Terenin Ilya M, Smirnova Victoria V, Andreev Dmitri E, Dmitriev Sergey E, Shatsky Ivan N
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119234, Russia.
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119334, Russia.
Cell Mol Life Sci. 2017 Apr;74(8):1431-1455. doi: 10.1007/s00018-016-2409-5. Epub 2016 Nov 16.
The idea of internal initiation is frequently exploited to explain the peculiar translation properties or unusual features of some eukaryotic mRNAs. In this review, we summarize the methods and arguments most commonly used to address cases of translation governed by internal ribosome entry sites (IRESs). Frequent mistakes are revealed. We explain why "cap-independent" does not readily mean "IRES-dependent" and why the presence of a long and highly structured 5' untranslated region (5'UTR) or translation under stress conditions cannot be regarded as an argument for appealing to internal initiation. We carefully describe the known pitfalls and limitations of the bicistronic assay and artefacts of some commercially available in vitro translation systems. We explain why plasmid DNA transfection should not be used in IRES studies and which control experiments are unavoidable if someone decides to use it anyway. Finally, we propose a workflow for the validation of IRES activity, including fast and simple experiments based on a single genetic construct with a sequence of interest.
内部起始的概念常被用于解释某些真核生物mRNA独特的翻译特性或异常特征。在本综述中，我们总结了用于处理由内部核糖体进入位点（IRES）调控的翻译情况时最常用的方法和论据。揭示了常见的错误。我们解释了为什么“不依赖帽子结构”并不容易等同于“依赖IRES”，以及为什么不能将长且高度结构化的5'非翻译区（5'UTR）的存在或应激条件下的翻译视为支持内部起始的论据。我们仔细描述了双顺反子检测已知的陷阱和局限性以及一些市售体外翻译系统的假象。我们解释了为什么在IRES研究中不应使用质粒DNA转染，以及如果有人无论如何决定使用它，哪些对照实验是不可避免的。最后，我们提出了一个验证IRES活性的工作流程，包括基于带有感兴趣序列的单个遗传构建体的快速且简单的实验。
