The Iron-Responsive Element Binding Protein: A Method for the Affinity Purification of a Regulatory RNA-Binding Protein

A method of affinity purification of a regulatory protein that binds specific RNA sequences is described. RNAs containing the regulatory sequences are transcribed in vitro from oligonucleotide templates, biotinylated, and incubated with unfractionated cytosol. Specific RNA-protein complexes are boun...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 86; no. 15; pp. 5768 - 5772
Main Authors Rouault, Tracey A., Hentze, Matthias W., Haile, David J., Harford, Joe B., Klausner, Richard D.
Format Journal Article
LanguageEnglish
Published Washington, DC National Academy of Sciences of the United States of America 01.08.1989
National Acad Sciences
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Summary:A method of affinity purification of a regulatory protein that binds specific RNA sequences is described. RNAs containing the regulatory sequences are transcribed in vitro from oligonucleotide templates, biotinylated, and incubated with unfractionated cytosol. Specific RNA-protein complexes are bound in solution to avidin, and the resulting complex is bound to biotin-agarose beads. The cytosolic binding protein is released from the RNA in high salt, and a second round of purification yields an essentially homogeneous protein. Using this method, we have identified the protein in human liver that binds iron-responsive RNA regulatory sequences. Iron-responsive elements (IREs) are RNA stem-loops present in the mRNAs encoding ferritin and the transferrin receptor. IREs form the basis for the translational regulation of ferritin gene expression and the regulation of transferrin receptor mRNA degradation rates. The IRE binding protein purified by this technique migrates as a 90-kDa polypeptide on SDS/PAGE. The interaction of the purified protein with IRE-containing RNAs can be detected by gel-mobility shift assays or by covalent crosslinking induced by UV irradiation.
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ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.86.15.5768