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Published online 23 February 2004

Nucleic Acids Research, 2004, Vol. 32, No. 4 1308-1317
© 2004 Oxford University Press

Identification of cellular proteins enhancing activities of internal ribosomal entry sites by competition with oligodeoxynucleotides

Kobong Choi, Jong Heon Kim, Xiaoyu Li1, Ki Young Paek, Sang Hoon Ha, Sung Ho Ryu, Eckard Wimmer1 and Sung Key Jang*

Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang, Kyungbuk 790-784, Korea and 1 Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794, USA

*To whom correspondence should be addressed. Tel: +82 54 279 2298; Fax: +82 54 279 8009; Email: sungkey{at}postech.ac.kr

The translation of numerous eukaryotic mRNAs is mediated by internal ribosomal entry sites (IRESs). IRES-dependent translation requires both canonical translation initiation factors and IRES-specific trans-acting factors (ITAFs). Here we report a strategy to identify and characterize ITAFs required for IRES-dependent translation. This process involves steps for identifying oligodeoxynucleotides affecting IRES-dependent translation, purifying proteins interacting with the inhibitory DNA, identifying the specific proteins with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, and confirming the roles of these proteins in IRES-dependent translation by depletion and repletion of proteins from an in vitro translation system. Using this strategy, we show that poly(rC)-binding proteins 1 and 2 enhance translation through polioviral and rhinoviral IRES elements.


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