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Abstract Transcriptional regulation of gene expression has been widely studied. More recently, there has been increasing appreciation of the role that translational regulation plays in gene expression, resulting in a number of new fields engaging in translational studies. Regulation of protein synthesis is critical for cell growth, development, and survival, and is primarily controlled at the initiation step. Eukaryotic cells utilize multiple mechanisms to initiate translation, depending on cell stress, growth conditions, viral infection, or the sequences present in the mRNA. While the vast majority of mRNAs are translated in a cap‐dependent manner, an important subset of mRNAs uses an alternative mechanism, whereby ribosomes are recruited internally to the message to initiate cap‐independent translation. Some of these mRNAs contain an internal ribosome entry site (IRES) located in the 5′ untranslated region (UTR). However, establishing that an RNA element is a functional IRES requires a number of carefully executed experiments with specific controls. This review will clearly explain the required experiments, and the pros and cons of various assays, used to determine whether (or not) an RNA element functions as an IRES to promote initiation of translation. We hope that demystifying the accepted methods for assaying IRES activity will open the study of this important mechanism to the broader community. WIREs RNA 2012 doi: 10.1002/wrna.1129 This article is categorized under: Translation > Translation Mechanisms

Mechanisms of cap‐dependent translation. (a) The standard cap‐dependent mechanism involves recognition of the 5′ cap (black) by a complex of eukaryotic initiation factors (eIF) eIF4E, eIF4G, and eIF4A that recruit the 43S pre‐initiation complex (40S subunit, eIF1, eIF5, eIF1A, eIF2•met‐tRNAi•GTP, and eIF3. This generates a 48S complex and the 40S ribosomal subunit scans down to the first AUG. The 60S subunit then joins and protein synthesis begins (not shown). (b) In leaky scanning, the 40S subunit can bypass AUGs that are in a poor sequence context to generate proteins (blue) that have alternate N‐terminal ends. Here, the darker the AUG, the better the sequence context, and thus the more likely that it will be used to initiate protein synthesis. (c) Ribosome shunting requires recruitment of the ribosome through a cap‐dependent mechanism as in (a), then, following scanning or translation of a short ORF the ribosome is shunted (no scanning) downstream to initiate protein synthesis. (d) The presence of upstream open reading frames (uORFs) can prevent translation of the major coding region unless levels of the eIF2•met‐tRNAi•GTP are low. The ribosome is brought to the 5′ end as in (a), then following translation of the first uORF the 40S subunit remains associated with the mRNA and continues to scan down the mRNA until it acquires another eIF2•met‐tRNAi•GTP, enabling it to initiate translation at a downstream ORF.

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Controls for the bicistronic reporter assay must be performed to rule out all possible reasons for second cistron expression aside from internal ribosome entry site (IRES) activity. (a) The sequences inserted within the bicistronic reporter may contain weak or cryptic promoter activity that would result in the expression of a monocistronic mRNA that can be translated by the cap‐dependent mechanism. (b) Readthrough of the stop codon in the first cistron would generate a chimeric reporter protein that would be translated using a cap‐dependent mechanism. (c) The inserted sequences in the bicistronic reporter may contain a cryptic splice site that would generate either a chimeric protein (as in (b)) or a monocistronic RNA that encodes the second cistron. These spliced products would result in cap‐dependent expression of the second cistron.

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Reporter assays used to assess internal ribosome entry site (IRES) activity. (a) In the bicistronic reporter assay the IRES is placed between two cistrons. Expression of the first cistron (red) is cap‐dependent whereas expression of the second cistron (green) is dependent upon a functional IRES. The ribosomal subunits are shown (brown). (b) Insertion of an IRES into a circular RNA ensures that the expression of the reporter is due to the internal initiation since there is no free 5′ end. The removal of all the stop codons in the circular RNA allows for the generation of a single continuous open reading frame (ORF), such that multiple rounds of translation around the circle demonstrate that the circle is intact. Insertion of a protease cleavage site (orange) allows for the collapse of the polyprotein into a single protein product consistent with the ribosome transversing around the circle once. (c) Insertion of a stable hairpin in the 5′UTR can be used to block cap‐dependent translation. Therefore, translation of the cistron would be dependent upon a functional IRES upstream of the reporter ORF.

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Mechanisms of cap‐independent translation. (a) Internal ribosome entry sites (IRESs) recruit the 40S subunit internally to the mRNA using anywhere from all to none of the translation initiation factors (defined in Fig. 1(a)). (b) A cap‐independent translation element (CITE) located in the 3′ untranslated region (UTR) binds to the cap‐binding complex (eukaryotic initiation factors, eIF4E, eIF4G, and eIF4A) and through circularization of the mRNA recruits a 43S pre‐initiation complex to 5′UTR of the mRNA.

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