In vitro analysis of bovine growth hormone pre-mRNA alternative splicing. Involvement of exon sequences and trans-acting factor(s).
Bovine growth hormone (bGH) pre-mRNA is alternatively spliced, resulting in retention of the last intron (intron D) in a fraction of the cytosolic bGH mRNA. To study the mechanism of this alternative splicing event, 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, reflecting the positive influence of exon sequences observed in transfected cells. No detectable spliceosome complex formation was observed using 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 by nonspecific RNAs. UV irradiation of the in vitro splicing reaction resulted in specific cross-linking of a 35-kDa protein(s) to the 115-nucleotide bGH exon sequence. These results suggest that terminal exon sequences are required at an early step of spliceosome complex formation and are consistent with a mechanism in which saturable, trans-acting factor(s) bind to these exon sequences to activate spliceosome complex formation and splicing of bGH intron D.
Sun
Q
Q
Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4960.
Hampson
R K
RK
Rottman
F M
FM
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D005091:Exons; D013006:Growth Hormone / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D015534:Trans-Activators
D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D012333:RNA, Messenger; D015534:Trans-Activators; D013006:Growth Hormone