La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease.

Uncapped reovirus mRNA extracted at late times from infected L-cells is preferentially translated in extracts from infected L-cells. However, translation of this uncapped, late, reovirus mRNA in extracts from infected cells is sensitive to inhibition by the cap analog m7GTP . These results imply that reovirus infection does not induce a transition from cap-dependent to cap-independent translation. Nevertheless, the results of in vitro translational competition experiments between L-cell mRNA and late viral mRNA were consistent with the view that reovirus does induce an alteration in the cap-dependent translational apparatus of L-cells. The reduced efficiency of translation of a variety of capped mRNAs in extracts from infected cells is also consistent with this notion. We further conclude that a factor exists in reovirus-infected L-cells that specifically stimulates translation of uncapped reovirus mRNAs.     

Expression of RUNX2 isoforms: involvement of cap-dependent and cap-independent mechanisms of translation

RUNX2, a major regulator of skeletogenesis, is expressed as type-I and type-II isoforms. Whereas most eukaryotic mRNAs are translated by the cap-dependent scanning mechanism, translation of many mRNAs including type-I and type-II RUNX2 mRNAs has been reported to be initiated by a cap independent internal ribosomal entry site (IRES). Since the dicistronic plasmid assay used to demonstrate IRES has been questioned, we investigated the presence of IRES in RUNX2 mRNAs using dicistronic plasmid and mRNA assays. Our results show that the dicistronic plasmid assay cannot be used to demonstrate IRES in RUNX2 mRNAs because the intercistronic region of dicistronic plasmids containing the 5'-UTRs of both RUNX2 mRNAs operates as a cryptic promoter. In dicistronic mRNA transfection studies the 5'-UTRs of both RUNX2 mRNAs exhibited no IRES activity. When transfected into osteoblastic cells, monocistronic reporter mRNA preceded by the 5'-UTR of type-II RUNX2 (Type-II-FLuc-A100) was translated to a high degree only in the presence of a functional cap (m(7)GpppG); in contrast, luciferase mRNA preceded by the 5'-UTR of type-I RUNX2 mRNA (Type-I-FLuc-A100) was translated poorly in the presence of either m(7)GpppG or a nonfunctional cap (ApppG). Notably, in transfected cells inhibitors of cap-dependent translation suppressed the translation of m(7)GpppG-capped Type-II-FLuc-A100, but not ApppG-capped reporter mRNA preceded by the IRES-containing hepatitis C virus (HCV) 5'-UTR. Our study demonstrates that type-II RUNX2 mRNA is translated by the cap-dependent mechanism. Although efficient translation of type-I RUNX2 mRNA appears to require a process other than cap-dependent, the mechanism of type-I RUNX2 mRNA translation remains to be resolved.     

Influenza Virus mRNA Translation Revisited: Is the eIF4E Cap-Binding Factor Required for Viral mRNA Translation?

Influenza virus mRNAs bear a short capped oligonucleotide sequence at their 5' ends derived from the host cell pre-mRNAs by a "cap-snatching" mechanism, followed immediately by a common viral sequence. At their 3' ends, they contain a poly(A) tail. Although cellular and viral mRNAs are structurally similar, influenza virus promotes the selective translation of its mRNAs despite the inhibition of host cell protein synthesis. The viral polymerase performs the cap snatching and binds selectively to the 5' common viral sequence. As viral mRNAs are recognized by their own cap-binding complex, we tested whether viral mRNA translation occurs without the contribution of the eIF4E protein, the cellular factor required for cap-dependent translation. Here, we show that influenza virus infection proceeds normally in different situations of functional impairment of the eIF4E factor. In addition, influenza virus polymerase binds to translation preinitiation complexes, and furthermore, under conditions of decreased eIF4GI association to cap structures, an increase in eIF4GI binding to these structures was found upon influenza virus infection. This is the first report providing evidence that influenza virus mRNA translation proceeds independently of a fully active translation initiation factor (eIF4E). The data reported are in agreement with a role of viral polymerase as a substitute for the eIF4E factor for viral mRNA translation.     

Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5'' noncoding region.

Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.     

Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.     

Enhancement of IRES-mediated translation of the c-myc and BiP mRNAs by the poly(A) tail is independent of intact eIF4G and PABP

The poly(A) tail at the 3' end of mRNAs enhances 5' cap-dependent translation initiation. We show that it also enhances IRES-directed translation of two cellular mRNAs in vitro and in vivo. The underlying mechanisms, however, differ fundamentally. In contrast to cap-dependent translation, IRES-driven translation continues to be enhanced by the poly(A) tail following proteolytic cleavage of eIF4G. Moreover, the poly(A) tail stimulates IRES-mediated translation even in the presence of PAIP2 or following effective depletion of the poly(A) binding protein (PABP) from HeLa cell extracts. The PABP-eIF4G bridging complex that is critical for cap-dependent translation is thus dispensable for the enhancement of the IRESs by the poly(A) tail. The polyadenylated mRNA translation from cellular IRESs is also profoundly sensitive to eIF4A activity in vitro. These mechanistic and molecular distinctions implicate the potential for a new layer of translational control mechanisms.     

Was the initiation of translation in early eukaryotes IRES-driven?

The initiation of translation in eukaryotes generally involves the recognition of a 'cap' structure at the 5' end of the mRNA. However, for some viral and cellular mRNAs, a cap-independent mechanism occurs through an mRNA structure known as the internal ribosome entry site (IRES). Here, I postulate that the first eukaryotic mRNAs were translated in a cap-independent, IRES-driven manner that was then superseded in evolution by the cap-dependent mechanism, rather than vice versa. This hypothesis is supported by the following observations: (i) IRES-dependent, but not cap-dependent, translation can take place in the absence of not only a cap, but also many initiation factors; (ii) eukaryotic initiation factor 4E (eIF4E) and eIF4G, molecules absolutely required for cap-dependent translation, are among the most recently evolved translation factors; and (iii) functional similarities suggest the evolution of IRESs from spliceosomal introns. Thus, the contemporary cellular IRESs might be relics of the past.     

DAP5 associates with eIF2β and eIF4AI to promote Internal Ribosome Entry Site driven translation

Initiation is a highly regulated rate-limiting step of mRNA translation. During cap-dependent translation, the cap-binding protein eIF4E recruits the mRNA to the ribosome. Specific elements in the 5′UTR of some mRNAs referred to as Internal Ribosome Entry Sites (IRESes) allow direct association of the mRNA with the ribosome without the requirement for eIF4E. Cap-independent initiation permits translation of a subset of cellular and viral mRNAs under conditions wherein cap-dependent translation is inhibited, such as stress, mitosis and viral infection. DAP5 is an eIF4G homolog that has been proposed to regulate both cap-dependent and cap-independent translation. Herein, we demonstrate that DAP5 associates with eIF2β and eIF4AI to stimulate IRES-dependent translation of cellular mRNAs. In contrast, DAP5 is dispensable for cap-dependent translation. These findings provide the first mechanistic insights into the function of DAP5 as a selective regulator of cap-independent translation.     

A cell cycle-dependent internal ribosome entry site

The eukaryotic mRNA 5' cap structure facilitates translation. However, cap-dependent translation is impaired at mitosis, suggesting a cap-independent mechanism for mRNAs translated during mitosis. Translation of ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, peaks twice during the cell cycle, at the G1/S transition and at G2/M. Here, we describe a cap-independent internal ribosome entry site (IRES) in the ODC mRNA that functions exclusively at G2/M. This ensures elevated levels of polyamines, which are implicated in mitotic spindle formation and chromatin condensation. c-myc mRNA also contains an IRES that functions during mitosis. Thus, IRES-dependent translation is likely to be a general mechanism to synthesize short-lived proteins even at mitosis, when cap-dependent translation is interdicted.     

Secondary structure in the 5'-leader or 3'-untranslated region reduces protein yield but does not affect the functional interaction between the 5'-cap and the poly(A) tail

The 5'-cap structure and poly(A) tail of eukaryotic mRNAs cooperate to promote translation initiation but whether this functional interaction benefits certain classes of mRNAs has not been investigated. In this study, we investigate whether a structured 5'-leader or 3'-untranslated region (UTR) affects the cap/poly(A) tail interaction. A structured leader reduced the degree to which the 5'-cap promoted translation in plant cells and inhibited translation from capped and uncapped mRNAs equally in yeast. Secondary structure within the 3'-UTR reduced translational efficiency when adjacent to the stop codon but had little effect on the cap/poly(A) tail synergy. The functional interaction between the cap and poly(A) tail was as important for an mRNA with a structured leader or 3'-UTR as it was for an unstructured mRNA in either species, suggesting that these structures can reduce translation without affecting the functional interaction between the cap and poly(A) tail. However, the loss of Xrn1p, the major 5'-->3' exoribonuclease in yeast, abolished cap-dependent translation and the functional interaction between the cap and poly(A) tail, suggesting that the cap/poly(A) tail synergy is of particular importance under conditions of active RNA turnover.     

Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.

Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.     

Opposite responses of rabbit and human globin mRNAs to translational inhibition by cap analogues

The translational efficiency of an mRNA may be determined at the step of translational initiation by the efficiency of its interaction with the cap binding protein complex. To further investigate the role of these interactions in translational control, we compare in vitro the relative sensitivities of rabbit and human alpha- and beta-globin mRNAs to translational inhibition by cap analogues. We find that rabbit beta-globin mRNA is more resistant to translational inhibition by cap analogues than rabbit alpha-globin mRNA, while in contrast, human beta-globin mRNA is more sensitive to cap analogue inhibition than human alpha-globin mRNA. This opposite pattern of translational inhibition by cap analogues of the rabbit and human alpha- and beta-globin mRNAs is unexpected as direct in vivo and in vitro comparisons of polysome profiles reveal parallel translational handling of the alpha- and beta-globin mRNAs from these two species. This discordance between the relative translational sensitivities of these mRNAs to cap analogues and their relative ribosome loading activities suggests that cap-dependent events may not be rate limiting in steady-state globin translation.     

The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro

The eukaryotic initiation factor eIF4E binds the mRNA 5' cap structure and has a central role during translational initiation. eIF4E and the mechanisms to control its activity have oncogenic properties and thus have become targets for anticancer drug development. A recent study (Kentsis et al. 2004) presented evidence that the antiviral nucleoside ribavirin and its phosphorylated derivatives were structural mimics of the mRNA cap, high-affinity ligands for eIF4E, and potent repressors of eIF4E-mediated cell transformation and tumor growth. Based on these findings, we tested ribavirin, ribavirin triphosphate (RTP), and the dinucleotide RpppG for their ability to inhibit translation in vitro. Surprisingly, the ribavirin-based compounds did not affect translation at concentrations where canonical cap analogs efficiently block cap-dependent translation. Using a set of reporter mRNAs that are translated via either cap-dependent or viral internal ribosome entry sites (IRES)-dependent initiation, we found that these ribavirin-containing compounds did inhibit translation at high (millimolar) concentrations, but there was no correlation of this inhibition with an eIF4E requirement for translation. The addition of a ribavirin-containing cap to mRNA did not stimulate translation. Fluorescence titration experiments with eIF4E and the nuclear cap-binding complex CBC indicated affinities for RTP and RpppG that were two to four orders of magnitude lower than those of m(7)GTP and m(7)GpppG. We conclude that, at least with respect to translation, ribavirin does not act in vitro as a functional mimic of the mRNA cap.     

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 new paradigm for translational control: inhibition via 5'-3' mRNA tethering by Bicoid and the eIF4E cognate 4EHP

Translational control is a key genetic regulatory mechanism implicated in regulation of cell and organismal growth and early embryonic development. Initiation at the mRNA 5' cap structure recognition step is frequently targeted by translational control mechanisms. In the Drosophila embryo, cap-dependent translation of the uniformly distributed caudal (cad) mRNA is inhibited in the anterior by Bicoid (Bcd) to create an asymmetric distribution of Cad protein. Here, we show that d4EHP, an eIF4E-related cap binding protein, specifically interacts with Bcd to suppress cad translation. Translational inhibition depends on the Bcd binding region (BBR) present in the cad 3' untranslated region. Thus, simultaneous interactions of d4EHP with the cap structure and of Bcd with BBR renders cad mRNA translationally inactive. This example of cap-dependent translational control that is not mediated by canonical eIF4E defines a new paradigm for translational inhibition involving tethering of the mRNA 5' and 3' ends.     

Adenovirus inhibition of cellular protein synthesis involves inactivation of cap-binding protein.

Adenovirus (Ad) infection results in a marked inhibition of cellular protein synthesis that initiates during the late phase of the viral infectious cycle. We show that the mechanism used for suppression of cellular protein synthesis during cell cycle progression is exploited by Ad to repress host and enhance late viral mRNA translation. Discrimination between cellular and late Ad mRNAs and inhibition of host protein synthesis are shown to involve viral-mediated underphosphorylation of cap-binding protein (CBP) and subsequent inactivation of CBP complex, a large enzymatic complex required for cap-dependent mRNA translation. Late Ad mRNAs, like those of poliovirus, possess the unique ability to translate independent of a normal cap recognition process and do not require the activity of CBP complex. Inhibition of cellular translation by these two viruses is quite similar, except that whereas CBP complex is proteolytically degraded by poliovirus, it is functionally inactivated by Ad.     

Synthesis and properties of mRNA cap analogs containing imidodiphosphate moiety--fairly mimicking natural cap structure, yet resistant to enzymatic hydrolysis

We describe synthesis and properties of eight dinucleotide mRNA 5' cap analogs containing imidodiphosphate moiety within 5',5'-tri- or tetraphosphate bridge (NH-analogs). The compounds were obtained by coupling an appropriate nucleoside 5'-imidodiphosphate with nucleotide P-imidazolide mediated by divalent metal chloride in anhydrous DMF. To evaluate the novel compounds as tools for studying cap-dependent processes, we determined their binding affinities for eukaryotic translation initiation factor 4E, susceptibilities to decapping pyrophosphatase DcpS and, for non-hydrolysable analogs, binding affinities to this enzyme. The results indicate that the O to NH substitution in selected positions of oligophosphate bridge ensures resistance to enzymatic decapping and suggest that interactions of NH-analogs with cap binding proteins fairly mimic interactions of unmodified parent compounds. Finally, we identified NH-analogs as potent inhibitors of cap-dependent translation in cell free system, and evaluated their utility as reagents for obtaining 5' capped mRNAs in vitro to be rather moderate.     

A hypoxia-controlled cap-dependent to cap-independent translation switch in breast cancer

Translational regulation is critical in cancer development and progression. Translation sustains tumor growth and development of a tumor vasculature, a process known as angiogenesis, which is activated by hypoxia. Here we first demonstrate that a majority of large advanced breast cancers overexpress translation regulatory protein 4E-BP1 and initiation factor eIF4G. Using model animal and cell studies, we then show that overexpressed 4E-BP1 and eIF4G orchestrate a hypoxia-activated switch from cap-dependent to cap-independent mRNA translation that promotes increased tumor angiogenesis and growth at the level of selective mRNA translation. Elevated levels of 4E-BP1 trigger hypoxia inhibition of cap-dependent mRNA translation at high-oxygen levels and, with eIF4G, increase selective translation of mRNAs containing internal ribosome entry sites (IRESs) that include key proangiogenic, hypoxia, and survival mRNAs. The switch from cap-dependent to cap-independent mRNA translation facilitates tumor angiogenesis and hypoxia responses in animal models.