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Originally published In Press as doi:10.1074/mcp.M600384-MCP200 on February 26, 2007.
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Molecular & Cellular Proteomics 6:1049-1058, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

The Myotonic Dystrophy Type 2 Protein ZNF9 Is Part of an ITAF Complex That Promotes Cap-independent Translation*,S

Vincent R. Gerbasi{ddagger} and Andrew J. Link§

From the Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

The 5'-untranslated region of the ornithine decarboxylase (ODC) mRNA contains an internal ribosomal entry site (IRES). Mutational analysis of the ODC IRES has led to the identification of sequences necessary for cap-independent translation of the ODC mRNA. To discover novel IRES trans-acting factors (ITAFs), we performed a proteomics screen for proteins that regulate ODC translation using the wild-type ODC mRNA and a mutant version with an inactive IRES. We identified two RNA-binding proteins that associate with the wild-type ODC IRES but not the mutant IRES. One of these RNA-binding proteins, PCBP2, is an established activator of viral and cellular IRESs. The second protein, ZNF9 (myotonic dystrophy type 2 protein), has not been shown previously to bind IRES-like elements. Using a series of biochemical assays, we validated the interaction of these proteins with ODC mRNA. Interestingly ZNF9 and PCBP2 biochemically associated with each other and appeared to function as part of a larger holo-ITAF ribonucleoprotein complex. Our functional studies showed that PCBP2 and ZNF9 stimulate translation of the ODC IRES. Importantly these results may provide insight into the normal role of ZNF9 and why ZNF9 mutations cause myotonic dystrophy.

§ Supported by National Institutes of Health Grants GM64779, HL68744, ES11993, and CA098131. Also funded in part with federal funds from the NIAID, National Institutes of Health, Department of Health and Human Services, under Contract HHSN266200400079C/N01-AI-40079. To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Vanderbilt University School of Medicine, 1161 21st Ave. S., Nashville, TN 37232-2363. Tel.: 615-343-6823; Fax: 615-343-7392; E-mail: andrew.link{at}vanderbilt.edu


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