Brown J T, Bai X, Johnson A W
Section of Molecular Genetics and Microbiology and The Institute for Cellular and Molecular Biology, The University of Texas at Austin, 78712-1095, USA.
RNA. 2000 Mar;6(3):449-57. doi: 10.1017/s1355838200991787.
The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A. XRN1 (SKI1) encodes a cytoplasmic 5'-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and for repression of translation of poly(A) minus RNA. Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats. An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex. Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation. The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8. Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic. These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 3'-mRNA degradation pathway.
酵母超级杀手（SKI）基因最初是从一些突变中鉴定出来的，这些突变使得由M“杀手”病毒（双链RNA病毒L - A的一种卫星病毒）编码的杀伤毒素产量增加。XRN1（SKI1）编码一种细胞质5'-外切核糖核酸酶，负责大部分细胞质RNA的周转，而SKI2、SKI3和SKI8是mRNA正常3'-降解以及抑制聚腺苷酸（poly(A)）缺失RNA翻译所必需的。Ski2p是一种推定的RNA解旋酶，Ski3p是一种四肽重复（TPR）蛋白，Ski8p包含五个WD - 40（β - 转导蛋白）重复序列。xrn1突变与ski2、ski3或ski8突变组合是致死的，这表明功能上存在冗余。使用功能性表位标记的Ski2、Ski3和Ski8蛋白，我们发现Ski2p、Ski3p和Ski8p可以作为一种明显的异源三聚体复合物被共免疫沉淀。对于表位标记的Ski2p，复合物中蛋白质的化学计量比为1:1:1。在没有Ski8p的情况下，Ski2p不与Ski3p结合，在没有Ski3p的情况下，Ski2p也不与Ski8p结合。然而，Ski3p/Ski8p相互作用不需要Ski2p。此外，ski6 - 2或ski4 - 1突变或SKI7的缺失并不影响复合物的形成。由Ski2p、Ski3p和Ski8p组成的复合物的鉴定解释了先前显示SKI2、SKI3和SKI8突变之间表型相似性的结果。Ski3p的间接免疫荧光以及Ski2p和Ski3p的亚细胞分级分离表明Ski2p和Ski3p位于细胞质中。这些数据支持了Ski2p、Ski3p和Ski8p在细胞质中参与3'-mRNA降解途径的观点。
