resulted in decreased expression level of RGS4. These 
results indicate that RGS4 expression is regulated by Tra2b in vivo. Tra2b Protein Interacts with RGS4 mRNAs To investigate whether Tra2b regulates RGS4 expression via its binding to RGS4 mRNAs, we performed RNA-binding protein immunoprecipitation assays to AMI-1 chemical information examine if RGS4 mRNAs are present in the immunoprecipitated complex of Tra2b. In the RIP assay, rabbit anti-Tra2b antibody and rabbit anti-GAP43 were incubated with equal amount of rat brain lysates, then pulled down by Protein A sepharose beads, and the antibody-precipitated mRNAs and proteins were analyzed by RTPCR and Western blot analyses, respectively. As shown in Fig. 2A, RGS4 mRNAs were precipitated by anti-Tra2b antibody, but not by the control antibody, suggesting an interaction between RGS4 mRNAs and Tra2b protein. In addition, we also examined the binding of recombinant Tra2b protein to RGS4 mRNAs in human SH-SY5Y cells. Anti-FLAG monoclonal antibody-agarose conjugates were incubated with the whole cell lysates from the cells transfected with plasmids expressing FLAG-Tra2b, FLAG-b-actin or FLAG plasmids. Results showed that RGS4 mRNAs were precipitated by FLAG-Tra2b, but not by FLAG-b-actin or FLAG alone. Tau mRNAs were detected as a positive control in this assay, because tau mRNAs have been reported to bind with Tra2b . Results Tra2b Regulates the Expression of RGS4 in Cultured Cells and in Rat LC To examine the effect of Tra2b on the exon 6 splicing, we used a GFP-fused minigene construct in which the sequence encoding green fluorescent protein is followed in frame with RGS4 minigene sequence. The RGS4 minigene consists of RGS4 exon 6 flanked by adjacent introns and constitutive exons, as illustrated in Fig. 1A. This minigene construct was used to transfect SH-SY5Y cells, a human derived neuroblastoma cell line, and generated two products. The major products were GFP-RGS4-1, whereas the minor were GFP-RGS4-4. The splicing pattern of this minigene is similar to that of endogenous RGS4 gene in vivo. Co-transfection of the minigene construct with the DNA construct expressing Tra2b protein altered the splicing pattern of the minigene. Tra2b overexpression significantly increased the level of GFP-RGS4-1 and reduced the level of GFP-RGS4-4. The results suggest that Tra2b promotes the exon 6 inclusive splicing. 8901831 To determine the effect of Tra2b on the expression of endogenous RGS4 gene, we directly examined the levels of RGS4 mRNAs and proteins in SH-SY5Y cells that endogenously express RGS4 gene. Real-time qPCR analysis showed that the relative level of RGS4-1 variant increased dramatically in the cells with Tra2b overexpression, while the relative level of RGS4-4 The SR Proteins ASF/SF2 and SRp30c Interact with Tra2b As an SR protein, Tra2b largely functions as a binding protein to its target mRNAs and also with other splicing factors, thereby contributing to spliceosome assembly and splicing site recognition. Here, we investigated the factors interacting with Tra2b protein by examining the precipitates of FLAG-Tra2b in whole cell lysates from SH-SY5Y cells transfected with plasmids expressing FLAG-Tra2b or FLAG plasmids. By silver staining of SDS-PAGE 20065018 gel, two bands were found in FLAG-Tra2b pull-down Tra2b Regulates the Expression of RGS4 Protein products, but not in the control. Mass spectrometry analysis demonstrated that they were ASF/SF2 and SRp30c, both belonging to SR protein family. Western blot analysis with speci
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