Benard L, Carroll K, Valle R C, Masison D C, Wickner R B
Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0830, USA.
J Virol. 1999 Apr;73(4):2893-900. doi: 10.1128/JVI.73.4.2893-2900.1999.
We mapped and cloned SKI7, a gene that negatively controls the copy number of L-A and M double-stranded RNA viruses in Saccharomyces cerevisiae. We found that it encodes a nonessential 747-residue protein with similarities to two translation factors, Hbs1p and EF1-alpha. The ski7 mutant was hypersensitive to hygromycin B, a result also suggesting a role in translation. The SKI7 product repressed the expression of nonpolyadenylated [non-poly(A)] mRNAs, whether capped or uncapped, thus explaining why Ski7p inhibits the propagation of the yeast viruses, whose mRNAs lack poly(A). The dependence of the Ski7p effect on 3' RNA structures motivated a study of the expression of capped non-poly(A) luciferase mRNAs containing 3' untranslated regions (3'UTRs) differing in length. In a wild-type strain, increasing the length of the 3'UTR increased luciferase expression due to both increased rates and duration of translation. Overexpression of Ski7p efficiently cured the satellite virus M2 due to a twofold-increased repression of non-poly(A) mRNA expression. Our experiments showed that Ski7p is part of the Ski2p-Ski3p-Ski8p antiviral system because a single ski7 mutation derepresses the expression of non-poly(A) mRNA as much as a quadruple ski2 ski3 ski7 ski8 mutation, and the effect of the overexpression of Ski7p is not obtained unless other SKI genes are functional. ski1/xrn1Delta ski2Delta and ski1/xrn1Delta ski7Delta mutants were viable but temperature sensitive for growth.
我们定位并克隆了SKI7基因，该基因在酿酒酵母中对L - A和M双链RNA病毒的拷贝数起负调控作用。我们发现它编码一种由747个氨基酸组成的非必需蛋白，与两种翻译因子Hbs1p和EF1 - α有相似性。ski7突变体对潮霉素B高度敏感，这一结果也表明其在翻译过程中发挥作用。SKI7产物可抑制非聚腺苷酸化[非poly(A)] mRNA的表达，无论其有无帽结构，这就解释了为什么Ski7p能抑制酵母病毒的增殖，因为这些病毒的mRNA缺乏poly(A)。Ski7p效应依赖于3' RNA结构，这促使我们研究含有不同长度3'非翻译区（3'UTR）的带帽非poly(A)荧光素酶mRNA的表达。在野生型菌株中，由于翻译速率和持续时间的增加，3'UTR长度的增加会提高荧光素酶的表达。Ski7p的过表达有效地治愈了卫星病毒M2，这是由于非poly(A) mRNA表达的抑制作用增强了两倍。我们的实验表明，Ski7p是Ski2p - Ski3p - Ski8p抗病毒系统的一部分，因为单个ski7突变对非poly(A) mRNA表达的去抑制作用与ski2、ski3、ski7、ski8四重突变一样大，而且除非其他SKI基因功能正常，否则无法获得Ski7p过表达的效果。ski1/xrn1Delta ski2Delta和ski1/xrn1Delta ski7Delta突变体是可存活的，但生长对温度敏感。
