title: Imaging dynamic interactions between spliceosomal proteins and pre-mRNA in living cells.
abstract: The ability to observe protein dynamics in living cells is critical for the mechanistic understanding of highly flexible biological processes such as pre-mRNA splicing by the spliceosome. Splicing relies on intricate RNA and protein networks that are repeatedly rearranged during spliceosome assembly. Here we describe a method based on fluorescence microscopy that has been used by our and other laboratories to study interaction of spliceosomal proteins with nascent pre-mRNA in living cells. The method involves co-expressing in mammalian cells the target pre-mRNA labeled with one color, and the spliceosomal protein tagged with another color. The diffusion coefficient of the protein as well as its association and dissociation rates with the pre-mRNA are estimated by fluorescence recovery after photobleaching (FRAP) or photoactivation.
authors: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
authors: José
authors: J
authors: Rino
authors: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
authors: Robert M
authors: RM
authors: Martin
authors: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
authors: Teresa
authors: T
authors: Carvalho
authors: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal. Electronic address: carmo.fonseca@fm.ul.pt.
authors: Maria
authors: M
authors: Carmo-Fonseca
mesh_terms: D000818:Animals; D036681:Fluorescence Recovery After Photobleaching; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D057054:Molecular Imaging / Q000379:methods*; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D013194:Staining and Labeling / Q000379:methods; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D000097573:Red Fluorescent Protein
keywords: Confocal microscopy; Fluorescence recovery after photobleaching; Live cell imaging; Photoactivation; Pre-mRNA splicing; Spliceosome