Page M F, Carr B, Anders K R, Grimson A, Anderson P
Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA.
Mol Cell Biol. 1999 Sep;19(9):5943-51. doi: 10.1128/MCB.19.9.5943.
mRNAs that contain premature stop codons are selectively degraded in all eukaryotes tested, a phenomenon termed "nonsense-mediated mRNA decay" (NMD) or "mRNA surveillance." NMD may function to eliminate aberrant mRNAs so that they are not translated, because such mRNAs might encode deleterious polypeptide fragments. In both yeasts and nematodes, NMD is a nonessential system. Mutations affecting three yeast UPF genes or seven nematode smg genes eliminate NMD. We report here the molecular analysis of smg-2 of Caenorhabditis elegans. smg-2 is homologous to UPF1 of yeast and to RENT1 (also called HUPF1), a human gene likely involved in NMD. The striking conservation of SMG-2, Upf1p, and RENT1/HUPF1 in both sequence and function suggests that NMD is an ancient system, predating the divergence of most eukaryotes. Despite similarities in the sequences of SMG-2 and Upf1p, expression of Upf1p in C. elegans does not rescue smg-2 mutants. We have prepared anti-SMG-2 polyclonal antibodies and identified SMG-2 on Western blots. SMG-2 is phosphorylated, and mutations of the six other smg genes influence the state of SMG-2 phosphorylation. In smg-1, smg-3, and smg-4 mutants, phosphorylation of SMG-2 was not detected. In smg-5, smg-6, and smg-7 mutants, a phosphorylated isoform of SMG-2 accumulated to abnormally high levels. In smg-2(r866) and smg-2(r895) mutants, which harbor single amino acid substitutions of the SMG-2 nucleotide binding site, phosphorylated SMG-2 accumulated to abnormally high levels, similar to those observed in smg-5, smg-6, and smg-7 mutants. We discuss these results with regard to the in vivo functions of SMG-2 and NMD.
在所有已检测的真核生物中，含有提前终止密码子的信使核糖核酸（mRNA）会被选择性降解，这一现象被称为“无义介导的mRNA降解”（NMD）或“mRNA监测”。NMD可能起到消除异常mRNA的作用，使其不被翻译，因为这类mRNA可能编码有害的多肽片段。在酵母和线虫中，NMD都是一个非必需系统。影响酵母三个UPF基因或线虫七个smg基因的突变会消除NMD。我们在此报告对秀丽隐杆线虫smg - 2的分子分析。smg - 2与酵母的UPF1以及RENT1（也称为HUPF1，一个可能参与NMD的人类基因）同源。SMG - 2、Upf1p和RENT1/HUPF1在序列和功能上的显著保守性表明NMD是一个古老的系统，在大多数真核生物分化之前就已存在。尽管SMG - 2和Upf1p的序列相似，但在秀丽隐杆线虫中表达Upf1p并不能挽救smg - 2突变体。我们制备了抗SMG - 2多克隆抗体，并在蛋白质免疫印迹中鉴定出了SMG - 2。SMG - 2被磷酸化，其他六个smg基因的突变会影响SMG - 2的磷酸化状态。在smg - 1、smg - 3和smg - 4突变体中，未检测到SMG - 2的磷酸化。在smg - 5、smg - 6和smg - 7突变体中，一种磷酸化的SMG - 2异构体积累到异常高水平。在携带SMG - 2核苷酸结合位点单氨基酸替换的smg - 2(r866)和smg - 2(r895)突变体中，磷酸化的SMG - 2积累到异常高水平，类似于在smg - 5、smg - 6和smg - 7突变体中观察到的情况。我们就这些结果讨论了SMG - 2和NMD在体内的功能。
