3P-12 PDF
Strategy for analysis of the splicing regulatory factors using high-resolution mass spectrometry
Splicing regulatory factors participate the regulation of splice site usage. To identify these factors bound to the targeted exon, we designed in vitro binding assay. The RNA probe was incubated with the cell nuclear extract. The separated band in PAGE, which contains the RNA-protein complex, was in-gel digested. This peptide mixture was applied to LC-MS/MS to identify the binding protein. To confirm the utility of this method, we designed an RNA probe consisting of the insulin receptor exon 11. We found that hnRNPA1 forms the complex with the exon11 sequence.