Identification of genes influencing synthetic lethality of genetic and epigenetic alterations in translation termination factors in yeast.
Identification of genes influencing synthetic lethality of genetic and epigenetic alterations in translation termination factors in yeast.
复制标题
鉴定影响酵母翻译终止因子遗传和表观遗传改变的合成致死性的基因。
DOI：
10.1134/s1607672911030021
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
Zhouravleva,GA
中科院分区：
文献类型：
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作者：
Kiktev,DA;Chernoff,YO;Archipenko,AV;Zhouravleva,GA
真核细胞中的翻译终止由蛋白质Sup 35（eRF 3）和Sup 45（eRF 1）[1]决定，它们与大量伴侣相互作用[2]。在酵母酿酒酵母中，蛋白Sup 35可以形成聚集的表观遗传遗传性构象异构体（朊病毒）[PSI+][3]。这种朊病毒通过细胞质携带，并导致翻译终止的干扰，这在表型上被鉴定为显性全能无义抑制。[PSI+]具有不同性质（无义抑制效率和有丝分裂中的传递稳定性）的变体可以在相同的酵母菌株中获得。朊病毒[PSI+]的存在导致在编码另一种终止因子Sup 45的基因中携带突变的单倍体酵母菌株的致死性[4]。我们已经证明，在二倍体品系中，杂合状态的SUP 45基因的某些突变等位基因与朊病毒[PSI+]的结合导致杂种的死亡[5]。朊病毒[PSI+]和sup 45基因的突变等位基因的合成致死性取决于突变等位基因和朊病毒变体的类型。变体[PSI+]是一种强抑制基因（“强”[PSI+]或[PSI+] S），在杂合子中引起所有无义突变和一些错义突变sup 45的合成致死性。我们的数据表明，杂种的致死率与Sup 45突变的情况下细胞中Sup 45蛋白的活性降低相关。本文描述了一种检测系统，该系统允许通过对朊病毒构象体Sup 35和SUP 45突变等位基因的合成致死率的影响，鉴定影响朊病毒[PSI+]稳定性和/或翻译终止效率的蛋白质。该试验系统适用于搜索影响酵母细胞中翻译终止效率和/或朊病毒维持的蛋白质。使用该测试系统进行基因文库筛选使我们能够鉴定CUR 1基因，该基因对另一种朊病毒[URE 3]的影响较早被证明，但对翻译终止因子的影响尚不清楚。该测试系统基于对同时含有（1）朊病毒[PSI+]，（2）杂合子中的sup 45突变，和（3）多拷贝质粒中的测试基因（图1）。合成致死性表现为具有基因型SUP 45/sup 45 [PSI+]的二倍体不生长（图2，突变101-107、111和116与“强”[PSI+] S不相容）。如果由于存在额外拷贝的测试基因（X）而使翻译终止的效率增加，则观察到具有基因型+/sup 45 [PSI+][基因X] n的活杂种的形成。相反，在X基因过表达的情况下，翻译终止效率的降低表现为含有突变等位基因sup 45的二倍体的死亡，其本身不是
Translation termination in eukaryotic cells is determined by proteins Sup35 (eRF3) and Sup45 (eRF1)[1], which interact with a large number of partners [2]. In yeast Saccharomyces cerevisiae, protein Sup35 can form an aggregating epigenetically inherited conformer (prion)[PSI+][3]. This prion is carried through the cytoplasm and causes disturbances in translation termination, which are phenotypically identified as the dominant omnipotent nonsense suppression.[PSI+] variants with different properties (nonsense suppression efficiency and transmission stability in mitosis) can be obtained in the same yeast strain. The presence of prion [PSI+] leads to lethality in the haploid yeast strain carrying mutations in the gene encoding another termination factor, Sup45 [4]. We have shown that the combination in the diploid strain of some mutant alleles of the SUP45 gene in the heterozygous state with prion [PSI+] entails the death of the hybrid [5]. The synthetic lethality of prion [PSI+] and mutant allele of the sup45 gene depends both on the type of mutant allele and the prion variant. Variant [PSI+], which is a strong suppressor (“strong”[PSI+], or [PSI+] S), causes synthetic lethality with all nonsense mutations and some missense mutations sup45 in the heterozygote. Our data indicate that the lethality of hybrids is correlated with a decreased activity of the Sup45 protein in the cell in case of sup45 mutations. This paper describes a test system that allows identification of proteins that affect the stability of prion [PSI+] and/or the efficiency of translation termination by their effect on the synthetic lethality of the prion conformer Sup35 and mutant alleles of SUP45. This test system is suitable to search for proteins that affect the translation termination efficiency and/or prion maintenance in yeast cells. Gene library screening using this test system allowed us to identify the CUR1 gene, whose influence on another prion,[URE3], was shown earlier but the effect on translation termination factors was not known.This test system is based on qualitative assessment of the viability of a diploid strain containing simultaneously (1) prion [PSI+],(2) sup45 mutation in the heterozygote, and (3) test gene (s) in a multicopy plasmid (Fig. 1). Synthetic lethality is manifested as the absence of growth of diploids with genotype SUP45/sup45 [PSI+](Fig. 2, mutations 101–107, 111, and 116 are incompatible with the “strong”[PSI+] S). If the efficiency of translation termination increases due to the presence of additional copies of the test gene (X), formation of viable hybrids with genotype+/sup45 [PSI+][gene X] n is observed. Conversely, a decrease in the translation termination efficiency in case of overexpression of the X gene is manifested as the death of diploids containing a mutant allele of sup45, which itself is not