Substrate Specificities of SR Proteins in Constitutive Splicing Are Determined by Their RNA Recognition Motifs and Composite Pre-mRNA Exonic Elements

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Molecular and Cellular Biology, March 1999, p. 1853-1863, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Substrate Specificities of SR Proteins in Constitutive Splicing Are Determined by Their RNA Recognition Motifs and Composite Pre-mRNA Exonic Elements

Akila Mayeda,¹^, Gavin R. Screaton,² Sharon D. Chandler,³ Xiang-Dong Fu,³ and Adrian R. Krainer¹^,^*

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208¹; Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom²; and Division of Cellular and Molecular Medicine, University of California $---$ San Diego, La Jolla, California 92093-0651³

Received 24 September 1998/Returned for modification 10 November 1998/Accepted 23 November 1998

We report striking differences in the substrate specificities of two human SR proteins, SF2/ASF and SC35, in constitutivesplicing. $beta$ -Globin pre-mRNA (exons 1 and 2) is spliced indiscriminatelywith either SR protein. Human immunodeficiency virus tat pre-mRNA(exons 2 and 3) and immunoglobulin µ-chain (IgM) pre-mRNA (exonsC3 and C4) are preferentially spliced with SF2/ASF and SC35, respectively.Using in vitro splicing with mutated or chimeric derivatives ofthe tat and IgM pre-mRNAs, we defined specific combinations ofsegments in the downstream exons, which mediate either positiveor negative effects to confer SR protein specificity. A seriesof recombinant chimeric proteins consisting of domains of SF2/ASFand SC35 in various combinations was used to localize trans-actingdomains responsible for substrate specificity. The RS domainsof SF2/ASF and SC35 can be exchanged without effect on substratespecificity. The RNA recognition motifs (RRMs) of SF2/ASF areactive only in the context of a two-RRM structure, and RRM2 hasa dominant role in substrate specificity. In contrast, the singleRRM of SC35 can function alone, but its substrate specificitycan be influenced by the presence of an additional RRM. The RRMsbehave as modules that, when present in different combinations,can have positive, neutral, or negative effects on splicing, dependingupon the specific substrate. We conclude that SR protein-specificrecognition of specific positive and negative pre-mRNA exonicelements via one or more RRMs is a crucial determinant of thesubstrate specificity of SR proteins in constitutivesplicing.

^* Corresponding author. Mailing address: Cold Spring Harbor Laboratory, P.O. Box 100, 1 Bungtown Rd., Cold Spring Harbor, NY11724-2208. Phone: (516) 367-8417. Fax: (516) 367-8453. E-mail:krainer{at}cshl.org.

Present address: Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33136-1019.

Molecular and Cellular Biology, March 1999, p. 1853-1863, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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