Buzina A, Shulman M J
Departments of Immunology and Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Mol Biol Cell. 1999 Mar;10(3):515-24. doi: 10.1091/mbc.10.3.515.
In many organisms nonsense mutations decrease the level of mRNA. In the case of mammalian cells, it is still controversial whether translation is required for this nonsense-mediated RNA decrease (NMD). Although previous analyzes have shown that conditions that impede translation termination at nonsense codons also prevent NMD, the residual level of termination was unknown in these experiments. Moreover, the conditions used to impede termination might also have interfered with NMD in other ways. Because of these uncertainties, we have tested the effects of limiting translation of a nonsense codon in a different way, using two mutations in the immunoglobulin mu heavy chain gene. For this purpose we exploited an exceptional nonsense mutation at codon 3, which efficiently terminates translation but nonetheless maintains a high level of mu mRNA. We have shown 1) that translation of Ter462 in the double mutant occurs at only approximately 4% the normal frequency, and 2) that Ter462 in cis with Ter3 can induce NMD. That is, translation of Ter462 at this low (4%) frequency is sufficient to induce NMD.
在许多生物体中，无义突变会降低mRNA的水平。对于哺乳动物细胞而言，这种无义介导的RNA降解（NMD）是否需要翻译仍存在争议。尽管先前的分析表明，阻碍无义密码子处翻译终止的条件也会阻止NMD，但在这些实验中终止的残留水平尚不清楚。此外，用于阻碍终止的条件也可能以其他方式干扰了NMD。由于存在这些不确定性，我们使用免疫球蛋白μ重链基因中的两个突变，以不同的方式测试了限制无义密码子翻译的效果。为此，我们利用了密码子3处的一个特殊无义突变，该突变可有效终止翻译，但仍保持高水平的μ mRNA。我们已经表明：1）双突变体中Ter462的翻译频率仅约为正常频率的4%；2）与Ter3顺式排列的Ter462可诱导NMD。也就是说，以这种低（4%）频率进行的Ter462翻译足以诱导NMD。
