Dmitri E. Andreev

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

ABSTRACT 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.

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 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.

Cap- and IRES-independent scanning mechanism of translation initiation as an alternative to the concept of cellular IRESs

During the last decade the concept of cellular IRES-elements has become predominant to explain the continued expression of specific proteins in eukaryotic cells under conditions when the cap-dependent translation initiation is inhibited. However, many cellular IRESs regarded as cornerstones of the concept, have been compromised by several recent works using a number of modern techniques. This review analyzes the sources of artifacts associated with identification of IRESs and describes a set of control experiments, which should be performed before concluding that a 5’ UTR of eukaryotic mRNA does contain an IRES. Hallmarks of true IRES-elements as exemplified by well-documented IRESs of viral origin are presented. Analysis of existing reports allows us to conclude that there is a constant confusion of the cap-independent with the IRES-directed translation initiation. In fact, these two modes of translation initiation are not synonymous. We discuss here not numerous reports pointing to the existence of a cap- and IRES-independent scanning mechanism of translation initiation based on utilization of special RNA structures called cap-independent translational enhancers (CITE). We describe this mechanism and suggest it as an alternative to the concept of cellular IRESs.

A Cross-Kingdom Internal Ribosome Entry Site Reveals a Simplified Mode of Internal Ribosome Entry

ABSTRACT 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 ∼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.

A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors

ABSTRACT 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.

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

Abstract 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′ m7G-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.

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 cells 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 tRNAGly 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.

Efficient cap-dependent translation of mammalian mRNAs with long and highly structured 5′-untranslated regions in vitro and in vivo

According to the generally accepted scanning model proposed by M. Kozak, the secondary structure of the 5′-untranslated region (5′-UTR) of eukaryotic mRNA can only inhibit the translation initiation by counteracting migration of the 40S ribosomal subunit along the mRNA polynucleotide chain. The existence of efficiently translatable mRNAs with long and highly structured 5′-UTRs is incompatible with the cap-dependent scanning mechanism. Such mRNAs are expected to use alternative ways of translation initiation to be efficiently translated, primarily the mechanism of internal ribosome entry mediated by internal ribosome entry sites (IRESs), special RNA structures that reside in the 5′-UTR. The paper shows that this viewpoint is incorrect and is probably based on experiments with mRNA translation in rabbit reticulocyte lysate. This cell-free system fails to adequately reflect the relative translation efficiencies observed for different mRNAs in vivo. Five structurally similar mRNAs with either short leaders of the β-globin and β-actin mRNAs or long and highly structured 5′-UTRs of the c-myc, LINE-1, and Apaf-1 mRNAs displayed comparable translation activities in transfected cells and an entire cytoplasmic extract of cultivated cells. Translation activity proved to strongly depend on the presence of a cap at the 5′ end.

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 m7G-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.

The 5′ untranslated region of Apaf-1 mRNA directs translation under apoptosis conditions via a 5′ end-dependent scanning mechanism

We have previously shown that translation driven by the 5′ UTR of Apaf‐1 mRNA is relatively efficient in the absence of m7G‐cap, but no IRES is involved. Nevertheless, it may be speculated that a “silent” IRES is activated under apoptosis conditions. Here, we show that translation of the mRNA with the Apaf‐1 5′ UTR is relatively resistant to apoptosis induced by etoposide when eIF4E is sequestered by 4E‐BP and eIF4G is partially cleaved. However, translation under these conditions remains governed by 5′ end‐dependent scanning. We hypothesize that the observed phenomenon is based on the intrinsic low cap‐dependence of the Apaf‐1 5′ UTR.