LOCALIZATION OF SEQUENCES REQUIRED FOR SIZE-SPECIFIC SPLICING OF A SMALL DROSOPHILA INTRON IN-VITRO

Many introns in Drosophila and other invertebrates are less than 80 nu cleotides in length, too small to be recognized by the vertebrate spli cing machinery Comparison of nuclear splicing extracts from human HeLa and Drosophila Kc cells has revealed species-specificity, consistent with the observed size differences. Here we present additional results with the 68 nucleotide fifth intron of the Drosophila myosin heavy ch ain gene. As observed with the 74 nucleotide second intron of the Dros ophila white gene, the wild-type myosin intron is accurately spliced i n a homologous extract, and increasing the size by 16 nucleotides both eliminates splicing in the Drosophila extract and allows accurate spl icing in the human extract. In contrast to previous results, however, an upstream cryptic 5' splice site is activated when the wild-type myo sin intron is tested in a human HeLa cell nuclear extract, resulting i n the removal of a 98 nucleotide intron. The size dependence of splici ng in Drosophila extracts is also intron-specific; we noted that a nat urally larger (150 nucleotide) intron from the ftz gene is efficiently spliced in Kc cell extracts that do not splice enlarged introns (of 8 4, 90, 150 or 350 nucleotides) derived from the 74 nucleotide white in tron. Here, we have exploited that observation, using a series of hybr id introns to show that a region of 46 nucleotides at the 3' end of th e white intron is sufficient to confer the species-specific size effec t. At least two sequence elements within this region, yet distinct fro m previously described branchpoint and pyrimidine tract signals, are r equired for efficient splicing of small hybrid introns in vitro. (C) 1 995 Academic Press Limited