Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover.
Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover.
复制标题
鉴定和表征 UPF1 基因中影响无义抑制和 Upf 蛋白复合物形成但不影响 mRNA 周转的突变。
DOI：
10.1128/mcb.16.10.5491
复制
发表时间：
1996
影响因子：
5.3
通讯作者：
Peltz,SW
中科院分区：
文献类型：
--
作者：
Weng,Y;Czaplinski,K;Peltz,SW
为了理解翻译与mRNA衰变之间的关系，我们一直在研究过早翻译终止如何加速mRNA的降解。在酵母酿酒酵母中，Upf1蛋白（Upflp）被证明是这一衰变途径中的一个反作用因子，它包含一个富含半胱氨酸和组氨酸的区域以及核苷三磷酸水解和解旋酶基序。aupf1基因的破坏导致含有无义mrna的稳定，并导致无义抑制表型。对野生型Upf1p的生化分析表明，它具有RNA依赖的atp酶、RNA解旋酶和RNA结合活性。在随附的论文中描述的工作(Y. Weng， K. Czaplinski和S. W. Peltz, Mol. Cell。在Upf1p的解旋酶区域发现了突变，使其mRNA衰变功能失活，但阻止了对leu2-2和tyr7-1无义等位基因的抑制。基于这些结果，我们认为Upf1p是一种参与调节mRNA衰变和无义密码子翻译终止的多功能蛋白。如果这是真的，我们预测具有相反表型的upf1突变应该被识别出来。在本报告中，我们描述了Upf1p的氨基端半胱氨酸和组氨酸丰富区域突变的鉴定和生化特征，这些突变具有正常的无义介导的mRNA衰变活性，但能够抑制sleu2-2和tyr7-1无义等位基因。这些突变蛋白的生化特性表明它们改变了RNA结合特性。此外，利用双混合系统，我们表征了Upf1p-Upf2p相互作用，并证明了Upf2p与Upf3p相互作用。在Upf1p富含半胱氨酸和组氨酸的区域发生突变，会破坏Upf1p- upf2p相互作用。在这些结果的基础上，Upf复合物在无义介导的mRNA衰变和无义抑制中的作用进行了讨论。
To understand the relationship between translation and mRNA decay, we have been studying how premature translation termination accelerates the degradation of mRNAs. In the yeastSaccharomyces cerevisiae, the Upf1 protein (Upflp), which contains a cysteine- and histidine-rich region and nucleoside triphosphate hydrolysis and helicase motifs, was shown to be atrans-acting factor in this decay pathway. AUPF1gene disruption results in the stabilization of nonsense-containing mRNAs and leads to a nonsense suppression phenotype. Biochemical analysis of the wild-type Upf1p demonstrated that it has RNA-dependent ATPase, RNA helicase, and RNA binding activities. In the work described in the accompanying paper (Y. Weng, K. Czaplinski, and S. W. Peltz, Mol. Cell. Biol. 16:5477–5490, 1996) mutations in the helicase region of Upf1p that inactivated its mRNA decay function but prevented suppression ofleu2-2andtyr7-1nonsense alleles are identified. On the basis of these results, we suggested that Upf1p is a multifunctional protein involved in modulating mRNA decay and translation termination at nonsense codons. If this is true, we predict thatUPF1mutations with the converse phenotype should be identified. In this report, we describe the identification and biochemical characterization of mutations in the amino-terminal cysteine- and histidine-rich region of Upf1p that have normal nonsense-mediated mRNA decay activities but are able to suppressleu2-2andtyr7-1nonsense alleles. Biochemical characterization of these mutant proteins demonstrated that they have altered RNA binding properties. Furthermore, using the two-hybrid system, we characterized the Upf1p-Upf2p interactions and demonstrated that Upf2p interacts with Upf3p. Mutations in the cysteine- and histidine-rich region of Upf1p abolish Upf1p-Upf2p interaction. On the basis of these results, the role of the Upf complex in nonsense-mediated mRNA decay and nonsense suppression is discussed.