The bicistronic reporter assay is the gold standard among the techniques that can be used to validate internal translation initiation. The unique feature, which discriminates internal initiation from other modes of translation initiation, is by definition that ribosomes are recruited to an internal part within the mRNA to be translated. To prove that the ribosome indeed enters the mRNA internally, one need to rule out potential ribosomal entry at the 5’end. Therefore, one could either use a circularized reporter that lacks any ends, a monocistronic reporter with a stable stem loop inserted at the 5’end or a bicistronic reporter. As generating circularized reporters is technically more challenging than generating linear ones, this method is not considered first choice. Further, a monocistronic reporter whose 5’end is blocked by a stable hairpin is still exposing the sequence under investigation at the 5’end and the stable element could introduce structural constraints that would interfere with the intrinsic initiation mechanism. Thus, the use of bicistronic reporters became the most conventional way to test IRES activity.
The working principle of such bicistronic reporter assays is simple: translation of the upstream cistron is cap-dependent, while translation of the downstream cistron is dependent on the intermediate sequence of interest. Due to the bicistronic setup, the reporter comes with its internal control and contribution of IRES-mediated translation can be set in relation to cap-dependent translation. However, the assay is prone to generate artifacts, wherefore additional control experiments are required to achieve unambiguous results.
When reporter assays are carried out by DNA transfection of reporter-encoding plasmids, reporter expression might not only be translationally but also transcriptionally controlled.
Monocistronic mRNAs might arise due to cryptic splice sites or promoters within the plasmid backbone or within the putative IRES-containing sequence under investigation itself. These aberrantly generated mRNAs will be capped and translationally competent in contrast to monocistronic mRNAs that would be uncapped and likely unstable when generated by breakage [72]. For example, firefly luciferase (sequence from the Photinus genus contained in pGL3-Basic, Promega) has cryptic promoter activity that is 10-16 times weaker than the cytomegalovirus (CMV) immediate-early promoter when expressed in human CCL13 and Huh7 cells [924]. When
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firefly luciferase is the 5’-reporter of a bicistronic construct, expression of the 3’-reporter will be a combination of IRES-dependent translation and firefly promoter activity generating truncated monocistronic transcripts. Another popular example of cryptic promoter activity is the 5’UTR of eIF4G, which was hypothesized to contain IRES activity. However, in a promoterless vector (SV40 promoter and chimeric intron sequence deleted from pRF (formerly pGL3R, Promega)), the eIF4G sequence yielded ~900-fold higher firefly activity than the promoterless pRF control vector in HeLa cells [53]. A series of deletion experiments mapped the promoter to a region of 168 bases upstream of the translation initiation site (TIS), with the most critical elements located between 69 and 49 bases upstream of the TIS [53]. But eIF4G was also shown to contain a cryptic splice site. Baranick et al. engineered a bicistronic vector eliminating all potential 5’ ss in the transcribed sequence upstream of the putative IRES by silent mutations [57]. That construct contains Gausia luciferase as 5’-reporter and GFP as 3’-reporter within a pGL4.75[hRluc/CMV]
backbone (Promega) [57]. Using this vector, eIF4G had similar translational activity as the non-IRES sequences of γ-actin or β-globin, which was about five times lower than EMCV-dependent or cap-dependent translation in Hela cells [57]. Deeper analysis identified a pyrimidine-rich tract and a 3’ss between 65 to 52 bases upstream of the TIS, which were used for splicing between the native murine leukemia virus 5’ss or a cryptic 5’ss of env within the upstream vector backbone [57].
Splicing between the untranslated vector backbone and the bicistronic reporter might happen more often as anticipated. Commonly used pRL vectors (Promega) are designed to contain a chimeric intron downstream of the constitutive promoter region and upstream of the RLuc sequence to increase the expression level of the reporter [925]. Although splice sites of the chimeric intron are modified to reflect consensus sequences that allow for optimal splicing efficiency, the splice donor site of the chimeric intron can unintentionally pair with a cryptic splice acceptor site of the putative IRES [925]. This was shown, for example, for the pRL-XIAP-FL construct in which the majority of the produced transcript contained the pRL-XIAP-FLuc reporter only, while the complete RLuc sequence was spliced out due to splicing between the donor splice site of the chimeric intron and the cryptic acceptor splice site in XIAP 5’UTR [65].
Not only chimeric introns but also other functional elements of the vector backbone can lead to aberrant reporter expression. For instance, the TATA-containing cryptic promoter within pMB1 origin of replication (ori) was shown to cause robust RNA expression when the inserted sequence of interest contained a 3’ss [481]. The authors of that study found a significant correlation between reporter activity and aberrant transcript expression from the ori, indicating that commonly used plasmids like pRF, pGL3 and pGL4 are prone to produce transcript artefacts promoting potentially incorrect interpretation of reporter assay results [481].
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Apart from aberrantly generated transcripts, another potential source of misinterpretation of reporter assays is the issue of background translation levels. In general, any kind of bicistronic reporter will yield expression of the downstream cistron [64]. The readout will never be zero.
Hence, translational activity of putative IRESs needs to be set in context of positive and negative controls in order to figure out from which value onwards one could consider the expression meaningful. Most of the time the natural context of a sequence under investigation is its 5’UTR.
In this setting putative IRES-dependent translation is usually in competition with 5’end or cap-dependent translation and in bicistronic reporters the translational activity of the putative IRES is usually inefficient compared to cap-dependent or viral IRES-dependent translation [27], [66], [73]. Inserting a sequence in the intercistronic space of a bicistronic reporter is typically yielding an expression ratio from first cistron/second cistron of about five- to twofold over the negative no IRES control [27]. As translation efficiency of this negative control should be considered background level, so in principle zero and very small, a stimulation of five to twofold is still small and putative IRES-activity substantially weaker than cap-dependent translation. [27]. It is important to keep this in mind. Further one needs to keep an eye on variations in the range of background expression, which are small but might appear meaningful when not properly controlled for. It was for example described that the design of a bicistronic reporter can affect expression levels of upstream and downstream cistrons. Both in vivo and in vitro, the expression from a downstream cistron increased with increasing lengths of the intercistronic region which lacked IRES activity [551], [926]. The expression level further increased in vitro by overexpression of PABP [926]. Different concentrations of translational regulators might explain as well why also in vivo expression levels from the second cistron of a bicistronic reporter without IRES in the intercistronic sequences can vary in between cell lines [551], [927]. Also, the order and the type of reporter can influence expression levels. A systematic analysis revealed that when inserted into pBS or pBC vectors (Agilent), poliovirus and EMCV IRESs work better when RLuc is in the upstream reporter position, while IRES activity is substantially weaker when FLuc is the upstream reporter [928]. The same was the case when RLuc was replaced by CAT [928].
Furthermore, FMCV IRES can enhance the translation of the upstream reporter, acting as a CITE by recruiting eIF4F, which makes the translation of the downstream reporter appear weaker [929]. Thus, the putative IRES activity of a sequence of interest cannot be compared between studies that used different cell lines, different intercistronic sequences, a different arrangement of reporters or different viral controls.
In general, the usage of viral control IRESs can have a huge impact on the interpretation of reporter assay results. HCV IRES for example has relatively weak activity in bicistronic reporter assays, being about ten times less efficient in HEK293T cells than EMCV IRES [67].
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The strengths of EMCV IRES can also vary by almost an order of magnitude depending which commercially available EMCV-coding plasmid is used [930]. The EMCV IRES activity depends on the inserted sequence (some sequences carry an additional adenine nucleotide in the bifurcation loop at the junction of J and K stems, some carry a minimal sequence) and how the sequence is modified around and connected with the AUG start codon of the protein coding gene [930]. Further the commonly used viral control IRESs EMCV and HCV have both been demonstrated to exhibit cryptic promoter activity so that proper control experiments are warranted not only for potential cellular IRESs but also for viral IRES reporters [931]–[933].
But not only the choice of the positive control is important, also the choice of the negative control is crucial for interpretation of results. Frequently a reverse complement of the sequence of interest is used. However, as the reversed sequence is not evolutionary selected against start codons, it almost always contains uAUGs that inhibit downstream reporter expression [64]. Hence antisense 5’UTRs are unreliable controls prone to produce aberrant ORFs which make it impossible to directly compare reporter expression between sense and antisense sequences [64].
To avoid all these pitfalls in the interpretation of bicistronic reporter assays a couple of validation experiments must be implemented. First, DNA transfection assays require validation by RNA transfection assays to unequivocally rule out regulation at the transcriptional instead of translational level. Four sets of mRNA reporters are sufficient to dissect the initiation mechanism promoting translation of a particular sequence of interest (Fig. 14) [64].
Cap-dependency of a sequence can be determined by comparing translational activity of a m7G- with A-capped mRNA. The contribution of 5’-end-dependent translation can be determined by adding another A-capped reporter carrying a stable hairpin at the 5’-end. The hairpin inhibits ribosome entry so that the comparison between translation efficiencies of an A-capped reporter with and without hairpin gives information on how efficiently the free 5’-end is recruiting
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Fig. 14: Sets of mRNA reporters required to dissect the contribution of different modes of translation initiation that are active for a sequence of interest. The contribution of cap-dependent, cap-independent and 5’end-independent translation initiation to the overall translational activity of a candidate sequence can be determined by directly comparing differentially capped monocistronic and bicistronic reporter expression levels. Introduction of stable hairpins close to the reporter 5’end can further help to elucidate the dependencies. Adapted from Terenin et al., 2017 [64].
ribosomes. Neither cap- nor 5’-end-dependent but internal translation can be determined by bicistronic reporter assays. Comparing translation efficiency of the downstream cistron of a bicistronic reporter with translation efficiencies of monocistronic m7G- and A-capped reporters identifies the contribution of IRES-dependent translation to the overall translation efficiency. To unequivocally rule out that translation of the second cistron is coupled to translation of the first cistron, by i.e. translation reinitiation or ribosome shunting, another bicistronic reporter carrying a stable hairpin at the 5’end can be included in the analyses.
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When the contribution of the cap to the overall translation is low and expression of the downstream cistron of the bicistronic reporter equals expression of the capped monocistronic reporter, the sequence might indeed function as an IRES. But also, less efficient putative IRESs might still promote meaningful expression levels under specific cellular conditions. This requires reporter analyses in the corresponding physiological context. When the bicistronic translation efficiency is appreciably lower than cap-dependent translation of a monocistronic reporter, the sequence is unlikely to function as an IRES. Instead it might act as a CITE, when 5’-end-dependent translation is high and cap-5’-end-dependent translation is low. But in most cases the studied sequence is translated two to three orders of magnitude higher in a capped monocistronic mRNA than in a bicistronic setting, which depicts cap-dependency and the absence of IRES activity [64].
In comparison, EMCV IRES can cause a 100 to 250-fold stimulation of the expressed downstream cistron compared to the upstream cistron, which begs the question of whether it is justified to call cellular IRESs, whose activity is not nearly as efficient, bona fide IRESs [27].
Moreover, it is important to provide absolute values of reporter expression instead of fold changes or ratios so that the strengths of cellular IRESs is not masked or artificially boosted by using background expression levels as a reference. As described above the translational activity of a sequence of interest should rather be compared to itself in a monocistronic context instead of an “empty” vector or a viral element. If IRES activity is tested under specific conditions like 4E-BP overexpression, protease-mediated eIF4G cleavage or in context of pharmacological inhibitors, expression levels of upstream and downstream cistrons should be displayed independently, as the calculation of a ratio can hide differential effects on cap-dependent and internal translation initiation.
When bicistronic reporter assays are carried out by DNA transfection in advance of RNA transfections, promoterless plasmid and RNAi tests are required to verify candidates. By deleting the promoter region but keeping the enhancer, it can be determined if bicistronic reporter vectors produce aberrant transcripts due to cryptic promoter activity. Usage of a different vector lacking the promoter and sometimes also the enhancer to screen for cryptic promoters is likely creating a source of error, as different plasmid backbones might generate different kinds of aberrant transcripts making it impossible to compare results obtained from different backbones.
As aberrant transcripts due to cryptic splicing will be missed by this approach, another RNAi test is required. Lloyd and colleagues invented a convenient control assay in which cells are co-transfected with siRNAs targeting the upstream cistron of a bicistronic reporter [65].
Expression of both cistrons will be reduced to similar extend if expression is indeed driven by the anticipated bicistronic reporter. But monocistronic reporters containing only the downstream
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cistron due to upstream splicing, won’t be affected by the knockdown and can easily be identified by this approach.
Other tests for RNA integrity like RNase protection assay, RT-PCR and northern blot can be applied as well but cannot replace promoterless and RNAi validation experiments.
Further, these assays should be designed to identify aberrantly produced shorter transcripts rather than the full-length bicistronic mRNA [64]. In RNase protections assays and RT-PCR experiments probes and primers need to be designed in a way that no potentially relevant incomplete reporter species is missed. Further, overexposed northern blots might still not be sensitive enough to detect monocistronic transcripts, which might be produced at low levels but could substantially contribute to the overall reporter expression [53], [59], [65]. Also, RT-PCR experiments might not provide enough sensitivity to show differential expression between the two cistrons of a reporter.
Cellular IRESs are also often studied by in vitro translation assays. However, the commonly used rabbit reticulocyte lysate (RRL) is only poorly reflecting physiological conditions.
Many putative IRESs seem to have weak activity in RRL, because weakly translated mRNAs are not inhibited by supplementation with m7G or 4E-BP or are not proportionally stimulated by capping or a poly(A) tail [64], [934]–[937].
To conclude, the gold standard bicistronic reporter assay is only as powerful in describing IRES activity as is the quality of associated validation experiments. Unfortunately, essential controls are often missing, so that to date a high number of reported cellular IRESs still requires conclusive verification, leaving these putative IRESs in an uncertain or not proven state [27], [59].
Some of the previously reported cellular IRESs have also been refuted during later in-depth examination. For future research it is important to not only stick to strict controls but also consider alternative explanations for translational phenomena, like CITE-like mechanisms, which so far seem to be less prominently addressed. It is vital to create greater awareness on the fact that cellular IRESs are not yet as firmly established as widely believed to enable unbiased investigation in all directions especially to researchers who are newly entering the field [64].