Determinants of initiation codon selection during translation in mammalian cells.
Determinants of initiation codon selection during translation in mammalian cells.
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DOI：
10.1371/journal.pone.0015057
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发表时间：
2010-11-24
期刊：
影响因子：
3.7
通讯作者：
Mauro VP
中科院分区：
文献类型：
--
作者：
Matsuda D;Mauro VP
影响mRNA翻译的因素增加了真核生物蛋白质组的复杂性。在某些情况下，特定mRNA的翻译可以产生多种蛋白质。然而，决定核糖体是从转录本中的第一个AUG密码子开始翻译，是从下游密码子开始翻译，还是从多个位点开始翻译的因素尚不完全清楚。各种mRNA的特性，包括AUG密码子可及性和5 '先导体长度被认为是影响核糖体在何处启动翻译的潜在决定因素。为了探讨这一问题，我们使用具有两个框架内AUG密码子的合成mrna进行了研究-两者都在良好的环境中。启动于AUG1和AUG2的开放阅读框分别编码报告蛋白的大、小同工型。在COS-7细胞中，这种mRNA的翻译被证明是5 '帽依赖的，并且有效地发生在两个AUG密码子上。利用反义锁定核酸寡核苷酸和外显子连接复合物两种不同的元件修饰AUG密码子的可及性。当任何一种元素被用来掩盖AUG1时，合成的蛋白质比例发生变化，有利于较小的（aug2启动的）蛋白质。此外，我们观察到引线长度的增加本身改变了蛋白质的比例，并有利于在AUG1起始。这些观察结果表明，起始密码子的选择受AUG密码子可及性和5 ‘先导子长度等多种因素的影响，并不一定取决于AUG密码子的顺序（5 ’→3 '）。AUG密码子可及性的调控可能为真核细胞翻译调控提供了一种强有力的手段。
Factors affecting translation of mRNA contribute to the complexity of eukaryotic proteomes. In some cases, translation of a particular mRNA can generate multiple proteins. However, the factors that determine whether ribosomes initiate translation from the first AUG codon in the transcript, from a downstream codon, or from multiple sites are not completely understood. Various mRNA properties, including AUG codon-accessibility and 5′ leader length have been proposed as potential determinants that affect where ribosomes initiate translation. To explore this issue, we performed studies using synthetic mRNAs with two in-frame AUG codons−both in excellent context. Open reading frames initiating at AUG1 and AUG2 encode large and small isoforms of a reporter protein, respectively. Translation of such an mRNA in COS-7 cells was shown to be 5′ cap-dependent and to occur efficiently from both AUG codons. AUG codon-accessibility was modified by using two different elements: an antisense locked nucleic acid oligonucleotide and an exon-junction complex. When either element was used to mask AUG1, the ratio of the proteins synthesized changed, favoring the smaller (AUG2-initiated) protein. In addition, we observed that increased leader length by itself changed the ratio of the proteins and favored initiation at AUG1. These observations demonstrate that initiation codon selection is affected by various factors, including AUG codon-accessibility and 5′ leader length, and is not necessarily determined by the order of AUG codons (5′→3′). The modulation of AUG codon accessibility may provide a powerful means of translation regulation in eukaryotic cells.
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