Jones M H, Frank D N, Guthrie C
Department of Biochemistry and Biophysics, University of California, San Francisco 94143, USA.
Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9687-91. doi: 10.1073/pnas.92.21.9687.
Temperature-sensitive alleles in four genes (slu7-1, prp16-2, prp17-1, and prp18-1) are known to confer a specific block to the second chemical step of pre-mRNA splicing in vivo in the yeast Saccharomyces cerevisiae. Previous studies showed that Prp16p and Prp18p are required solely for the second step in vitro. The RNA-dependent ATPase, Prp16p, functions at a stage in splicing when ATP is required, whereas Prp18p functions at an ATP-independent stage. Here we use immunodepletion to show that the roles of Slu7p and Prp17p are also confined to the second step of splicing. We find that extracts depleted of Prp17p require both Prp17p and ATP for slicing complementation, whereas extracts depleted of Slu7p require only the addition of Slu7p. These different ATP requirements suggest that Prp16p and Prp17p function before Prp18p and Slu7p. Although SLU7 encodes an essential gene product, we find that a null allele of prp17 is temperature-sensitive for growth and has a partial splicing defect in vitro. Finally, high-copy suppression experiments indicate functional interactions between PRP16 and PRP17, PRP16 and SLU7, and SLU7 and PRP18. Taken together, the results suggest that these four factors may function within a multi-component complex that has both an ATP-dependent and an ATP-independent role in the second step of pre-mRNA splicing.
已知四个基因（slu7-1、prp16-2、prp17-1和prp18-1）中的温度敏感等位基因会在酿酒酵母体内对前体mRNA剪接的第二步化学反应产生特定阻碍。先前的研究表明，Prp16p和Prp18p仅在体外剪接的第二步中是必需的。RNA依赖性ATP酶Prp16p在剪接过程中需要ATP的阶段发挥作用，而Prp18p在不依赖ATP的阶段发挥作用。在这里，我们使用免疫耗竭法来表明Slu7p和Prp17p的作用也局限于剪接的第二步。我们发现，耗尽Prp17p的提取物在剪接互补时既需要Prp17p也需要ATP，而耗尽Slu7p的提取物只需要添加Slu7p。这些不同的ATP需求表明Prp16p和Prp17p在Prp18p和Slu7p之前发挥作用。尽管SLU7编码一种必需的基因产物，但我们发现prp17的无效等位基因对生长是温度敏感的，并且在体外有部分剪接缺陷。最后，高拷贝抑制实验表明PRP16与PRP17、PRP16与SLU7以及SLU7与PRP18之间存在功能相互作用。综上所述，结果表明这四个因子可能在一个多组分复合物中发挥作用，该复合物在前体mRNA剪接的第二步中既具有依赖ATP的作用，也具有不依赖ATP的作用。
