PMID: 2106665Jan 11, 1990Paper

A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta

Nucleic Acids Research
B NeupertL C Kühn

Abstract

We describe a simple method for the affinity purification of specific RNA-binding proteins. DNA sequences corresponding to the protein-binding site of the RNA are subcloned into an in vitro transcription vector between the T7 viral promoter and a poly(A) track. A polyadenylated RNA transcript is bound to poly(U)-Sepharose and subsequently incubated with a cellular extract prepurified on heparin-agarose. Specifically adsorbed proteins are recovered in high yield and purity from the affinity matrix by high salt elution. Using this method we isolated the iron regulatory factor (IRF), a cytoplasmic protein which binds to specific palindromic elements in the 5' and 3' untranslated sequences of ferritin and transferrin receptor mRNA, respectively. Activation and binding of this regulatory factor correlates with increased transferrin receptor mRNA stability and inhibition of ferritin translation. The purified factor from human placenta migrates as a monomer in gel chromatography, but is present in equimolar amounts of two proteins with molecular weights of 95 and 100 kDa when analysed by SDS/PAGE. The two proteins are highly related as judged by the identity of their isoelectric points and their specificity to form RNA-protein complexes.

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Citations

Nov 1, 1996·Veterinary Immunology and Immunopathology·M P MurtaughG Lin
Oct 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·T A RouaultR D Klausner
May 15, 1992·Proceedings of the National Academy of Sciences of the United States of America·A ConstableM W Hentze
Dec 15, 1992·Proceedings of the National Academy of Sciences of the United States of America·D J HaileR D Klausner
Mar 14, 1995·Proceedings of the National Academy of Sciences of the United States of America·J NakagawaC Moroni
Aug 6, 1996·Proceedings of the National Academy of Sciences of the United States of America·M W Hentze, L C Kühn
Jul 1, 1992·The Journal of Cell Biology·C Dargemont, L C Kühn
Jan 1, 1993·Molecular Biology of the Cell·R D Klausner, T A Rouault
Sep 1, 1991·British Journal of Haematology·L C Kühn
Jan 1, 1992·Biometals : an International Journal on the Role of Metal Ions in Biology, Biochemistry, and Medicine·T A RouaultJ B Harford
Jan 1, 1992·Critical Reviews in Biochemistry and Molecular Biology·L C Eriksson, G N Andersson
May 9, 2013·Wiley Interdisciplinary Reviews. RNA·Carina C Clingman, Sean P Ryder
Dec 1, 1992·Archives of Biochemistry and Biophysics·L ZhengH Zalkin
Dec 1, 1994·Baillière's Clinical Haematology·L C Kühn
Mar 30, 2001·The Journal of Biological Chemistry·A I Lalev, R N Nazar
Feb 1, 1992·Arteriosclerosis and Thrombosis : a Journal of Vascular Biology·Z C GarciaT L Innerarity
Jan 22, 2013·Biotechnology and Bioengineering·Spyridon KonstantinidisNigel Titchener-Hooker
Sep 1, 1991·European Journal of Haematology·M Worrall, M Worwood
Aug 15, 1992·Journal of Inorganic Biochemistry·L C Kühn, M W Hentze
Jul 31, 1996·Biochimica Et Biophysica Acta·P M Harrison, P Arosio
Nov 11, 1991·Biochimica Et Biophysica Acta·M W Hentze

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