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

作者：Unterberger Alexander; Torrisani Jerome; Szyf Moshe^*

来源：Biological Procedures Online, 2008, 10: 47-57.

DOI：10.1251/bpo142

• DNA (cytosine-5)-methyltransferase 1
• DNA methylation
• RNA-binding proteins

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 similar to 40 kDa on DNMT1 3'-UTR triggered the destabilization of DNMT1 mRNA transcript during G(0)/G(1) 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.

• 出版日期2008-3-28