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Spatial mapping of splicing factor complexes involved in exon and intron definition
Correspondence to Javier F. Caceres: Javier.Caceres{at}hgu.mrc.ac.uk
We have analyzed the interaction between serine/arginine-rich (SR) proteins and splicing components that recognize either the 5' or 3' splice site. Previously, these interactions have been extensively characterized biochemically and are critical for both intron and exon definition. We use fluorescence resonance energy transfer (FRET) microscopy to identify interactions of individual SR proteins with the U1 small nuclear ribonucleoprotein (snRNP)–associated 70-kD protein (U1 70K) and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) in live-cell nuclei. We find that these interactions occur in the presence of RNA polymerase II inhibitors, demonstrating that they are not exclusively cotranscriptional. Using FRET imaging by means of fluorescence lifetime imaging microscopy (FLIM), we map these interactions to specific sites in the nucleus. The FLIM data also reveal a previously unknown interaction between HCC1, a factor related to U2AF65, with both subunits of U2AF. Spatial mapping using FLIM-FRET reveals differences in splicing factors interactions within complexes located in separate subnuclear domains.
M. Denegri's present address is Centro Ricerca E. Menni, Fondazione Poliambulanza Istituto Ospedaliero, 25124 Brescia, Italy.
Abbreviations used in this paper: 5-FU, 5 Fluorouracil; co-IP, coimmunoprecipitation; DRB, 5,6-dichloro-1-b-d-ribofuranosylbenzimidazole; FLIM, fluorescence lifetime imaging microscopy; FRET, fluorescence resonance energy transfer; IGC, interchromatin granule cluster; IP, immunoprecipitation; RRM, RNA recognition motif; RS, arginine/serine rich; SF2/ASF, splicing factor 2/alternative splicing factor; snRNP, small nuclear RNP; SR, serine/arginine rich; TCSPC, time-correlated single-photon counting.
© 2008 Ellis et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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