title: A researcher's guide to the galaxy of IRESs.
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 "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.
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119234, Russia. terenin@genebee.msu.ru.|Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119334, Russia. terenin@genebee.msu.ru.
authors.forename: Ilya M
authors.identifier: 0000-0002-3384-0589
authors.initials: IM
authors.lastname: Terenin
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119234, Russia.|Department of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119234, Russia.
authors.forename: Victoria V
authors.identifier: 0000-0002-1472-3555
authors.initials: VV
authors.lastname: Smirnova
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119234, Russia.
authors.forename: Dmitri E
authors.identifier: 0000-0001-5320-3045
authors.initials: DE
authors.lastname: Andreev
authors.affiliation: 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.|Department of Biochemistry, Biological Faculty, Lomonosov Moscow State University, Moscow, 119234, Russia.
authors.forename: Sergey E
authors.identifier: 0000-0002-1774-8475
authors.initials: SE
authors.lastname: Dmitriev
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119234, Russia. shatsky@genebee.msu.ru.
authors.forename: Ivan N
authors.identifier: 0000-0002-4428-9905
authors.initials: IN
authors.lastname: Shatsky
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D039642:Eukaryotic Initiation Factors / Q000378:metabolism; D006801:Humans; D000067556:Internal Ribosome Entry Sites; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D020121:5' Untranslated Regions; D039642:Eukaryotic Initiation Factors; D000067556:Internal Ribosome Entry Sites; D012333:RNA, Messenger
keywords: Bicistronic vector; CITE; Cap-independent translation; Cellular IRES; RNA transfection; RRL