Splicing Factors SF1 and U2AF Associate in Extraspliceosomal Complexes

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 analysis in live cells shows that the mobilities of SF1 and the two subunits of U2AF (U2AF⁶⁵ and U2AF³⁵) are correlated with the abilities of these proteins to interact with each other. Direct binding of SF1 to U2AF⁶⁵ was demonstrated by fluorescence resonance energy transfer in both 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 extraspliceosomal complexes before and after taking part in the assembly of catalytic spliceosomes.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We are grateful to Angus Lamond, Juan Valcárcel, and Angela Kramer for kindly providing reagents and to Eduardo Pinho e Melo for the use of the gel filtration facilities at Instituto Superior Técnico, Portugal. We thank Erik B. van Munster for his assistance with the FLIM experiments. We are also thankful to our colleagues José Braga and Margarida Gama-Carvalho for help and stimulating discussions.

This work was supported by grants from Fundação para a Ciência e Tecnologia, Portugal (POCI/SAU-MMO/57700/2004) and the European Commission (LSHG-CT-2003-503259 and LSHG-CT-2005-518238).