Selection of Alternative 5' Splice Sites: Role of U1 snRNP and Models for the Antagonistic Effects of SF2/ASF and hnRNP A1

doi:10.1128/MCB.20.22.8303-8318.2000

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Molecular and Cellular Biology, November 2000, p. 8303-8318, Vol. 20, No. 22
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Selection of Alternative 5' Splice Sites: Role of U1 snRNP and Models for the Antagonistic Effects of SF2/ASF and hnRNP A1

Ian C. Eperon,¹^,^* Olga V. Makarova,¹^, Akila Mayeda,²^,³ Stephen H. Munroe,⁴ Javier F. Cáceres,²^,⁵ Daniel G. Hayward,¹ and Adrian R. Krainer²

Department of Biochemistry, University of Leicester, Leicester LE1 7RH,¹ and MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, United Kingdom⁵; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208²; Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33136-1019³; and Department of Biology, Marquette University, Milwaukee, Wisconsin 53233⁴

Received 26 May 2000/Returned for modification 13 July 2000/Accepted 24 August 2000

The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP.However, the relative levels of U1 snRNP binding to alternative5'SSs do not necessarily determine the splicing outcome. Strikingly,SF2/ASF, one of the essential SR protein-splicing factors, causesa dose-dependent shift in splicing to a downstream (intron-proximal)site, and yet it increases U1 snRNP binding at upstream and downstreamsites simultaneously. We show here that hnRNP A1, which shiftssplicing towards an upstream 5'SS, causes reduced U1 snRNP bindingat both sites. Nonetheless, the importance of U1 snRNP bindingis shown by proportionality between the level of U1 snRNP bindingto the downstream site and its use in splicing. With purifiedcomponents, hnRNP A1 reduces U1 snRNP binding to 5'SSs by bindingcooperatively and indiscriminately to the pre-mRNA. Mutationsin hnRNP A1 and SF2/ASF show that the opposite effects of theproteins on 5'SS choice are correlated with their effects on U1snRNP binding. Cross-linking experiments show that SF2/ASF andhnRNP A1 compete to bind pre-mRNA, and we conclude that this competitionis the basis of their functional antagonism; SF2/ASF enhancesU1 snRNP binding at all 5'SSs, the rise in simultaneous occupancycausing a shift in splicing towards the downstream site, whereashnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancyis lower and the affinities of U1 snRNP for the individual sitesdetermine the site ofsplicing.

^* Corresponding author. Mailing address: Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.Phone: 44 116 2523482. Fax: 44 116 2523369. E-mail: eci{at}le.ac.uk.

Present address: Max-Planck-Institut fur Biophysikalische Chemie, Abt. Zellulare Biochemie, 37077 Gottingen,Germany.

Molecular and Cellular Biology, November 2000, p. 8303-8318, Vol. 20, No. 22
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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