The Drosophila repressor splicing factor 1 (RSF1) comprises an N-terminal RNA-binding region and a C-terminal domain rich in glycine, arginine and serine residues, termed the GRS domain. Recently, RSF1 has been shown to antagonize splicing factors of the serine/arginine-rich (SR) family and it is, therefore, expected to play a role in processing of a subset of Drosophila pre-mRNAs through specific interactions with RNA. To investigate the RNA-binding specificity of RSF1, we isolated RSF1-binding RNAs using an in vitro selection approach. We have identified two RNA target motifs recognized by RSF1, designated A (CAACGACGA)- and B (AAACGCGCG)-type sequences. We show here that the A-type cognate sequence behaves as an SR protein-dependent exonic splicing enhancer. Namely, three copies of the A-type ligand bind SR proteins, stimulate the efficiency of splicing of reporter pre-mRNAs several fold and lead to inclusion of a short internal exon both in vitro and in vivo. However, three copies of a B-type ligand were much less active. The finding that RSF1 acts as a potent repressor of pre-mRNA splicing in vitro led us to propose that the equilibrium between a limited number of structurally-related general splicing activators or repressors, competing for common or promiscuous binding sites, may be a major determinant of the underlying mechanisms controlling many alternative pre-mRNA process-ing events.
Recognition of exonic splicing enhancer sequences by the Drosophila splicing repressor RSF1
Labourier, E.; Allemand, E.; Brand, S.; Fostier, M.; Tazi, J.; Bourbon, H. M.
Nucleic Acids Res
1999-06-01 / vol 27 / pages 2377-86
Humans; Animals; Amino Acid Sequence; Molecular Sequence Data; Nuclear Proteins/metabolism; Base Sequence; Cell Line; Binding Sites; Recombinant Fusion Proteins/genetics/metabolism; Phosphoproteins/metabolism; Sequence Homology, Amino Acid; Drosophila; Hela Cells; *Exons; Globins/genetics; *RNA Splicing; RNA-Binding Proteins/genetics/*metabolism; *Drosophila Proteins; *Enhancer Elements (Genetics); Insect Proteins/genetics/*metabolism; Repressor Proteins/genetics/*metabolism