Localization of Sequences Required for Size-specific Splicing of a SmallDrosophilaIntronin Vitro

Many introns inDrosophilaand other invertebrates are less than 80 nucleotides in length, too small to be recognized by the vertebrate splicing machinery. Comparison of nuclear splicing extracts from human HeLa andDrosophilaKc cells has revealed species-specificity, consistent with the observed size differences. Here we present additional results with the 68 nucleotide fifth intron of theDrosophilamyosin heavy chain gene. As observed with the 74 nucleotide second intron of theDrosophilawhite gene, the wild-type myosin intron is accurately spliced in a homologous extract, and increasing the size by 16 nucleotides both eliminates splicing in theDrosophilaextract and allows accurate splicing in the human extract. In contrast to previous results, however, an upstream cryptic 5′ splice site is activated when the wild-type myosin intron is tested in a human HeLa cell nuclear extract, resulting in the removal of 98 nucleotide intron.The size dependence of splicing inDrosophilaextracts is also intron-specific; we noted that a naturally larger (150 nucleotide) intron from theftzgene is efficiently spliced in Kc cell extracts that do not splice enlarged introns (of 84, 90, 150 or 350 nucleotides) derived from the 74 nucleotidewhiteintron. Here, we have exploited that observation, using a series of hybrid introns to show that a region of 46 nucleotides at the 3′ end of thewhiteintron is sufficient to confer the species-specific size effect. At least two sequence elements within this region, yet distinct from previously described branchpoint and pyrimidine tract signals, are required for efficient splicing of small splicing of small hybrid intronsin vitro.