Specific mechanisms of translation initiation in higher eukaryotes: the eIF4G2 story.

The eukaryotic initiation factor 4G2 (eIF4G2, DAP5, Nat1, p97) was discovered in 1997. Over the past two decades, dozens of papers have presented contradictory data on eIF4G2 function. Since its identification, eIF4G2 has been assumed to participate in noncanonical translation initiation mechanisms, but recent results indicate that it can be involved in scanning as well. In particular, eIF4G2 provides leaky scanning through some upstream open reading frames (uORFs), which are typical for long 5' UTRs of mRNAs from higher eukaryotes. It is likely the protein can also help the ribosome overcome other impediments during scanning of the 5' UTRs of animal mRNAs. This may explain the need for eIF4G2 in higher eukaryotes, as many mRNAs that encode regulatory proteins have rather long and highly structured 5' UTRs. Additionally, they often bind to various proteins, which also hamper the movement of scanning ribosomes. This review discusses the suggested mechanisms of eIF4G2 action, denotes obscure or inconsistent results, and proposes ways to uncover other fundamental mechanisms in which this important protein factor may be involved in higher eukaryotes.

Ribosomal leaky scanning through a translated uORF requires eIF4G2.

eIF4G2 (DAP5 or Nat1) is a homologue of the canonical translation initiation factor eIF4G1 in higher eukaryotes but its function remains poorly understood. Unlike eIF4G1, eIF4G2 does not interact with the cap-binding protein eIF4E and is believed to drive translation under stress when eIF4E activity is impaired. Here, we show that eIF4G2 operates under normal conditions as well and promotes scanning downstream of the eIF4G1-mediated 40S recruitment and cap-proximal scanning. Specifically, eIF4G2 facilitates leaky scanning for a subset of mRNAs. Apparently, eIF4G2 replaces eIF4G1 during scanning of 5' UTR and the necessity for eIF4G2 only arises when eIF4G1 dissociates from the scanning complex. In particular, this event can occur when the leaky scanning complexes interfere with initiating or elongating 80S ribosomes within a translated uORF. This mechanism is therefore crucial for higher eukaryotes which are known to have long 5' UTRs with highly frequent uORFs. We suggest that uORFs are not the only obstacle on the way of scanning complexes towards the main start codon, because certain eIF4G2 mRNA targets lack uORF(s). Thus, higher eukaryotes possess two distinct scanning complexes: the principal one that binds mRNA and initiates scanning, and the accessory one that rescues scanning when the former fails.

A Solitary Stalled 80S Ribosome Prevents mRNA Recruitment to Stress Granules.

In response to stress stimuli, eukaryotic cells typically suppress protein synthesis. This leads to the release of mRNAs from polysomes, their condensation with RNA-binding proteins, and the formation of non-membrane-bound cytoplasmic compartments called stress granules (SGs). SGs contain 40S but generally lack 60S ribosomal subunits. It is known that cycloheximide, emetine, and anisomycin, the ribosome inhibitors that block the progression of 80S ribosomes along mRNA and stabilize polysomes, prevent SG assembly. Conversely, puromycin, which induces premature termination, releases mRNA from polysomes and stimulates the formation of SGs. The same effect is caused by some translation initiation inhibitors, which lead to polysome disassembly and the accumulation of mRNAs in the form of stalled 48S preinitiation complexes. Based on these and other data, it is believed that the trigger for SG formation is the presence of mRNA with extended ribosome-free segments, which tend to form condensates in the cell. In this study, we evaluated the ability of various small-molecule translation inhibitors to block or stimulate the assembly of SGs under conditions of severe oxidative stress induced by sodium arsenite. Contrary to expectations, we found that ribosome-targeting elongation inhibitors of a specific type, which arrest solitary 80S ribosomes at the beginning of the mRNA coding regions but do not interfere with all subsequent ribosomes in completing translation and leaving the transcripts (such as harringtonine, lactimidomycin, or T-2 toxin), completely prevent the formation of arsenite-induced SGs. These observations suggest that the presence of even a single 80S ribosome on mRNA is sufficient to prevent its recruitment into SGs, and the presence of extended ribosome-free regions of mRNA is not sufficient for SG formation. We propose that mRNA entry into SGs may be mediated by specific contacts between RNA-binding proteins and those regions on 40S subunits that remain inaccessible when ribosomes are associated.

The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA.

Upstream open reading frames (uORFs) are a frequent feature of eukaryotic mRNAs. Upstream ORFs govern main ORF translation in a variety of ways, but, in a nutshell, they either filter out scanning ribosomes or allow downstream translation initiation via leaky scanning or reinitiation. Previous reports concurred that eIF4G2, a long-known but insufficiently studied eIF4G1 homologue, can rescue the downstream translation, but disagreed on whether it is leaky scanning or reinitiation that eIF4G2 promotes. Here, we investigated a unique human mRNA that encodes two highly conserved proteins (POLGARF with unknown function and POLG, the catalytic subunit of the mitochondrial DNA polymerase) in overlapping reading frames downstream of a regulatory uORF. We show that the uORF renders the translation of both POLGARF and POLG mRNAs reliant on eIF4G2. Mechanistically, eIF4G2 enhances both leaky scanning and reinitiation, and it appears that ribosomes can acquire eIF4G2 during the early steps of reinitiation. This emphasizes the role of eIF4G2 as a multifunctional scanning guardian that replaces eIF4G1 to facilitate ribosome movement but not ribosome attachment to an mRNA.

Cap-Independent Translation: What's in a Name?

Eukaryotic translation initiation relies on the m7G cap present at the 5' end of all mRNAs. Some viral mRNAs employ alternative mechanisms of initiation based on internal ribosome entry. The 'IRES ideology' was adopted by researchers to explain the differential translation of cellular mRNAs when the cap recognition is suppressed. However, some cellular IRESs have already been challenged and others are awaiting their validation. As an alternative cap-independent mechanism, we propose adopting the concept of cap-independent translation enhancers (CITEs) for mammalian mRNAs. Unlike IRESs, CITEs can be located both within 5' and 3' UTRs and bind mRNA-recruiting translational components. The respective 5' UTRs are then inspected by the scanning machinery essentially in the same way as under cap-dependent translation.

eIF4G2 balances its own mRNA translation via a PCBP2-based feedback loop.

Poly(rC)-binding protein 2 (PCBP2, hnRNP E2) is one of the most abundant RNA-binding proteins in mammalian cells. In humans, it exists in seven isoforms, which are assumed to play similar roles in cells. The protein is shown to bind 3'-untranslated regions (3'-UTRs) of many mRNAs and regulate their translation and/or stability, but nothing is known about the functional consequences of PCBP2 binding to 5'-UTRs. Here we show that the PCBP2 isoform f interacts with the 5'-UTRs of mRNAs encoding eIF4G2 (a translation initiation factor with a yet unknown mechanism of action, also known as DAP5) and Cyclin I, and inhibits their translation in vitro and in cultured cells, while the PCBP2 isoform e only affects Cyclin I translation. Furthermore, eIF4G2 participates in a cap-dependent translation of the PCBP2 mRNA. Thus, PCBP2 and eIF4G2 seem to regulate one another's expression via a novel type of feedback loop formed by the translation initiation factor and the RNA-binding protein.

Non-Canonical Translation Initiation Mechanisms Employed by Eukaryotic Viral mRNAs.

Viruses exploit the translation machinery of an infected cell to synthesize their proteins. Therefore, viral mRNAs have to compete for ribosomes and translation factors with cellular mRNAs. To succeed, eukaryotic viruses adopt multiple strategies. One is to circumvent the need for m7G-cap through alternative instruments for ribosome recruitment. These include internal ribosome entry sites (IRESs), which make translation independent of the free 5' end, or cap-independent translational enhancers (CITEs), which promote initiation at the uncapped 5' end, even if located in 3' untranslated regions (3' UTRs). Even if a virus uses the canonical cap-dependent ribosome recruitment, it can still perturb conventional ribosomal scanning and start codon selection. The pressure for genome compression often gives rise to internal and overlapping open reading frames. Their translation is initiated through specific mechanisms, such as leaky scanning, 43S sliding, shunting, or coupled termination-reinitiation. Deviations from the canonical initiation reduce the dependence of viral mRNAs on translation initiation factors, thereby providing resistance to antiviral mechanisms and cellular stress responses. Moreover, viruses can gain advantage in a competition for the translational machinery by inactivating individual translational factors and/or replacing them with viral counterparts. Certain viruses even create specialized intracellular "translation factories", which spatially isolate the sites of their protein synthesis from cellular antiviral systems, and increase availability of translational components. However, these virus-specific mechanisms may become the Achilles' heel of a viral life cycle. Thus, better understanding of the unconventional mechanisms of viral mRNA translation initiation provides valuable insight for developing new approaches to antiviral therapy.

Functional Cyclization of Eukaryotic mRNAs.

The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions that brings 5' and 3' ends together. This circularization is thought to promote the engagement of terminating ribosomes to a new round of translation at the same mRNA molecule, thus enhancing protein synthesis. Despite the general acceptance and the elegance of the hypothesis, it has never been proved experimentally. Using continuous in situ monitoring of luciferase synthesis in a mammalian in vitro system, we show here that the rate of translation initiation at capped and polyadenylated reporter mRNAs increases after the time required for the first ribosomes to complete mRNA translation. Such acceleration strictly requires the presence of a poly(A)-tail and is abrogated by the addition of poly(A) RNA fragments or m7GpppG cap analog to the translation reaction. The optimal functional interaction of mRNA termini requires 5' untranslated region (UTR) and 3' UTR of moderate lengths and provides stronger acceleration, thus a longer poly(A)-tail. Besides, we revealed that the inhibitory effect of the dominant negative R362Q mutant of initiation factor eIF4A diminishes in the course of translation reaction, suggesting a relaxed requirement for ATP. Taken together, our results imply that, upon the functional looping of an mRNA, the recycled ribosomes can be recruited to the start codon of the same mRNA molecule in an eIF4A-independent fashion. This non-canonical closed-loop assisted reinitiation (CLAR) mode provides efficient translation of the functionally circularized mRNAs.

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 (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.

Sliding of a 43S ribosomal complex from the recognized AUG codon triggered by a delay in eIF2-bound GTP hydrolysis.

During eukaryotic translation initiation, 43S ribosomal complex scans mRNA leader unless an AUG codon in an appropriate context is found. Establishing the stable codon-anticodon base-pairing traps the ribosome on the initiator codon and triggers structural rearrangements, which lead to Pi release from the eIF2-bound GTP. It is generally accepted that AUG recognition by the scanning 43S complex sets the final point in the process of start codon selection, while latter stages do not contribute to this process. Here we use translation reconstitution approach and kinetic toe-printing assay to show that after the 48S complex is formed on an AUG codon, in case GTP hydrolysis is impaired, the ribosomal subunit is capable to resume scanning and slides downstream to the next AUG. In contrast to leaky scanning, this sliding is not limited to AUGs in poor nucleotide contexts and occurs after a relatively long pause at the recognized AUG. Thus, recognition of an AUG per se does not inevitably lead to this codon being selected for initiation of protein synthesis. Instead, it is eIF5-induced GTP hydrolysis and Pi release that irreversibly trap the 48S complex, and this complex is further stabilized by eIF5B and 60S joining.

Transcriptome-wide studies uncover the diversity of modes of mRNA recruitment to eukaryotic ribosomes.

The conventional paradigm of translation initiation in eukaryotes states that the cap-binding protein complex eIF4F (consisting of eIF4E, eIF4G and eIF4A) plays a central role in the recruitment of capped mRNAs to ribosomes. However, a growing body of evidence indicates that this paradigm should be revised. This review summarizes the data which have been mostly accumulated in a post-genomic era owing to revolutionary techniques of transcriptome-wide analysis. Unexpectedly, these techniques have uncovered remarkable diversity in the recruitment of cellular mRNAs to eukaryotic ribosomes. These data enable a preliminary classification of mRNAs into several groups based on their requirement for particular components of eIF4F. They challenge the widely accepted concept which relates eIF4E-dependence to the extent of secondary structure in the 5' untranslated regions of mRNAs. Moreover, some mRNA species presumably recruit ribosomes to their 5' ends without the involvement of either the 5' m(7)G-cap or eIF4F but instead utilize eIF4G or eIF4G-like auxiliary factors. The long-standing concept of internal ribosome entry site (IRES)-elements in cellular mRNAs is also discussed.

A novel mechanism of eukaryotic translation initiation that is neither m7G-cap-, nor IRES-dependent.

Resistance of translation of some eukaryotic messenger RNAs (mRNAs) to inactivation of the cap-binding factor eIF4E under unfavorable conditions is well documented. To date, it is the mechanism of internal ribosome entry that is predominantly thought to underlay this stress tolerance. However, many cellular mRNAs that had been considered to contain internal ribosome entry sites (IRESs) failed to pass stringent control tests for internal initiation, thus raising the question of how they are translated under stress conditions. Here, we show that inserting an eIF4G-binding element from a virus IRES into 5'-UTRs of strongly cap-dependent mRNAs dramatically reduces their requirement for the 5'-terminal m(7)G-cap, though such cap-independent translation remains dependent on a vacant 5'-terminus of these mRNAs. Importantly, direct binding of eIF4G to the 5'-UTR of mRNA makes its translation resistant to eIF4F inactivation both in vitro and in vivo. These data may substantiate a new paradigm of translational control under stress to complement IRES-driven mechanism of translation.

Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation.

Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5' end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNA(Gly) anticodon stem-loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus-host cell interaction are discussed.

GTP-independent tRNA delivery to the ribosomal P-site by a novel eukaryotic translation factor.

During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNA(i)(Met) occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.

Differential contribution of the m7G-cap to the 5' end-dependent translation initiation of mammalian mRNAs.

Many mammalian mRNAs possess long 5' UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5' UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5' UTRs with so-called 'cellular IRESes' demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5' UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5' UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated.

Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated.

Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.

A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors.

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5' leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.

A novel uORF-based regulatory mechanism controls translation of the human MDM2 and eIF2D mRNAs during stress.

Short upstream open reading frames (uORFs) are the most prevalent cis-acting regulatory elements in the mammalian transcriptome which can orchestrate mRNA translation. Apart from being "passive roadblocks" that decrease expression of the main coding regions, particular uORFs can serve as specific sensors for changing conditions, thus regulating translation in response to cell stress. Here we report a novel uORF-based regulatory mechanism that is employed under conditions of hyperosmotic stress by at least two human mRNAs, coding for translation reinitiation/recycling factor eIF2D and E3 ubiquitin ligase MDM2. This novel mode of translational control selectively downregulates their expression and requires as few as one uORF. Using a set of reporter mRNAs and fleeting mRNA transfection (FLERT) technique, we provide evidence that the phenomenon does not rely on delayed reinitiation, altered AUG recognition, ribosome stalling, mRNA destabilization or other known mechanisms. Instead, it is based on events taking place at uORF stop codon or immediately downstream. Functional aspects and implications of the novel regulatory mechanism to cell physiology are discussed.

Translatome and transcriptome analysis of TMA20 (MCT-1) and TMA64 (eIF2D) knockout yeast strains.

TMA20 (MCT-1), TMA22 (DENR) and TMA64 (eIF2D) are eukaryotic translation factors involved in ribosome recycling and re-initiation. They operate with P-site bound tRNA in post-termination or (re-)initiation translation complexes, thus participating in the removal of 40S ribosomal subunit from mRNA stop codons after termination and controlling translation re-initiation on mRNAs with upstream open reading frames (uORFs), as well as de novo initiation on some specific mRNAs. Here we report ribosomal profiling data of S.cerevisiae strains with individual deletions of TMA20, TMA64 or both TMA20 and TMA64 genes. We provide RNA-Seq and Ribo-Seq data from yeast strains grown in the rich YPD or minimal SD medium. We illustrate our data by plotting differential distribution of ribosomal-bound mRNA fragments throughout uORFs in 5'-untranslated region (5' UTR) of GCN4 mRNA and on mRNA transcripts encoded in MAT locus in the mutant and wild-type strains, thus providing a basis for investigation of the role of these factors in the stress response, mating and sporulation. We also document a shift of transcription start site of the APC4 gene which occurs when the neighboring TMA64 gene is replaced by the standard G418-resistance cassette used for the creation of the Yeast Deletion Library. This shift results in dramatic deregulation of the APC4 gene expression, as revealed by our Ribo-Seq data, which can be probably used to explain strong genetic interactions of TMA64 with genes involved in the cell cycle and mitotic checkpoints. Raw RNA-Seq and Ribo-Seq data as well as all gene counts are available in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE122039 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE122039).

A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry.

Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5' untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay. There is an absolute requirement for the initiation factors eIF2 and eIF3 and the scanning factor eIF1 to form 48S complexes on the IRES. In addition, eIF1A, eIF4F (or the C-terminal fragment of eIF4G), and eIF4A strongly stimulated the assembly of this complex, whereas eIF4B had no effect. Although the eIF4-dependent pathway is dominant in the RhPV IRES-directed cell-free translation, omission of either eIF4G or eIF4A or both still allowed the assembly of 48S complexes from purified components with approximately 23% of maximum efficiency. Deletions of up to 100 nucleotides throughout the 5'-UTR sequence produced at most a marginal effect on the IRES activity, suggesting the absence of specific binding sites for initiation factors. Only deletion of the U-rich unstructured 380-nucleotide region proximal to the initiation codon resulted in a complete loss of the IRES activity. We suggest that the single-stranded nature of the RhPV IRES accounts for its strong but less selective potential to bind key mRNA recruiting components of the translation initiation apparatus from diverse origins.