Chemical shift perturbation studies of the interactions of the second RNA-binding domain of the Drosophila sex-lethal protein with the transformer pre-mRNA polyuridine tract and 3' splice-site sequences

Abstract

The interactions of the second RNA-binding domain of the Drosophila melanogaster Sex-lethal protein (Sx1 RBD2) with the oligoribonucleotides, GUUUUUUUU (GU8) and CUAGUG, representing the sequences surrounding an alternative 3'-splicing site of the transformer pre-mRNA (GU8CUAGUG), were studied using heteronuclear two-dimensional NMR techniques. The 1H and 15N chemical shifts of the backbone amide resonances upon titration of Sx1 RBD2 with each of these RNAs were recorded. It was found that Sx1 RBD2 can bind not only to the polyuridine tract, GU8, but also to the downstream 3' splice-site sequence, CUAGUG, with similar affinities. In contrast, a nonspecific sequence, C8, did not bind to Sx1 RBD2. This result is consistent with previous in vitro RNA-selection and UV-cross-linking results which indicated that the Sex-lethal protein binds to the uridine stretch and the AG dinucleotide in the consensus sequence, AU(n)N(n)AGU. In both cases, the chemical-shift perturbations were significant for almost the same amino acid residues, including the two central β-strands formed by the RNP2-motif and RNP1-motif with the two highly conserved aromatic residues (Y214 and F256) in the middle. As the first RNA-binding domain of Sex-lethal (Sx1 RBD1) has a characteristic aliphatic residue at one of the two corresponding positions (I128 and F170), Y214 of Sx1 RBD2 was replaced by Ile using site- directed mutagenesis. On the one hand, the 1H and 15N chemical-shift perturbations indicated that GU8 binds to the same interface of mutant Sx1 RBD2 as of wild-type Sx1 RBD2, although its binding affinity was decreased significantly. On the other hand, the specific binding of Sx1 RBD2 to CUAGUG was abolished almost completely by the Y→I mutation. Taken together, the present results indicate that the interface residues that bind with GU8 and CUAGUG are much the same, but the role of the Y214 residue is clearly different between these two target sequences.