Lin C H, Patton J G
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
RNA. 1995 May;1(3):234-45.
We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection.
我们设计了一种体外剪接试验，其中α-原肌球蛋白的互斥外显子2和3作为相互竞争的3'剪接位点与外显子1连接。在正常的HeLa细胞核提取物中进行剪接，几乎只能得到外显子1和3的连接产物。在降低细胞核提取物浓度和离子强度的情况下进行剪接，会使外显子1-2的连接增加。我们利用该试验来确定三种组成型前体mRNA剪接因子在3'剪接位点选择中的作用。发现多嘧啶序列结合蛋白（PTB）可抑制含有该因子强结合位点的内含子的剪接。然而，如果在前体mRNA在补充了PTB的提取物中进行剪接之前，先与U2辅助因子（U2AF）预孵育，PTB的抑制作用可部分逆转。对于α-原肌球蛋白，PTB和U2AF对剪接的调节主要影响外显子1-3的连接，未检测到外显子1-2连接的显著增加。前体mRNA与SR蛋白预孵育会使外显子1-2的连接略有增加。然而，如果前体mRNA先与SR蛋白预孵育，然后在补充了PTB的提取物中进行剪接，正常的外显子1-2与1-3剪接比例几乎完全逆转。因此，组成型前体mRNA剪接因子与前体mRNA之间的多种两两相互作用，有时是拮抗作用，可调节3'剪接位点的选择。
