EVIDENCE FOR A ROLE OF THE DROSOPHILA-MELANOGASTER-SUPPRESSOR-OF-SABLE GENE IN THE PREMESSENGER RNA SPLICING PATHWAY

Recessive mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene result in elevated accumulation of RNA from vermilion (v ) mutant alleles that have an insertion of the 7.5-kb retrotransposon 412 in the first exon of the v gene. During transcription of such a v mutant gene, the 412 sequences are incorporated into the primary trans cripts and are subsequently removed by splicing at cryptic sites withi n 412 sequences. In a su(s)+ background, the level of these unusually spliced transcripts is exceedingly low, and su(s) mutations increase t heir accumulation. We previously proposed that v R-NA levels are eleva ted in su(s) mutants because of increased recognition of the cryptic s plice sites, and the aim of this study was to test this hypothesis. We generated a v mutant derivative with a smaller 412 insertion, introdu ced alterations into the 412-associated splice sites, and examined the effect of su(s) mutations on expression of these derivatives after ge rm line transformation. To increase overall expression levels, the v p romoter was replaced with the stronger Metallothionein (Mtn) gene prom oter. We found that transformants bearing a v derivative with 480 bp o f 412 sequences accumulate both transcripts, with 412 sequences splice d out and transcripts that retain 412 sequences. Mutations of su(s) in crease the levels of both transcript classes without affecting the rel ative amounts of the two forms. Strikingly, replacement of the cryptic 5' splice sites with a 5' consensus produces the same effect as, and eliminates the response to, a su(s) mutation. In addition, we demonstr ated that mutations of su(s) lead to increased accumulation of v trans cripts even when the previously identified cryptic 412 5' and 3' splic e sites were destroyed and that other cryptic splice sites reside with in Mtn and 412 sequences. These results indicate that the v mutant tra nscripts are stabilized by assembly of the 412 sequences into splicing complexes and support the hypothesis that splicing complexes more rea dily assemble on cryptic splice sites in su(s) mutants.