INTERACTION OF THE SEX-LETHAL RNA-BINDING DOMAINS WITH RNA

Sex determination and X chromosome dosage compensation in Drosophila m elanogaster are directed by the Sex-lethal (Sxl) protein, In part, Sxl functions by regulating the splicing of the transformer pre-mRNA by b inding to a 3' splice site polypyrimidine tract. Polypyrimidine tracts are essential for splicing of metazoan pre-mRNAs. To unravel the mech anism of splicing regulation at polypyrimidine tracts we analyzed the interaction of Sxl with RNA. The RNA binding activity of Sxl was mappe d to the two ribonucleoprotein consensus sequence domains of the prote in. Quantitation of binding showed that both RNA binding domains (RBDs ) were required in cis for site-specific RNA binding. Individual RBDs interacted with RNA more weakly and had lost the ability to discrimina te between wild-type and mutant transformer polypyrimidine tracts. Str uctural elements in one of the RBDs that are likely to interact with a polypyrimidine tract were identified using nuclear magnetic resonance techniques. In addition, our data suggest that multiple imino protons of the transformer polypyrimidine tract were involved in hydrogen bon ding. Interestingly, in vitro Sxl bound with equal affinity to polypyr imidine tracts of pre-mRNAs that it does not regulate in vivo. We disc uss the implications of this finding for the mechanism through which S xl may gain selectivity for particular polypyrimidine tracts in vivo.