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

Lookup NU author(s): Dr Ian Mickleburgh, Dr Herve Chabanon, David Nury, Brian Burtle, Professor Zofia Chrzanowska-Lightowlers, Professor John Hesketh

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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.


Publication metadata

Author(s): Mickleburgh I, Chabanon H, Nury D, Fan K, Burtle B, Chrzanowska-Lightowlers Z, Hesketh J

Publication type: Article

Publication status: Published

Journal: RNA

Year: 2006

Volume: 12

Issue: 7

Pages: 1397-1407

ISSN (print): 1355-8382

ISSN (electronic): 1469-9001

Publisher: Cold Spring Harbor Laboratory Press

URL: http://dx.doi.org/10.1261/rna.2730106

DOI: 10.1261/rna.2730106

PubMed id: 16723660


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