Cap-independent translation initiation at the 5’end: CITE-like mechanisms

Alternative translation initiation mechanisms can be independent of the cap structure but still dependent on a free 5‘-end from which the ribosome enters the mRNA for scanning towards the start codon. This mechanism contrasts with IRES-mediated translation in which the ribosome is recruited to an internal site and it contrasts as well with the canonical initiation mechanism in which the 43S PIC is recruited by the eIF4F-bound cap. Although the cap is highly stimulatory and efficient in promoting translation, any mRNA can be translated to a certain extent in mammalian cells even if it lacks a cap [179]. In the 90s Gunnery and Mathews found that this is not only true for in vitro transcribed mRNAs, but also for uncapped reporters synthesized by polymerase III (pol III) [180]. They introduced the tat gene of HIV-1 under a pol III promoter, resulting in a mRNA that was neither capped nor polyadenylated and that was expressed in vitro in a pol III transcription system and translated in vivo in transfected HeLa cells [180]. Mutation analysis of short uORFs and stable stem loops revealed that the uncapped mRNA was scanned from the 5’end suggesting that cap-independent translation still depends on the 5’end [180], [181]. Also uncapped and leaderless mRNA was translated by mammalian ribosomes after the 80S complex and initiator Met-tRNA assembled on the mRNA in the absence of translation initiation factors in toeprinting assays [182]. The mRNA analyzed was cIlacZ from the Λ phage.

It had only one nt (G) upstream of the AUG start codon and lacked all regulatory elements inherent to eukaryotic translation initiation. Comparing a capped to an uncapped version of it

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(starting with ApppN instead of m⁷G to stabilize the mRNA by preventing degradation by 5’

exonucleases), revealed that cIlacZ had a low cap-dependency compared to conventional mRNAs containing a 5’UTR [183]. In principle the leaderless mRNA can be translated by four different mechanisms in which the initiator tRNA is delivered either by eIF2, eIF2D and its homologs, eIF5B or initiation might even proceed without any of these factors involved [183]. However, it hasn’t been investigated whether all four mechanisms are equally efficient in cap-dependent and cap-independent translation initiation on the leaderless mRNA.

But cap-independent translation initiation has also been described for natural mammalian mRNAs. One mechanism resembles the translation initiation of plant positive-strand RNA viruses that employ CITEs to promote protein synthesis from uncapped RNAs. CITEs are usually located at the 3’ UTR of viral RNAs. They can bind a component of the eIF4F complex and form RNA-RNA kissing-loop interactions with a hairpin loop close to the RNA 5’ end [184].

CITEs work as well when they are placed in the 5’UTR [185]. After initiation factors are recruited, ribosomes enter at or near the free 5’ end from where they scan towards the start codon [184]. A similar mechanism has been suggested for mammalian mRNAs like for instance Apaf-1. Feasibility of this mechanism was experimentally demonstrated by inserting the J-K domain of the EMCV IRES, which binds eIF4G, into the 5’UTR of the cap-dependent mRNA of β-globin, rendering β-globin translation cap-independent [186]. Apaf-1 was found to contain a domain, which reduces its dependency on the cap so that an ApppN-capped Apaf-1 mRNA still displays ~15 % of the translational activity of a m⁷G-capped Apaf-1 mRNA in RNA transfection assays in HEK293T cells [67], [187]. However, further analysis revealed that Apaf-1 mRNA translation is 5’-end dependent and involves ribosome scanning, as insertion of an uAUG or uORF reduces its translational activity, which is only around background level in bicistronic reporters, where it is also not stimulated under stress conditions [67], [187]. Thus the 5’UTR of Apaf-1 is acting like CITEs in plant RNA viruses and careful analyses were required to demonstrate that CITES are functionally different from IRESs in terms of that a free 5’end is required for CITE-mediated cap-independent translation.

Roughly two decades ago the Hentze lab performed similar assays by directly tethering IRP-1 fused to the central domain (AA 642-1091) of eIF4G to three repeating iron response elements (IREs) in the intercistronic space of a bicistronic reporter [188]. Like in the proof of principle experiments for CITEs by Terenin et al., recruitment of eIF4G stimulated the translation of both upstream and downstream cistrons, with downstream translation being 2.3 – 5.5 % of cap-dependent translation and negligible compared with viral IRES-mediated reporter translation (~14 % of HCV in DNA transfection assays) [188].

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Apart from eIF4G recruitment during CITE-like translation, other eIFs could play a role in this mechanism. For example, eIF3 could be another potentially recruited factor. eIF3 had been shown to play a role in N⁶-methyladenosine (m⁶A)-promoted cap-independent translation and can activate or repress mRNAs of cell proliferation regulators through different modes of binding stem-loops within the respective 5’UTRs [189], [190]. eIF3 encompasses 13 subunits and it can bind the 40S subunit, stimulates 43S formation and 43S attachment to the mRNA as well as subsequent scanning [13]. Two subunits, eIF3d and eIF3l, were reported to have cap-binding activity and eIF3d was even reported to promote an alternative translation initiation mechanism, when eIF4E recruitment to the mRNA is blocked [191]. Hence, eIF3 seems a promising candidate to contribute to CITE-like translation, which could coexist with canonical cap-dependent translation at corresponding mRNAs.

Another factor which was frequently reported to play a role in cap-independent or IRES-dependent translation is eIF4G2, also known as NAT1, p97 or DAP5. It was simultaneously discovered by four different research groups in 1997 and is one out of three eIF4G homologues that is N-terminally truncated and therefore misses the binding sites for eIF4E and PABP [192]–

[195]. First, it was regarded as a factor that promotes non-canonical translation initiation after caspase cleavage and during cellular stress conditions, but later it was described to activate translation also under physiological conditions [196]–[202]. It’s non-canonical translation regulation was also reported to play a role in the retinoic acid pathway and ESC differentiation [203], [204]. Although eIF4G2 was described to play a role in translation of several putative IRES-containing mRNAs (Apaf-1, Bcl-2, CDK1, c-IAP1/HIAP2, c-Myc, HMGN3, p53, XIAP and its own transcript), it is unclear whether it actually does so, as some of these IRES evaluating experiments would require additional experimental support, while other putative IRESs have been refuted later (Apaf-1, XIAP) [57], [149], [197], [199], [204]–[209]. So, it is unclear if and to what extend eIF4G2 is involved in non-canonical translation initiation. However, eIF4G2 plays role in translation of not only a few selected, but rather a larger group of mRNAs, as depletion of eIF4G2 leads to a reduction in global protein synthesis of about 20-30 % (siRNA silencing in human breast cancer cells) [210]. Interestingly, the depletion of eIF4G1 also leads to a reduction in protein synthesis of about 20-30 %, while simultaneous depletion of both eIF4G1 and eIF4G2 leads to a reduction of about 60 % [210]. Overexpression of eIF4G2 could not compensate eIF4G1 depletion, showing that the factors could not physiologically substitute for each other and that they regulate translation of different mRNA subgroups [210]. Further, it was shown in vitro that eIF4G2 can stimulate translation of distinct m⁷G- and A-capped reporters and also stimulates their translation when cap-dependent translation is repressed by the presence of 4E-BP1 [206]. Due to these properties and its homology to eIF4G1 it might well be that eIF4G2

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directs cap-independent or CITE-like mediated translation of distinct mRNAs. This might likely include as well other auxiliary factors or alternative eIF isoforms, but further analyses are required validate this hypothesis.

There have also been a few approaches to detect cap-independently translated sequences genome wide. Wellensiek and colleagues for example used an mRNA display strategy by which in vitro-transcribed and in vitro-translated uncapped mRNA reporter fragments of 150 nt length were photo-ligated to a DNA-puromycin linker so that the mRNA got covalently linked to a peptide affinity tag encoded in their own ORF during translation [131]. Fusion products were isolated and functional RNA reporters underwent another selection cycle. After six rounds, more than 12,000 cap-independent translation-enhancing elements (TEEs) were identified within the ~10¹³ fragments from total human DNA that were included in the assay [131]. Roughly 12 % of these TEEs mapped to genomic regions of known genes. Selected TEEs were validated in vivo by monocistronic reporters with 5-terminal hairpins which were expressed by the vaccinia virus expression system. Roughly 20 % of the sequences passed this test and roughly 60 % of sequences passed the promoterless reporter test [131]. Apart from the fact that a substantial proportion of false positives was identified, the experimental design of this validation assay was not targeted for cap-independent translation. The comparison between m⁷G- and A-capped reporters would have been straightforward. Insertion of hairpins close to the cap instead evaluates if a free 5’end is required for translation initiation. A follow-up study characterized some putative TEEs in greater detail and a conserved 13-mer motif was identified that is partially complementary to the 18S rRNA [211]. This 13-mer is part of a larger conserved region that spans ~80 nts and contains also a conserved AUG triplet [211]. When the 13-mer is separately tested for its translation efficiency in luciferase reporter assays (HeLa cells transfected with the vaccinia virus expression system) without the larger conserved sequence context, it has only 10 % of the activity of the full length sequence [211]. Further, one of the exemplarily examined TEEs (HGL6.985) didn’t display substantial cap-independent translation efficiency in a mRNA transfection assay (~25 % of capped mRNA reporter) and the 13-mer motif did not enhance translation of the authentic start codon but enhanced uAUG translation generating an elongated protein instead [211]. Thus, it’s unclear whether the TEE would produce a functional protein, when introduced in the 5’UTR of natural mRNAs. One other drawback of this study is that AUGs of the conserved AUG triplet within putative TEEs were generally considered single start codons and not regarded as an entity together with their respective ORF generating patterns of overlapping and non-overlapping uORFs, which themselves could constitute a regulatory unit.

Overall, the question of whether the putative cap-independent TEEs work in their genomic context remains unanswered.

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Another high-throughput approach to decipher cis-regulatory elements driving cap-independent translation was performed by Weingarten-Gabby and colleagues. Sequences with the length of ~170 nt from viruses, human genes, systematically mutated previously reported IRESs and native 5’UTRs were introduced into a synthetic oligo library comprising 55,000 oligonucleotides in total [135]. The library was cloned into a lentiviral bicistronic reporter plasmid and infected H1299 cells, each with a single oligo integration, were sorted into different reporter expression bins by FACS followed by deep sequencing. According to this high-throughput analysis, about 10 % of human 5’UTRs harbor cap-independent translation activity [135]. The assay demonstrates one of the major problems in identifying non-canonical translation elements:

the activity levels of the 583 human sequences, which harbor translational activity, range from 1.5 to 8 on a log2 scale relative to an empty vector. Hence, there exist a huge variety of activity and the focus of such analyses should not be primarily on identifying this activity above a selected threshold but rather on showing if this is meaningful in its natural context or during a stress condition. Therefore, a comparison with the monocistronic mRNA would be necessary to evaluate the stimulation from the 5’cap, showing if cap-independent translational activity compared to cap-dependent translational activity could lead to relevant protein output.

In a high-throughput assay, thresholds need to be defined for splicing and promoter activity. When it comes to reporter assays, this is an issue as already tiny amounts of aberrant transcripts can lead to significant read-out. Thus, the use of a cut-off value for promoter activity of more than 20 % of reads coming from the cell population that expressed the second-cistron although the expression vector lacked an intrinsic promoter and a cut-off value for splicing activity of ≤-2.5 (log2) of cDNA compared to gDNA reads likely leaves room for false positives [135]. Hence, additional control experiments were performed, but qRT-PCR assays are not the best suitable control, as they are not sensitive enough to rule out the presence of weaker splice sites. The investigators also performed control reporter assays for selected candidates using DNA transfection, which is prone to generate artefacts from cryptic promoter or splice sites, wherefore only mRNA transfection assays are suitable control.

Despite the lack of such controls, the identification of novel and previously described short U-rich IRES elements was reported (UACUCCC, UUCCUUU) [135]. The activity of these elements varied between reporter transcripts and also between different sites within a single transcript, indicating that the element on its own might not be a fully functional IRES but might require surrounding structures [135]. This also leads to the question of how active the short motifs are in their natural context that should be quite different from the artificial intercistronic one used in reporter assays. All in all, the authors didn’t clearly distinguish between

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independent and IRES-dependent translation within their assays and the identified translation elements require further experiments to validate and clarify their mode of action.

Taken together, eukaryotic mRNAs can in principle be translated in the absence of the cap structure, although cap-dependent translation is much stronger. Translation enhancing elements in the 5’UTR have been described that can boost cap-independent but 5’end-dependent translation. So-called CITES have been proposed to act in concert with canonical eIFs or specific variants of canonical eIFs like eIF4G, eIF4G2 or subunits of eIF3. The best studied mRNA with efficient independent but 5’end-dependent translation is Apaf-1. Other mRNAs that are cap-independently translated but unable to mediate significant IRES-dependent translation (translation levels around background values), might be translated through a CITE-like translation initiation mechanism under distinct cellular conditions. High-throughput genome-wide assays are not well suited to study the details of non-canonical translation initiation mechanisms as stringent validation experiments are required to rule-out false positives which can easily arise with gold-standard bicistronic reporters. Hence, tedious experimental settings are required to investigate single candidates, which is likely a reason why only few examples of well-described cap-independently translated mRNAs exist and outstanding questions remain. Future analyses might uncover if other isoforms of canonical eIFs are involved in CITE-mediated translation and if CITEs can also promote cap-independent translation when located within the 3’UTR as it has previously been shown in plant viruses.