ABSTRACT
The localization of metallothionein-1 (MT-1) mRNA to the perinuclear cytoskeleton is determined by a signal in the 3'untranslated region (3'UTR) and trans-acting binding proteins. The present study carried out detailed mapping of this signal and further characterized the binding to elongation factor 1 alpha (eEF1alpha) and other interacting proteins. Electrophoresis mobility shift assays demonstrated that shortening of a stem region proximal to nucleotides 66-76 abrogated binding. Full length recombinant rat eEF1alpha, and independently domains I and III, formed complexes with the mRNA. Proteins binding to biotinylated MT-1 3'UTR sequences were isolated using RNA-affinity techniques, and mass spectrometry identified histidine-tRNA ligase as one of the major MT-1 3'UTR binding proteins. We conclude that a 5-bp internal stem in the MT-1 3'UTR is critical for binding of eEF1alpha and histidine-tRNA ligase, and that binding of eEF1alpha is facilitated through domains I and III.
Subject(s)
3' Untranslated Regions/metabolism , Metallothionein/genetics , Peptide Elongation Factor 1/metabolism , 3' Untranslated Regions/chemistry , Animals , CHO Cells , Cricetinae , Cricetulus , Electrophoretic Mobility Shift Assay , Nucleic Acid Conformation , Rats , Transcription, GeneticABSTRACT
In eukaryotic cells, mRNA localization can provide local protein synthesis. Metallothionein-1 (MT-1) mRNA is associated with the perinuclear cytoskeleton, and this is essential for subsequent nuclear import of the protein. The present study defines the cis-acting localization signal and a trans-acting binding protein. Gel retardation and UV cross-linking assays using MT-1 3'UTR transcripts and CHO cell extracts revealed formation of a complex containing a approximately 50-kDa protein. Only localization-positive mutant transcripts competed for binding of this protein. Using an RNA affinity technique, Western blotting, mass spectrometry, and a supershift assay, the protein was identified as Elongation factor 1alpha (eEF1alpha). Mutation and deletion analysis showed that two regions, nucleotides 21-36 and 66-76, were required for both binding and localization. RNA-folding prediction combined with chemical and enzymatic probing experiments suggest that these regions are in juxtaposition within a stem/internal loop structure. Mutations that are predicted to alter this structure abrogate protein binding. Our hypothesis is that the cis-acting signal in MT-1 3'UTR is formed by this stem/internal loop, that it binds eEF1alpha, and that eEF1alpha-cytoskeleton interactions play a role in perinuclear mRNA localization.
