Nuclear protein TIA-1 regulates COL2A1 alternative splicing and interacts with precursor mRNA and genomic DNA.

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.