A researcher's guide to the galaxy of IRESs.
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.
['Terenin|Ilya M|IM|0000-0002-3384-0589', 'Smirnova|Victoria V|VV|0000-0002-1472-3555', 'Andreev|Dmitri E|DE|0000-0001-5320-3045', 'Dmitriev|Sergey E|SE|0000-0002-1774-8475', 'Shatsky|Ivan N|IN|0000-0002-4428-9905']
D020121:5' Untranslated Regions D000818:Animals D039642:Eukaryotic Initiation Factors D006801:Humans D000067556:Internal Ribosome Entry Sites D010442:Peptide Chain Initiation, Translational D014176:Protein Biosynthesis D012333:RNA, Messenger D012270:Ribosomes D014162:Transfection
Bicistronic vector CITE Cap-independent translation Cellular IRES RNA transfection RRL