Authors: Ilya M Terenin Victoria V Smirnova Dmitri E Andreev Sergey E Dmitriev Ivan N Shatsky
Publish Date: 2016/11/16
Volume: 74, Issue: 8, Pages: 1431-1455
Abstract
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 “capindependent” does not readily mean “IRESdependent” 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
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