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dc.contributor.advisorCaceres, Javier
dc.contributor.authorEllis, Jonathan
dc.date.accessioned2010-11-22T14:22:10Z
dc.date.available2010-11-22T14:22:10Z
dc.date.issued2008
dc.identifier.urihttp://hdl.handle.net/1842/4397
dc.description.abstractI have analyzed the interactions between SR proteins and splicing components that are bound at the 5’ or 3’ splice site using fluorescence resonance energy transfer (FRET) microscopy. The SR proteins interact with the U1 snRNP-associated 70 kDa protein (U170K) at the 5’splice site and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) at the 3’ splice site. These interactions have been extensively characterized biochemically in the past, and are proposed to play roles in both intron and exon definition. We employed FRET acceptor photobleaching and fluorescence lifetime imaging microscopy (FLIM) to identify and spatially localise sites of direct interactions of SF2/ASF, and other SR proteins, with U2AF35 and U1-70K in live cell nuclei. These interactions were shown to occur more strongly in interchromatin granule clusters (IGCs). They also occur in the presence of the RNA polymerase II inhibitor, DRB, demonstrating that they are not exclusively co-transcriptional. FLIM data have also revealed a novel interaction between HCC1, a factor highly related to the large subunit of the U2AF splicing factor, with both subunits of U2AF that occur in discrete domains within the nucleoplasm but not within IGCs. These data demonstrate that the interactions defining intron and exon definition do occur in living cells in a transcription-independent manner.en
dc.language.isoenen
dc.publisherThe University of Edinburghen
dc.subjectFRETen
dc.subjectFLIMen
dc.subjectfluorescence resonance energy transferen
dc.subjectfluorescence lifetime imaging microscopyen
dc.titleFRET analysis of splicing factors involved in exon and intron definition in living cellsen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


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