Nucleic Acids Research, 1990, Vol. 18, No. 1 51-55
© 1990
MOLECULAR BIOLOGY |
A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta
Swiss Institute for Experimental Cancer Research, Genetics Unit CH-1066 Epalinges, Switzerland
* To whom correspondence should be addressed
Received October 26, 1989. Accepted November 28, 1989.
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.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. R. ALLERSON, A. MARTINEZ, E. YIKILMAZ, and T. A. ROUAULT A high-capacity RNA affinity column for the purification of human IRP1 and IRP2 overexpressed in Pichia pastoris RNA, March 1, 2003; 9(3): 364 - 374. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wu and J. Bag Negative Control of the Poly(A)-binding Protein mRNA Translation Is Mediated by the Adenine-rich Region of Its 5'-Untranslated Region J. Biol. Chem., December 18, 1998; 273(51): 34535 - 34542. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Tanguay and D. R. Gallie Isolation and Characterization of the 102-Kilodalton RNA-binding Protein That Binds to the 5' and 3' Translational Enhancers of Tobacco Mosaic Virus RNA J. Biol. Chem., June 14, 1996; 271(24): 14316 - 14322. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Guo, F. M. Brown, J. D. Phillips, Y. Yu, and E. A. Leibold Characterization and Expression of Iron Regulatory Protein 2 (IRP2) J. Biol. Chem., July 14, 1995; 270(28): 16529 - 16535. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Peyret, P. Perez, and M. Alric Structure, Genomic Organization, and Expression of the Arabidopsis thaliana Aconitase Gene J. Biol. Chem., April 7, 1995; 270(14): 8131 - 8137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C W Siebel, R Kanaar, and D C Rio Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI. Genes & Dev., July 15, 1994; 8(14): 1713 - 1725. [Abstract] [PDF]
|
|
|
|
 |
|
 L. Goessling, S Daniels-McQueen, M Bhattacharyya-Pakrasi, J. Lin, and R. Thach Enhanced degradation of the ferritin repressor protein during induction of ferritin messenger RNA translation Science, May 1, 1992; 256(5057): 670 - 673. [Abstract] [PDF]
|
|
|
|
|
|
A. I. Lalev and R. N. Nazar A Chaperone for Ribosome Maturation J. Biol. Chem., May 11, 2001; 276(20): 16655 - 16659. [Abstract] [Full Text] [PDF]
|
|