A SPECIFIC RNA HAIRPIN LOOP STRUCTURE BINDS THE RNA RECOGNITION MOTIFS OF THE DROSOPHILA SR PROTEIN B52

B52, also known as SRp55, is a member of the Drosophila melanogaster S R protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR protei ns, B52 contains two RNA recognition motifs in the N terminus and a C- terminal domain rich in serine-arginine dipeptide repeats, Since B52 i s an essential protein and is expected to play a role in splicing a su bset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specif icity of B52, we isolated B52-binding RNAs by selection and amplificat ion from a pool of random RNA sequences by using full length B52 prote in as the target. These RNAs contained a conserved consensus motif tha t constitutes the core of a secondary structural element predicted by energy minimization, Deletion and substitution mutations defined the B 52-binding site on these RNAs as a hairpin loop structure covering abo ut 20 nucleotides, which was confirmed by structure-specific enzymatic probing, Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.