Identification of RNA-binding proteins that regulate FGFR2 splicing through the use of sensitive and specific dual color fluorescence minigene assays
| Autor: | Richard B. Jones, Stephanie J. Muh, Ruben H. Hovhannisyan, Emily A. Newman, Claude C. Warzecha, Wallace L. McKeehan, Russ P. Carstens |
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| Jazyk: | angličtina |
| Rok vydání: | 2006 |
| Předmět: |
Recombinant Fusion Proteins
Green Fluorescent Proteins Exonic splicing enhancer Method Color RNA-binding protein Biology Transfection Green fluorescent protein Exon Genes Reporter Receptor Fibroblast Growth Factor Type 2 Molecular Biology Gene Cells Cultured Fluorescent Dyes Models Genetic Alternative splicing RNA-Binding Proteins Flow Cytometry Molecular biology Introns Cell biology Alternative Splicing Luminescent Proteins Enhancer Elements Genetic RNA splicing Trans-Activators Biological Assay Minigene |
| Popis: | We have developed a series of fluorescent splicing reporter minigenes for the establishment of cell-based screens to identify splicing regulatory proteins. A key technical advance in the application of these reporters was the use of two different fluorescent proteins: EGFP and monomeric Red Fluorescent Protein (mRFP). Through establishment of stable cell lines expressing such dual color fluorescent reporters, these minigenes can be used to perform enhanced screens for splicing regulatory proteins. As an example of such applications we generated fluorescent minigenes that can be used to determine the splicing of mutually exclusive FGFR2 exons IIIb and IIIc by flow cytometry. One minigene contained a coding sequence for EGFP whose translation was dependent on splicing of exon IIIb, whereas a second minigene required exon IIIc splicing for translation of an mRFP coding sequence. Stable incorporation of both minigenes into cells that express endogenous FGFR2-IIIb or FGFR2-IIIc resulted in EGFP or mRFP fluorescence, respectively. Cells stably transfected with both minigenes were used to screen a panel of cDNAs encoding known splicing regulatory proteins, and several were identified that induced a switch in splicing that could be detected specifically by an increase in green, but not red, fluorescence. We further demonstrated additional minigenes that can be used in dual color fluorescent screens for identification of splicing regulatory proteins that function through specific intronic splicing enhancer elements (ISEs). The methods and minigene designs described here should be adaptable for broader applications in identification of factors and mechanisms involved in alternative splicing of numerous other gene transcripts. |
| Databáze: | OpenAIRE |
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