IDENTIFICATION OF A NOVEL COMPONENT OF THE NONSENSE-MEDIATED MESSENGER-RNA DECAY PATHWAY BY USE OF AN INTERACTING PROTEIN SCREEN
IDENTIFICATION OF A NOVEL COMPONENT OF THE NONSENSE-MEDIATED MESSENGER-RNA DECAY PATHWAY BY USE OF AN INTERACTING PROTEIN SCREEN
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DOI：
10.1101/gad.9.4.437
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发表时间：
1995-02-15
影响因子：
10.5
通讯作者：
JACOBSON, A
中科院分区：
文献类型：
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作者：
HE, F;JACOBSON, A
酵母中无义mRNA的快速周转依赖于UPF 1基因（Upf 1 p）的产物。UPF 1的突变导致含有早期无义突变的mRNA的选择性稳定，而不影响大多数其他mRNA的衰变率。为了确定这种衰变途径的其他组成部分，我们采用了双杂交筛选，寻找那些与Upf 1 p特异性相互作用的细胞因子。筛选酵母基因组文库确定了6个基因编码潜在的Upf 1 p相互作用蛋白。这些包括四个以前未表征的基因，NMD 1 -4（无义介导的mRNA衰变），DBP 2，一个编码与哺乳动物p68 RNA解旋酶同源的推定RNA解旋酶的基因，和SNP 1，一个编码U1 snRNP 70-kD蛋白同源物的基因。在本文中，我们报告的NMD 2，酵母基因编码一个特定的Upf 1 p相互作用蛋白的鉴定和表征。破坏NMD 2产生的无义介导的mRNA衰变表型相同的UPF 1缺失菌株中获得的，表明NMD 2基因产物（Nmd 2 p）是一个新的因素，在无义介导的mRNA衰变途径。缺失分析表明，Nmd 2 p的酸性羧基端构成Upf 1 p相互作用域。高水平表达的Nmd 2 p片段包含这个域有一个显性负效应无义介导的mRNA衰减时，蛋白质被定位到细胞质，但不是当它被定位到细胞核，表明这种衰变途径有细胞质的组成部分。在双杂交筛选中鉴定的基因片段与显性阴性表型的关联表明了一种确认在这种筛选中鉴定的基因的功能的通用方法。
Rapid turnover of nonsense-containing mRNAs in yeast is dependent on the product of the UPF1 gene (Upf1p). Mutations in UPF1 lead to the selective stabilization of mRNAs containing early nonsense mutations without affecting the decay rates of most other mRNAs. To identify other integral components of this decay pathway, we have employed a two-hybrid screen, seeking those cellular factors that specifically interact with Upf1p. Screening of yeast genomic libraries identified six genes encoding potential Upf1p-interacting proteins. These include four previously uncharacterized genes, NMD1-4 (nonsense-mediated mRNA decay), DBP2, a gene encoding a putative RNA helicase with homology to mammalian p68 RNA helicase, and SNP1, a gene encoding a U1 snRNP 70-kD protein homolog. In this paper we report the identification and characterization of NMD2, a yeast gene that encodes a specific Upf1p-interacting protein. Disruption of NMD2 yields a nonsense-mediated mRNA decay phenotype identical to that obtained in UPF1-deletion strains, indicating that the NMD2 gene product (Nmd2p) is a new factor in the nonsense-mediated mRNA decay pathway. Deletion analysis demonstrated that the acidic carboxyl terminus of Nmd2p constituted the Upf1p-interacting domain. High-level expression of a fragment of Nmd2p containing this domain had a dominant-negative effect on nonsense-mediated mRNA decay when the protein was localized to the cytoplasm but not when it was localized to the nucleus, indicating that this decay pathway has a cytoplasmic component. The association of a dominant-negative phenotype with a gene fragment identified in a two-hybrid screen suggests a generalized approach to confirming the function of genes identified in such screens.