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Molecular and Cellular Biology, March 1999, p. 1853-1863, Vol. 19, No. 3
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
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 constitutive splicing.
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

San Diego, La Jolla,
California 92093-06513
-Globin pre-mRNA (exons 1 and 2) is spliced indiscriminately with
either SR protein. Human immunodeficiency virus tat
pre-mRNA (exons 2 and 3) and immunoglobulin µ-chain (IgM) pre-mRNA
(exons C3 and C4) are preferentially spliced with SF2/ASF and SC35,
respectively. Using in vitro splicing with mutated or chimeric
derivatives of the tat and IgM pre-mRNAs, we defined
specific combinations of segments in the downstream exons, which
mediate either positive or negative effects to confer SR protein
specificity. A series of recombinant chimeric proteins consisting of
domains of SF2/ASF and SC35 in various combinations was used to
localize trans-acting domains responsible for substrate
specificity. The RS domains of SF2/ASF and SC35 can be exchanged
without effect on substrate specificity. The RNA recognition motifs
(RRMs) of SF2/ASF are active only in the context of a two-RRM
structure, and RRM2 has a dominant role in substrate specificity. In
contrast, the single RRM of SC35 can function alone, but its substrate
specificity can be influenced by the presence of an additional RRM. The
RRMs behave as modules that, when present in different combinations, can have positive, neutral, or negative effects on splicing, depending upon the specific substrate. We conclude that SR protein-specific recognition of specific positive and negative pre-mRNA exonic elements
via one or more RRMs is a crucial determinant of the substrate
specificity of SR proteins in constitutive splicing.
*
Corresponding author. Mailing address: Cold Spring
Harbor Laboratory, P.O. Box 100, 1 Bungtown Rd., Cold Spring Harbor, NY 11724-2208. 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.
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