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.

Keywords: Bicistronic vector; CITE; Cap-independent translation; Cellular IRES; RNA transfection; RRL.

Fig. 1

a When different bicistronic mRNAs are compared to each other, a choice of whether or not to call the sequence an IRES strongly relies on what the latter is compared to. Inevitably, the choice becomes very subjective, especially when different negative controls are used. Note that the y-axis is split to show the relative efficiencies of EMCV and HCV IRESs. b A comparison of m⁷G- and A-capped mRNAs discriminates cap-dependent from cap-independent translation, while a comparison of monocistronic and bicistronic mRNAs reveals the contribution of internal (5′ end-independent) initiation to the overall level of translation. Such segregation should be made for a particular condition under which IRES is thought to be active

Fig. 2

Plasmid transfection leads to spurious transcription and splicing. When assayed by RT-PCR, most of these aberrant mRNA species will pass unnoticed

Fig. 3

a Single bicistronic plasmid is sufficient to provide PCR-made templates for the transcription of monocistronic and the bicistronic mRNAs. b Four sets of mRNAs are sufficient to dissect translation initiation mechanisms operating on a particular 5′UTR

Fig. 4

A generalized workflow of how to address internal initiation. Notably, all DNA-based experiments are inevitably followed by RNA-based experiments; thus, it is advisable to start with the latter