Mutations in conserved secondary structures of Adh pre-mRNA effect splicing efficiencies. S. Tanda 1, W. Stephan 2. 1) Dept Biological Sci, Ohio Univ, Athens, OH; 2) Dept. Biology, Univ Rochester, Rochester, N.Y.
Phylogenetic studies suggest the existence of RNA secondary structures in introns of Drosophila Adh genes. A single eight-bp long stem with the branchpoint in a loop is predicted in Intron 2 of D. melanogaster Adh pre-mRNA. Two covariations were identified in this stem structure (Kirby et al. 1995), thus this structure may have been selected in evolution. In order to explore if this structure possesses a biological function, we have made mutations in vitro and assessed their effects on splicing of Intron 2 in vitro. Mutant constructs were introduced to Schneider L2 cells by transfection, and the ratios of spliced mRNA molecules to unspliced pre-mRNA were measured. Mutations that weaken the stem showed a slight increase in splicing, and their compensatory mutations restored the splicing efficiency to the wild type level. On the other hand, mutations that strengthen the stem resulted in significant reductions in splicing, and their compensatory mutations restored these reduced levels to wild type. Our result suggests that a tighter stem structure may have a negative effect on Adh activity. Thus, an appropriate stem structure might be necessary for the proper function of Adh.