Kuo B A, Norton P A
Department of Medicine, Division of Gastroenterology and Hepatology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Nucleic Acids Res. 1999 Oct 1;27(19):3945-52. doi: 10.1093/nar/27.19.3945.
Inclusion of fibronectin alternative exon B in mRNA is developmentally regulated. Here we demonstrate that exon B contains two unique purine-rich sequence tracts, PRE1 and PRE2, that are important for proper 5' splice site selection both in vivo and in vitro. Targeted mutations of both PREs decreased the inclusion of exon B in the mRNA by 50% in vivo. Deletion or mutation of the PREs reduced removal of the downstream intron, but not the upstream intron, and induced the activation of cryptic 5' splice sites in vitro. PRE-mediated 5' splice selection activity appears sensitive to position and sequence context. A well characterized exon sequence enhancer that normally acts on the upstream 3' splice site can partially rescue proper exon B 5' splice site selection. In addition, we found that PRE 5' splice selection activity was preserved when exon B was inserted into a heterologous pre-mRNA substrate. Possible roles of these unique activities in modulating exon B splicing are considered.
纤连蛋白可变外显子B包含在mRNA中受发育调控。在此我们证明外显子B含有两个独特的富含嘌呤的序列区域，即PRE1和PRE2，它们对于体内和体外正确的5'剪接位点选择都很重要。两个PRE的靶向突变在体内使外显子B在mRNA中的包含率降低了50%。PRE的缺失或突变减少了下游内含子的去除，但未减少上游内含子的去除，并在体外诱导了隐蔽5'剪接位点的激活。PRE介导的5'剪接选择活性似乎对位置和序列背景敏感。一个通常作用于上游3'剪接位点的特征明确的外显子序列增强子可以部分挽救外显子B正确的5'剪接位点选择。此外，我们发现当外显子B插入到异源前体mRNA底物中时，PRE的5'剪接选择活性得以保留。我们考虑了这些独特活性在调节外显子B剪接中的可能作用。
