Recognition of exonic splicing enhancer sequences by the Drosophila splicing repressor RSF1

The Drosophila repressor splicing factor 1 (RSF1) comprises an N-terminal R NA-binding region and a C-terminal domain rich in glycine, arginine and ser ine residues, termed the GRS domain. Recently, RSF1 has been shown to antag onize splicing factors of the serine/arginine-rich (SR) family and it is, t herefore, expected to play a role in processing of a subset of Drosophila p re-mRNAs through specific interactions with RNA. To investigate the RNA-bin ding specificity of RSF1, we isolated RSF1-binding RNAs using an in vitro s election approach. We have identified two RNA target motifs recognized by R SF1,designated A (CAACGACGA)- and B (AAACGCGCG)-type sequences. We show her e that the A-type cognate sequence behaves as an SR protein-dependent exoni c splicing enhancer. Namely, three copies of the A-type ligand bind SR prot eins, stimulate the efficiency of splicing of reporter pre-mRNAs several fo ld 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 findi ng that RSF1 acts as a potent repressor of pre-mRNA splicing in vitro led u s 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 mec hanisms controlling many alternative pre-mRNA processing events.