McDonald W H, Ohi R, Smelkova N, Frendewey D, Gould K L
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Mol Cell Biol. 1999 Aug;19(8):5352-62. doi: 10.1128/MCB.19.8.5352.
Myb-related cdc5p is required for G(2)/M progression in the yeast Schizosaccharomyces pombe. We report here that all detectable cdc5p is stably associated with a multiprotein 40S complex. Immunoaffinity purification has allowed the identification of 10 cwf (complexed with cdc5p) proteins. Two (cwf6p and cwf10p) are members of the U5 snRNP; one (cwf9p) is a core snRNP protein. cwf8p is the apparent ortholog of the Saccharomyces cerevisiae splicing factor Prp19p. cwf1(+) is allelic to the prp5(+) gene defined by the S. pombe splicing mutant, prp5-1, and there is a strong negative genetic interaction between cdc5-120 and prp5-1. Five cwfs have not been recognized previously as important for either pre-mRNA splicing or cell cycle control. Further characterization of cwf1p, cwf2p, cwf3p, and cwf4p demonstrates that they are encoded by essential genes, cosediment with cdc5p at 40S, and coimmunoprecipitate with cdc5p. We further show that cdc5p associates with the U2, U5, and U6 snRNAs and that cells lacking cdc5(+) function are defective in pre-mRNA splicing. These data raise the possibility that the cdc5p complex is an intermediate in the assembly or disassembly of an active S. pombe spliceosome.
酵母粟酒裂殖酵母中,与Myb相关的cdc5p对于G(2)/M期进程是必需的。我们在此报告,所有可检测到的cdc5p都与一个多蛋白40S复合物稳定结合。免疫亲和纯化已鉴定出10种与cdc5p复合的cwf蛋白。其中两种(cwf6p和cwf10p)是U5 snRNP的成员;一种(cwf9p)是核心snRNP蛋白。cwf8p明显是酿酒酵母剪接因子Prp19p的直系同源物。cwf1(+)与粟酒裂殖酵母剪接突变体prp5-1所定义的prp5(+)基因等位,并且cdc5-120与prp5-1之间存在强烈的负向遗传相互作用。此前未认识到五种cwf蛋白对于前体mRNA剪接或细胞周期调控很重要。对cwf1p、cwf2p、cwf3p和cwf4p的进一步表征表明,它们由必需基因编码,在40S时与cdc5p共沉降,并与cdc5p共免疫沉淀。我们进一步表明,cdc5p与U2、U5和U6 snRNAs结合,并且缺乏cdc5(+)功能的细胞在前体mRNA剪接方面存在缺陷。这些数据增加了cdc5p复合物是活跃的粟酒裂殖酵母剪接体组装或拆卸中间体的可能性。