Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences
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Discussion
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Implication
In vitro selection identifies degenerate RNA splicing enhancers activated by distinct SR proteins, revealing broad sequence diversity in splice site regulation.
Key Points
- Identify and characterize novel functional pre-mRNA splicing enhancer sequences and evaluate their interaction with specific serine/arginine-rich (SR) splicing factors.
- Inserted randomized 18-nucleotide RNA sequences downstream of a Drosophila doublesex enhancer-dependent pre-mRNA splicing substrate.
- Performed iterative cycles of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification to isolate functional enhancers.
- Evaluated individual enhancer motifs using S100 complementation assays with specific SR factors (SC35, 9G8, SF2/ASF) and binding assays for a 20-kDa SR protein.
- Identified six classes of recurring degenerate sequence motifs (5 to 7 nucleotides in length) that function as splicing enhancers.
- Demonstrated that distinct enhancer classes are selectively activated by specific SR proteins (SC35, 9G8, and SF2/ASF) despite showing significant sequence degeneracy.
- Isolated a potent splicing enhancer that selectively binds an SRp20-like protein and shares sequence homology with an SRp20-immunoprecipitated RNA-protein adduct.
Cite This Study
Schaal et al. (1999) studied this question.