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Mol. Cell. Biol. doi:10.1128/MCB.02015-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Splicing factors SF1 and U2AF associate in extra-spliceosomal complexes

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal; Molecular Cytology Section, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM, Amsterdam, the Netherlands

^* To whom correspondence should be addressed. Email: carmo.fonseca{at} fm.ul.pt.

	Abstract

Splicing factors SF1 and U2AF associate cooperatively with pre-mRNA and play a crucial role in 3' splice site recognition during early steps of spliceosome assembly. Formation of the active spliceosome subsequently displaces SF1 in a remodeling process that stabilizes the association of U2 snRNP with pre-mRNA. Fluorescence microscopy shows SF1 and U2AF distributed throughout the nucleoplasm, where transcription occurs, with additional concentration in nuclear speckles, where splicing factors accumulate when not engaged in splicing. Fluorescence recovery after photobleaching (FRAP) analysis in live cells shows that the mobility of SF1 and the two subunits of U2AF (U2AF⁶⁵ and U2AF³⁵) is correlated with the ability of these proteins to interact with each other. Direct binding of SF1 to U2AF⁶⁵ was demonstrated by fluorescence resonance energy transfer (FRET) both in the nucleoplasm and nuclear speckles. This interaction persisted after transcription inhibition, suggesting that SF1 associates with U2AF in a splicing-independent manner. We propose that SF1 and U2AF form extra-spliceosomal complexes before and after taking part in the assembly of catalytic spliceosomes.

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