Recent proteogenomic studies revealed extensive translation outside of annotated protein coding regions, such as non-coding RNAs and untranslated regions of mRNAs. This non-canonical translation is ...largely due to start codon plurality within the same RNA. This plurality is often due to the failure of some scanning ribosomes to recognize potential start codons leading to initiation downstream-a process termed leaky scanning. Codons other than AUG (non-AUG) are particularly leaky due to their inefficiency. Here we discuss our current understanding of non-AUG initiation. We argue for a near-ubiquitous role of non-AUG initiation in shaping the dynamic composition of mammalian proteomes.
Abstract
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.
Cap-Independent Translation: What’s in a Name? Shatsky, Ivan N.; Terenin, Ilya M.; Smirnova, Victoria V. ...
Trends in biochemical sciences (Amsterdam. Regular ed.),
November 2018, 2018-11-00, 20181101, Volume:
43, Issue:
11
Journal Article
Peer reviewed
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.
The eukaryotic translation initiation relies not only on the cap-dependent mechanism of mRNA binding to the ribosome, but also on poorly studied cap-independent ways of recruiting mRNAs to ribosomes. The latter mode plays an important role in cell differentiation and cellular response to abnormal conditions.
The widely adopted way to explain the cap-independent translation of cellular mRNAs is the use of internal ribosome entry sites (IRESs). However, some of the cap-independent mechanisms cannot be explained with the IRES concept.
This review proposes an alternative mechanism for cap-independent initiation. It is based on the presence (in the UTRs of mRNAs) of structural elements that bind the factors recruiting mRNAs to the ribosome cap-independent translational enhancers (CITEs). This mechanism is supported by recent reports.
By binding components of the translation machinery, CITEs help specific mRNAs to win the competition for ribosomes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The phenomenon of internal initiation of translation was discovered in 1988 on poliovirus mRNA. The prototypic
-acting element in the 5' untranslated region (5'UTR) of poliovirus mRNA, which is able ...to direct initiation at an internal start codon without the involvement of a cap structure, has been called an IRES (Internal Ribosome Entry Site or Segment). Despite its early discovery, poliovirus and other related IRES elements of type I are poorly characterized, and it is not yet clear which host proteins (a.k.a. IRES trans-acting factors, ITAFs) are required for their full activity in vivo. Here we discuss recent and old results devoted to type I IRESes and provide evidence that Poly(rC) binding protein 2 (PCBP2), Glycyl-tRNA synthetase (GARS), and Cold Shock Domain Containing E1 (CSDE1, also known as UNR) are major regulators of type I IRES activity.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, ...eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.
The development of Ribosome Profiling (RiboSeq) has revolutionized functional genomics. RiboSeq is based on capturing and sequencing of the mRNA fragments enclosed within the translating ribosome and ...it thereby provides a 'snapshot' of ribosome positions at the transcriptome wide level. Although the method is predominantly used for analysis of differential gene expression and discovery of novel translated ORFs, the RiboSeq data can also be a rich source of information about molecular mechanisms of polypeptide synthesis and translational control. This review will focus on how recent findings made with RiboSeq have revealed important details of the molecular mechanisms of translation in eukaryotes. These include mRNA translation sensitivity to drugs affecting translation initiation and elongation, the roles of upstream ORFs in response to stress, the dynamics of elongation and termination as well as details of intrinsic ribosome behavior on the mRNA after translation termination. As the RiboSeq method is still at a relatively early stage we will also discuss the implications of RiboSeq artifacts on data interpretation.
During protein synthesis, a ribosome moves along the mRNA template and, using aminoacyl-tRNAs, decodes the template nucleotide triplets to assemble a protein amino acid sequence. This movement is ...accompanied by shifting of mRNA–tRNA complexes within the ribosome in a process called translocation. In living cells, this process proceeds in a unidirectional manner, bringing the ribosome to the 3′ end of mRNA, and is catalyzed by the GTPase translation elongation factor 2 (EF-G in prokaryotes and eEF2 in eukaryotes). Interestingly, the possibility of spontaneous backward translocation has been shown in vitro for bacterial ribosomes, suggesting a potential reversibility of this reaction. However, this possibility has not yet been tested for eukaryotic ribosomes. Here, using a reconstituted mammalian translation system, we show that the eukaryotic elongation factor eEF2 catalyzes ribosomal reverse translocation at one mRNA triplet. We found that this process requires a cognate tRNA in the ribosomal E-site and cannot occur spontaneously without eEF2. The efficiency of this reaction depended on the concentrations of eEF2 and cognate tRNAs and increased in the presence of nonhydrolyzable GTP analogues. Of note, ADP-ribosylation of eEF2 domain IV blocked reverse translocation, suggesting a crucial role of interactions of this domain with the ribosome for the catalysis of the reaction. In summary, our findings indicate that eEF2 is able to induce ribosomal translocation in forward and backward directions, highlighting the universal mechanism of tRNA–mRNA movements within the ribosome.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Unlike bacteria, a specialized eukaryotic initiation factor (eIF)-2, in the form of the ternary complex eIF2-GTP-Met-tRNA(i) (Met), is used to deliver the initiator tRNA to the ribosome in all ...eukaryotic cells. Here we show that the hepatitis C virus (HCV) internal ribosome entry site (IRES) can direct translation without eIF2 and its GTPase-activating protein eIF5. In addition to the general eIF2- and eIF5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a bacterial-like pathway requiring as initiation factors only eIF5B (an analog of bacterial IF2) and eIF3. The switch from the conventional eukaryotic mode of translation initiation to the eIF2-independent mechanism occurs when eIF2 is inactivated by phosphorylation under stress conditions.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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 tRNAiMet 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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