IN-VITRO ANALYSIS OF BOVINE GROWTH-HORMONE PREMESSENGER RNA ALTERNATIVE SPLICING - INVOLVEMENT OF EXON SEQUENCES AND TRANS-ACTING FACTOR(S)

Bovine growth hormone (bGH) pre-mRNA is alternatively spliced, resulti ng in retention of the last intron (intron D) in a fraction of the cyt osolic bGH mRNA. To study the mechanism of this alternative splicing e vent, we examined the splicing of bGH pre-mRNA in vitro. The splicing of bGH intron D in vitro required a 115-base pair segment of exon 5, r eflecting the positive influence of exon sequences observed in transfe cted cells. No detectable spliceosome complex formation was observed u sing bGH pre-mRNA containing the 115-base pair deletion in exon 5. The in vitro splicing of the wild type bGH pre-mRNA was inhibited by the addition of RNA containing the 115-nucleotide exon sequence, but not b y nonspecific RNAs. UV irradiation of the in vitro splicing reaction r esulted in specific cross-linking of a 35-kDa protein(s) to the 115-nu cleotide bGH exon sequence. These results suggest that terminal exon s equences are required at an early step of spliceosome complex formatio n and are consistent with a mechanism in which saturable, trans-acting factor(s) bind to these exon sequences to activate spliceosome comple x formation and splicing of bGH intron D.