Information Services banner Edinburgh Research Archive The University of Edinburgh crest

Edinburgh Research Archive >
Molecular, Genetic and Population Health Sciences, School of >
Molecular, Genetic and Population Health Sciences thesis and dissertation collection >

Please use this identifier to cite or link to this item:

This item has been viewed 19 times in the last year. View Statistics

Files in This Item:

File Description SizeFormat
Ellis2008.docFile not available for download26.54 MBMicrosoft Word
Ellis2008.pdfPhD thesis8.01 MBAdobe PDFView/Open
Title: FRET analysis of splicing factors involved in exon and intron definition in living cells
Authors: Ellis, Jonathan
Supervisor(s): Caceres, Javier
Issue Date: 2008
Publisher: The University of Edinburgh
Abstract: I 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.
Keywords: FRET
fluorescence resonance energy transfer
fluorescence lifetime imaging microscopy
Appears in Collections:Molecular, Genetic and Population Health Sciences thesis and dissertation collection

Items in ERA are protected by copyright, with all rights reserved, unless otherwise indicated.


Valid XHTML 1.0! Unless explicitly stated otherwise, all material is copyright © The University of Edinburgh 2013, and/or the original authors. Privacy and Cookies Policy