuclear cytoplasm has been investigated. Different 3'-UTRs were also used in UV cross-linking experiments that highlighted two distinct regions (nt 26-30 and 66-76) necessary for the binding of a protein of approx. 50 kDa, presumably involved in the mRNA targeting. The poor sequence homology between the MT-1 3'-UTR of various species, together with the bipartite nature of the required cis-element, indicates the involvement of a particular structure in the localization signal. The secondary structure of the MT-1 3'-UTR was investigated using enzymic and chemical probing. Current structural analysis of mutant 3'-UTRs will allow the critical structural features of the MT-1 mRNA perinuclear localization signal to be defined.
Subject(s)
Metallothionein/biosynthesis , Metallothionein/genetics , RNA, Messenger/chemistry , 3' Untranslated Regions , Actins/genetics , Animals , Base Sequence , Cell Nucleus/metabolism , Cricetinae , Cross-Linking Reagents/pharmacology , Cytoplasm/metabolism , Cytoskeleton/metabolism , Humans , Mice , Molecular Sequence Data , Mutation , Polyribosomes/chemistry , Protein Structure, Secondary , RNA, Messenger/metabolism , Response Elements , Sequence Homology, Nucleic Acid , Ultraviolet RaysABSTRACT
mRNA localization provides a mechanism for localized protein synthesis. mRNAs encoding certain proteins, including c-MYC, c-FOS, MT-1 (Metallothionein-1) and vimentin, are localized around the nuclei of mammalian cells and are associated with the cytoskeleton. Targeting of these mRNAs to the perinuclear cytoplasm is mediated by elements within their 3'-UTRs (3'-untranslated regions), but many of the trans-acting proteins remain unidentified. UV cross-linking assays using radiolabelled transcripts indicated that a protein of approx. 50 kDa (from the Chinese-hamster ovary cell extracts) bound to the MT-1 3'-UTR sequence. Competition experiments using unlabelled mutant 3'-UTR RNAs revealed that the binding of this protein is specific to localization-positive mutants. Isolation of a 50 kDa protein was achieved by an RNA affinity-based method in which biotinylated MT-1 3'-UTR RNA was anchored to paramagnetic beads. Bound proteins were eluted and analysed by SDS/PAGE. The 50 kDa protein was extracted from the gel, subjected to trypsin digestion and identified by matrix-assisted laser-desorption/ionization-time-of-flight mass spectrometry as eukaryote elongation factor 1alpha.