Cells were also stained with antibodies against SR protein kinases

rior to stimulation with 20% serum for 4 h. The quantitative band intensity relative to the control is indicated at the bottom. Alternatively spliced products affected by RANBP2 knockdown are marked with asterisks. RANBP2 and GAPDH were used to confirm the knockdown efficiency of RANBP2 and unaffected transcription, respectively. Quantitative RT-PCR of the C-FOS gene under serum induction. The expression of C-FOS, an archetypal immediate early gene, was unaffected by RANBP2 knockdown. RANBP2 and ACTB served as controls for the knockdown efficiency and constant transcription, respectively. The values are the means and SDs of three independent experiments. Molecular Biology of the Cell Transcription of the proto-oncogene C-FOS is markedly upregulated upon serum induction through a signaling cascade including the Rho family GTPase RHOA and phosphatidylinositol 3-kinase. Quantitative RT-PCR showed that the kinetics of this gene activation was almost identical in control and RANBP2-knockdown cells, implying that transcriptional induction and serum-responsive signal transduction were intact in the RANBP2-knockdown cells. In conclusion, we found that alternative splicing is affected by the physical segregation of phosphorylated and hypophosphorylated SR proteins that is induced by RANBP2 knockdown. This suggests that the speckled distribution of phosphorylated SR proteins in the nucleus is important for regulation of alternative splicing of pre-mRNA. DISCUSSION The spatial organization of the eukaryotic nucleus AVE-8062 site reflects its gene expression profile, and the distribution pattern of the nuclear substructures relative to genes may govern genome function. In the present study, we demonstrated that the nucleoporin protein RANBP2 plays a specific role in nuclear speckle formation. Its loss resulted in the absence of SC35-positive nuclear speckles and, instead, the generation of CGs, novel granular structures in the cytoplasm of interphase cells. Phosphorylated forms of a subset of SR proteins and RNAPII, together with SRPKs, accumulated in CGs, and SRPKs were required for CG formation. CGs occurred mostly in G1-phase cells, suggesting that CGs are the remnants of MIGs, which are induced by dysfunction of the nucleocytoplasmic transport of phosphorylated SR proteins. More important, CG-containing cells were capable of constitutive transcription and pre-mRNA splicing, but their alternative splicing patterns were altered. This suggests a specific function of the speckled distribution of phosphorylated SR proteins in determining alternative pre-mRNA splicing patterns. Indeed, the distribution of SR proteins was developmentally regulated, and CGs existed in the mouse testis with reduced levels of Ranbp2 and high alternative splicing activity. Alternative pre-mRNA splicing provides one of the most powerful ways of gene expression regulation by selecting a combination of exons, leading to the generation of many distinct protein isoforms from a common primary transcript. Genome-wide studies indicated that alternative splicing is fundamental in vivo and that 95% of human multiexon genes are alternatively spliced. Many pre-mRNA splicing factors, including SR proteins, are phosphorylated; phosphorylation plays a crucial role in both constitutive and alternative splicing. It has been suggested that phosphorylation modulates proteinprotein interactions in the spliceosome, which results in switches in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19793655 splice-site recognition. Alternative splice-site choice is also