Elongation factor 1α binds to the region of the metallothionein-1 mRNA implicated in perinuclear localization - Importance of an internal stem-loop

Abstract

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 cisacting 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 ∼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 1α (eEF1α). 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 eEF1α, and that eEF1α-cytoskeleton interactions play a role in perinuclear mRNA localization. Copyright © 2006 RNA Society.