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Molecular and Cellular Biology, March 1999, p. 1705-1719, Vol. 19, No. 3
Department of Molecular and Cellular Biology,
Harvard University, Cambridge, Massachusetts 02138
Received 14 September 1998/Returned for modification 28 October
1998/Accepted 23 November 1998
Splicing enhancers are RNA sequences required for accurate splice
site recognition and the control of alternative splicing. In this
study, we used an in vitro selection procedure to identify and
characterize novel RNA sequences capable of functioning as pre-mRNA
splicing enhancers. Randomized 18-nucleotide RNA sequences were
inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear
extracts, reverse transcription, and selective PCR amplification of the
spliced products. Characterization of the selected splicing enhancers
revealed a highly heterogeneous population of sequences, but we
identified six classes of recurring degenerate sequence motifs five to
seven nucleotides in length including novel splicing enhancer sequence
motifs. Analysis of selected splicing enhancer elements and other
enhancers in S100 complementation assays led to the identification of
individual enhancers capable of being activated by specific
serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In
addition, a potent splicing enhancer sequence isolated in the selection
specifically binds a 20-kDa SR protein. This enhancer sequence has a
high level of sequence homology with a recently identified RNA-protein
adduct that can be immunoprecipitated with an SRp20-specific antibody.
We conclude that distinct classes of selected enhancers are activated
by specific SR proteins, but there is considerable sequence degeneracy
within each class. The results presented here, in conjunction with
previous studies, reveal a remarkably broad spectrum of RNA sequences
capable of binding specific SR proteins and/or functioning as
SR-specific splicing enhancers.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Selection and Characterization of Pre-mRNA Splicing
Enhancers: Identification of Novel SR Protein-Specific
Enhancer Sequences
*
Corresponding author. Mailing address: Department of
Molecular and Cellular Biology, Harvard University, 7 Divinity Ave., Cambridge, MA 02138. Phone: (617) 495-1811. Fax: (617) 495-3537. E-mail: maniatis{at}biohp.harvard.edu.
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