Direct isolation of specific RNA-interacting proteins using a novel affinity medium.
Isolation of proteins that specifically interact with a given RNA or RNA regulation element is essential for studies on the molecular mechanisms of gene expression. Here, a novel method for direct isolation of such interacting proteins is described. It uses an affinity medium that consists of an interacting RNA with an artificially added 'tail', which is annealed to one end of a DNA 'arm', the other end of which is fixed covalently on the surface of aminosilanized glass powder. Thus the RNA itself is fully suspending, facilitating its interactions with proteins in its natural conformation. The proteins bound on the interacting RNA are eluted and subjected to SDS-PAGE, and the Coomassie-stained protein bands are cut and subjected to mass spectrometry (MS) analysis. Using this method, three proteins specifically interacting with the C/EBPbeta 3'-untranslated region (3'-UTR) RNA were isolated and identified. This method is simple and convenient, and the DNA-glass powder medium can be used repeatedly.
Liu
Ding-Gan
DG
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China. dgliu@sibs.ac.cn
Sun
Li
L
D020413:3' Untranslated Regions / Q000378:metabolism; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000302:isolation & purification; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005898:Glass / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D015347:RNA Probes / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000302:isolation & purification*
D020413:3' Untranslated Regions; D022782:CCAAT-Enhancer-Binding Protein-beta; D015347:RNA Probes; D016601:RNA-Binding Proteins; D004247:DNA