STRUCTURAL AND FUNCTIONAL CONSERVATION OF THE DROSOPHILA DOUBLESEX SPLICING ENHANCER REPEAT ELEMENTS

We have compared the RNA sequences and secondary structures of the Dro sophila melanogaster and Drosophila virilis doublesex (dsx) splicing e nhancers. The sequences of the two splicing enhancers are highly diver gent except for the presence of nearly identical 13-nt repeat elements (six in D. melanogaster and four in D. virilis) and a stretch of nucl eotides at the 5' and 3' ends of the enhancers. In vitro RNA structure probing of the two enhancers revealed that the 13-nt repeats are pred ominantly single-stranded. Thus, both the primary sequences and single -stranded nature of the repeats are conserved between the two species. The significance of the primary sequence conservation was demonstrate d by showing that the two enhancers are functionally interchangeable i n Tra-/Tra2-dependent in vitro splicing. In addition, inhibition of sp licing enhancer activity by antisense oligonucleotides complementary t o the repeats demonstrated the importance of the conserved single-stra nded structure of the repeats. In vitro binding studies revealed that Tra2 interacts with each of the D. melanogaster repeat elements, excep t for repeat 2, with affinities that are indistinguishable, whereas Tr a binds nonspecifically to the enhancer. Taken together, these observa tions indicate that the organization of sequences within the dsx splic ing enhancers of D. melanogaster and D. virilis results in a structure in which each of the repeat elements is single-stranded and therefore accessible for specific recognition by the RNA-binding domain of Tra2 .