Sun Q, Hampson R K, Rottman F M
Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4960.
J Biol Chem. 1993 Jul 25;268(21):15659-66.
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.
牛生长激素（bGH）前体mRNA存在可变剪接，导致一部分胞质bGH mRNA中保留了最后一个内含子（内含子D）。为了研究这种可变剪接事件的机制，我们在体外检测了bGH前体mRNA的剪接。体外bGH内含子D的剪接需要外显子5的一个115碱基对片段，这反映了在转染细胞中观察到的外显子序列的积极影响。使用外显子5中含有115碱基对缺失的bGH前体mRNA未观察到可检测到的剪接体复合物形成。野生型bGH前体mRNA的体外剪接受到含有115个核苷酸外显子序列的RNA的抑制，但不受非特异性RNA的抑制。体外剪接反应的紫外线照射导致一种35 kDa蛋白质与115核苷酸bGH外显子序列发生特异性交联。这些结果表明，外显子末端序列在剪接体复合物形成的早期步骤中是必需的，并且与一种机制一致，即饱和的反式作用因子与这些外显子序列结合以激活bGH内含子D的剪接体复合物形成和剪接。
