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 1α.
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November 2004
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Conference Article|
October 26 2004
Isolation and identification of a protein binding to the localization element of Metallothionein-1 mRNA
I. Mickleburgh;
I. Mickleburgh
*Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, U.K.
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B. Burtle;
B. Burtle
*Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, U.K.
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D. Nury;
D. Nury
*Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, U.K.
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H. Chabanon;
H. Chabanon
*Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, U.K.
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Z. Chrzanowska-Lightowlers;
Z. Chrzanowska-Lightowlers
†School of Neurology, Neurobiology and Psychiatry, University of Newcastle upon Tyne, U.K.
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J.E. Hesketh
J.E. Hesketh
1
*Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, U.K.
1To whom correspondence should be addressed (email J.E.Hesketh@ncl.ac.uk).
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Publisher: Portland Press Ltd
Received:
July 19 2004
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© 2004 The Biochemical Society
2004
Biochem Soc Trans (2004) 32 (5): 705–706.
Article history
Received:
July 19 2004
Citation
I. Mickleburgh, B. Burtle, D. Nury, H. Chabanon, Z. Chrzanowska-Lightowlers, J.E. Hesketh; Isolation and identification of a protein binding to the localization element of Metallothionein-1 mRNA. Biochem Soc Trans 1 November 2004; 32 (5): 705–706. doi: https://doi.org/10.1042/BST0320705
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