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 (IR...

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Bibliographic Details
Published in:Cellular and molecular life sciences : CMLS 2017-04, Vol.74 (8), p.1431-1455
Main Authors: Terenin, Ilya M., Smirnova, Victoria V., Andreev, Dmitri E., Dmitriev, Sergey E., Shatsky, Ivan N.
Format: Article
Language:English
Subjects:
5' Untranslated Regions
Animals
Binding sites
Biochemistry
Biomedical and Life Sciences
Biomedicine
Cell Biology
Eukaryotic Initiation Factors - metabolism
Experiments
Gene expression
Humans
Internal Ribosome Entry Sites
Kinases
Life Sciences
Molecular biology
Peptide Chain Initiation, Translational
Plasmids
Protein Biosynthesis
Protein synthesis
Proteins
Review
Ribonucleic acid
Ribosomes - genetics
Ribosomes - metabolism
RNA
RNA polymerase
RNA, Messenger - genetics
RNA, Messenger - metabolism
Transfection
Translation
Translation systems
Viruses
Workflow
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Summary: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.
ISSN:1420-682X
1420-9071
DOI:10.1007/s00018-016-2409-5