NMR ANALYSIS OF THE HYDROGEN-BONDING INTERACTIONS OF THE RNA-BINDING DOMAINS OF THE DROSOPHILA SEX-LETHAL PROTEIN WITH TARGET RNA FRAGMENTSWITH SITE-SPECIFIC [3-N-15]URIDINE SUBSTITUTIONS

It has been reported that a 183 residue fragment, consisting of the tw o RNA-binding domains (RBD1-RBD2) of the Drosophila melanogster Sex-le thal (Sri) protein, strongly binds an oligonucleotide of the target RN A sequence (5'-GUUUUUUUUC-3') that regulates alternative splicing, and forms four or five hydrogen bonds with the imino groups of the RNA, I n the present study, we used site-directed mutagenesis to improve the solubility of the didomain fragment of Sri, and confirmed that this mu tant fragment forms hydrogen bonds with the target RNA in the same man ner as that of the wild-type fragment, The mutant fragment was shown t o bind the cognate RNA sequences GUUUUUUUUC and AUUUUUUUUC more tightl y than UUUUUUUUC, By using a [3-N-15]uridine phosphoramidite, we synth esized a series of N-15-labeled target RNAs, in which one of the uridi ne residues was specifically replaced by [3-N-15]uridine, By observing the imino H-1-N-15 coupling of the labeled uridine residue, we assign ed all four of the hydrogen-bonded imino protons to U1, U2, U5 and U6, respectively, of the target RNA, The imino protons of U2 and U6 exhib ited nuclear Overhauser effects with aliphatic protons of the protein, All these results indicate that the A/G, U1, U2, U5 and U6 residues i n the target sequence of (G/A)UUUUUUUU are specifically recognized by the two RNA-binding domains of the Sri protein.