title: Identification of cells deficient in signaling-induced alternative splicing by use of somatic cell genetics.
abstract: In recent years, a growing number of mammalian genes have been shown to undergo alternative splicing in response to extracellular stimuli. However, the factors and pathways involved in such signal-induced alternative splicing are almost entirely unknown. Here we describe a novel method for identifying candidate trans-acting factors that are involved in regulating mammalian alternative splicing, using the activation-induced alternative splicing of the human CD45 gene in T cells as a model system. We generated a cell line that stably expresses a CD45 minigene-based GFP reporter construct, such that the levels of green-fluorescent protein (GFP) expressed in the cell reflect the splicing state of the endogenous CD45 gene. Following mutagenesis of this cell line, and multiple rounds of selection for cells that displayed aberrant levels of GFP expression, we isolated several cell lines that are at least partially defective in their ability to support regulated alternative splicing of endogenous CD45 pre-mRNA in response to cell stimulation. Thus we have successfully isolated mutants in a mammalian alternative splicing pathway through use of a somatic cell-based genetic screen. This study clearly demonstrates the feasibility of using genetic screens to further our understanding of the regulation of mammalian splicing, particularly as it occurs in response to environmental cues.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Sheives
authors.forename: Kristen W
authors.initials: KW
authors.lastname: Lynch
mesh_terms: D017398:Alternative Splicing; D015703:Antigens, CD / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005434:Flow Cytometry; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins; D006801:Humans; D037181:Lectins, C-Type; D017493:Leukocyte Common Antigens / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology; D010766:Phosphorylation; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D005020:Ethyl Methanesulfonate; D049452:Green Fluorescent Proteins; D037181:Lectins, C-Type; D017493:Leukocyte Common Antigens; D008164:Luminescent Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009153:Mutagens; D012322:RNA Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013755:Tetradecanoylphorbol Acetate; D000099192:CD69 Antigens