Moriarty P M, Reddy C C, Maquat L E
Department of Human Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Mol Cell Biol. 1998 May;18(5):2932-9. doi: 10.1128/MCB.18.5.2932.
The mammalian mRNA for selenium-dependent glutathione peroxidase 1 (Se-GPx1) contains a UGA codon that is recognized as a codon for the nonstandard amino acid selenocysteine (Sec). Inadequate concentrations of selenium (Se) result in a decrease in Se-GPx1 mRNA abundance by an uncharacterized mechanism that may be dependent on translation, independent of translation, or both. In this study, we have begun to elucidate this mechanism. We demonstrate using hepatocytes from rats fed either a Se-supplemented or Se-deficient diet for 9 to 13 weeks that Se deprivation results in an approximately 50-fold reduction in Se-GPx1 activity and an approximately 20-fold reduction in Se-GPx1 mRNA abundance. Reverse transcription-PCR analyses of nuclear and cytoplasmic fractions revealed that Se deprivation has no effect on the levels of either nuclear pre-mRNA or nuclear mRNA but reduces the level of cytoplasmic mRNA. The regulation of Se-GPx1 gene expression by Se was recapitulated in transient transfections of NIH 3T3 cells, and experiments were extended to examine the consequences of converting the Sec codon (TGA) to either a termination codon (TAA) or a cysteine codon (TGC). Regardless of the type of codon, an alteration in the Se concentration was of no consequence to the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. The ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA from the wild-type (TGA-containing) allele was reduced twofold when cells were deprived of Se for 48 h after transfection, which has been shown to be the extent of the reduction for the endogenous Se-GPx1 mRNA of cultured cells incubated as long as 20 days in Se-deficient medium. In contrast to the TGA allele, Se had no effect on expression of either the TAA allele or the TGC allele. Under Se-deficient conditions, the TAA and TGC alleles generated, respectively, 1.7-fold-less and 3-fold-more cytoplasmic Se-GPx1 mRNA relative to the amount of nuclear Se-GPx1 mRNA than the TGA allele. These results indicate that (i) under conditions of Se deprivation, the Sec codon reduces the abundance of cytoplasmic Se-GPx1 mRNA by a translation-dependent mechanism and (ii) there is no additional mechanism by which Se regulates Se-GPx1 mRNA production. These data suggest that the inefficient incorporation of Sec at the UGA codon during mRNA translation augments the nonsense-codon-mediated decay of cytoplasmic Se-GPx1 mRNA.
哺乳动物硒依赖性谷胱甘肽过氧化物酶1（Se-GPx1）的信使核糖核酸（mRNA）含有一个UGA密码子，该密码子被识别为非标准氨基酸硒代半胱氨酸（Sec）的密码子。硒（Se）浓度不足会导致Se-GPx1 mRNA丰度下降，其机制尚不明确，可能依赖于翻译、独立于翻译或两者皆有。在本研究中，我们已开始阐明这一机制。我们使用喂食硒补充或缺硒饮食9至13周的大鼠肝细胞进行实验，结果表明，缺硒导致Se-GPx1活性降低约50倍，Se-GPx1 mRNA丰度降低约20倍。对细胞核和细胞质组分进行逆转录聚合酶链反应（RT-PCR）分析显示，缺硒对细胞核前体mRNA或细胞核mRNA水平均无影响，但会降低细胞质mRNA水平。在NIH 3T3细胞的瞬时转染实验中重现了硒对Se-GPx1基因表达的调控作用，并进一步开展实验，研究将Sec密码子（TGA）转换为终止密码子（TAA）或半胱氨酸密码子（TGC）的后果。无论密码子类型如何，硒浓度的改变对细胞核Se-GPx1 mRNA与细胞核Se-GPx1前体mRNA的比例均无影响。转染后若细胞缺硒48小时，野生型（含TGA）等位基因的细胞质Se-GPx1 mRNA与细胞核Se-GPx1 mRNA的比例会降低两倍，而这一降低幅度已被证明与在缺硒培养基中培养长达20天的培养细胞内源性Se-GPx1 mRNA的降低幅度相同。与TGA等位基因不同，硒对TAA等位基因或TGC等位基因的表达均无影响。在缺硒条件下，相对于细胞核Se-GPx1 mRNA的量，TAA和TGC等位基因产生的细胞质Se-GPx1 mRNA分别比TGA等位基因少1.7倍和多3倍。这些结果表明：（i）在缺硒条件下，Sec密码子通过依赖于翻译的机制降低细胞质Se-GPx1 mRNA的丰度；（ii）不存在硒调控Se-GPx1 mRNA产生的其他机制。这些数据表明，在mRNA翻译过程中，UGA密码子处Sec的低效掺入增强了细胞质Se-GPx1 mRNA的无义密码子介导的衰变。
