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J. Biol. Chem., Vol. 282, Issue 33, 24444-24454, August 17, 2007

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Nuclear Protein TIA-1 Regulates COL2A1 Alternative Splicing and Interacts with Precursor mRNA and Genomic DNA^*

Audrey McAlinden¹, Li Liang, Yoshiki Mukudai, Toshihiro Imamura², and Linda J. Sandell

From the Departments of Orthopaedic Surgery and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

The RNA-binding protein TIA-1 (T-cell-restricted intracellular antigen-1) functions in regulating post-transcriptional mechanisms, including precursor mRNA (pre-mRNA) alternative splicing and mRNA translation. Utilizing a mini-gene consisting of part of the type II procollagen gene (COL2A1), we show that TIA-1 interacts with a conserved AU-rich cis element in COL2A1 intron 2 and modulates alternative splicing of exon 2. This unique, highly conserved cis element containing stem-loop secondary structure was previously identified in our laboratory as an essential motif that controls the developmentally regulated exon 2 splicing switch during chondrogenesis (McAlinden, A., Havlioglu, N., Liang, L., Davies, S. R., and Sandell, L. J. (2005) J. Biol. Chem. 280, 32700-32711). In vivo binding of endogenous TIA-1 to the AU-rich cis element in COL2A1 pre-mRNA was confirmed by the ribonucleoprotein immunoprecipitation assay. Importantly, we also show that TIA-1 interacts with the equivalent DNA sequence with a preference for single-stranded rather than double-stranded DNA. Chromatin immunoprecipitation assays (including an additional RNase step) confirmed this interaction in vivo. Competition assays showed that TIA-1 apparently binds with higher affinity to DNA than to RNA. Finally, we show that this strong DNA-TIA-1 interaction can be disrupted by an RNA polymerase during active transcription. This suggests a potentially novel, dual role for TIA-1 in shuttling between DNA and RNA ligands to co-regulate COL2A1 expression at the level of transcription and pre-mRNA alternative splicing.

Received for publication, March 29, 2007 , and in revised form, June 5, 2007.

^* This work was supported by NIAMS, National Institutes of Health Grant AR036994 (to L. J. S. and A. M.) and an Arthritis Foundation Investigator grant (to A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Current Address: Dept. of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

¹ To whom correspondence should be addressed: Dept. of Orthopaedic Surgery, Washington University School of Medicine, 660 South Euclid Ave., Yalem 704, Box 8233, St Louis, MO 63110. Tel.: 314-454-8860; Fax: 314-454-5900; E-mail: mcalindena{at}wustl.edu.

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