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A method for purification, identification and validation of DNMT1 mRNA binding proteins

Biological Procedures Online volume 10, pages 47–57 (2008)Cite this article

Abstract

DNA methyltransferase 1 (DNMT1) is the enzyme responsible for the maintenance of DNA methylation patterns during cell division. DNMT1 expression is tightly regulated within the cell cycle. Our previous study showed that the binding of a protein with an apparent size of ∼40 kDa on DNMT1 3′-UTR triggered the destabilization of DNMT1 mRNA transcript during Go/G1 phase. Using RNA affinity capture with the 3′-UTR of DNMT1 mRNA and matrix- assisted laser desorption-time of flight tandem mass spectrometry (MALDI-TOF-MS-MS) analysis, we isolated and identified AUF 1 (AU-rich element ARE:poly-(U)-binding/degradation factor) as the binding protein. We then validated the role of this protein in the destabilization of DNMT1 mRNA. In this report, we detail the different approaches used for the isolation, the identification of a RNA binding protein and the validation of its role.

Abbreviations

AUF1:

AU-rich element ARE:poly-(U)-binding/degradation factor

DNMT1:

DNA methyltransferase 1

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Authors and Affiliations

  1. Department of Pharmacology and Therapeutics, McGill University, 3655, Sir William-Osler Promenade, H3G 1Y6, Montreal, QC

    Moshe Szyf Ph.D.

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  1. Alexander Unterberger
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  2. Jérôme Torrisani
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  3. Moshe Szyf Ph.D.
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Correspondence to Moshe Szyf Ph.D..

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These authors contributed equally to this manuscript

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Unterberger, A., Torrisani, J. & Szyf, M. A method for purification, identification and validation of DNMT1 mRNA binding proteins. Biol. Proced. Online 10, 47–57 (2008). https://doi.org/10.1251/bpo142

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  • DOI: https://doi.org/10.1251/bpo142

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Biological Procedures Online

ISSN: 1480-9222

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