Cap-independent Internal Initiation Research Articles

Exploring Internal Ribosome Entry Sites as Therapeutic Targets.

Initiation of eukaryotic mRNA translation may proceed via several different routes, each requiring a different subset of factors and relying on different and specific interactions between the mRNA and the ribosome. Two modes predominate: (i) so-called cap-dependent initiation, which requires all canonical initiation factors and is responsible for about 95–97% of all initiation events in eukaryotic cells; and (ii) cap-independent internal initiation, which requires a reduced subset of initiation factors and accounts for up to 5% of the remaining initiation events. Internal initiation relies on the presence of so-called internal ribosome entry site (IRES) elements in the 5′ UTRs of some viral and cellular mRNAs. These elements (often possessing complex secondary and tertiary structures) promote efficient interaction of the mRNA with the 40S ribosome and allow for internal ribosome entry. Internal initiation of translation of specific mRNAs may contribute to development of severe disease and pathological states, such as hepatitis C and cancer. Therefore, this cellular mechanism represents an attractive target for pharmacological modulation. The purpose of this review is to provide insight into current strategies used to target viral and cellular IRESs and discuss the physiological consequences (and potential therapeutic implications) of abrogation/modulation of IRES-mediated translation.

IL-1-induced Post-transcriptional Mechanisms Target Overlapping Translational Silencing and Destabilizing Elements in IκBζ mRNA*[S

The inflammatory cytokine IL-1 induces profound changes in gene expression. This is contributed in part by activating translation of a distinct set of mRNAs, including IκBζ, as indicated by genome-wide analysis of changes in ribosomal occupancy in IL-1α-treated HeLa cells. Polysome profiling of IκBζ mRNA and reporter mRNAs carrying its 3' UTR indicated poor translation in unstimulated cells. 3' UTR-mediated translational silencing was confirmed by suppression of luciferase activity. Translational silencing was unaffected by replacing the poly(A) tail with a histone stem-loop, but lost under conditions of cap-independent internal initiation. IL-1 treatment of the cells caused profound shifts of endogenous and reporter mRNAs to polysome fractions and relieved suppression of luciferase activity. IL-1 also inhibited rapid mRNA degradation. Both translational activation and mRNA stabilization involved IRAK1 and -2 but occurred independently of the p38 MAPK pathway, which is known to target certain other post-transcriptional mechanisms. The translational silencing RNA element contains the destabilizing element but requires additional 5' sequences and is impaired by mutations that leave destabilization unaffected. These differences in function are associated with differential changes in protein binding in vitro. Thus, rapid degradation occurs independently of the translational silencing effect. The results provide evidence for a novel mode of post-transcriptional control by IL-1, which impinges on the time course and pattern of IL-1-induced gene expression.

Contribution of internal initiation to translation of cellular mRNAs containing IRESs

A broad range of cellular stresses lead to the inhibition of translation. Despite this, some cellular mRNAs are selectively translated under these conditions. It is widely supposed that cap-independent internal initiation may maintain efficient translation of particular cellular mRNAs under a variety of stresses and other special conditions when cap-dependent protein synthesis is impaired. However, in spite of a large number of reports focused on the investigation of the regulation of IRES (internal ribosome entry site) activity in different tissues and under various stresses, only rarely is the real efficiency of IRES-driven translation in comparison with cap-dependent translation evaluated. When precisely measured, the efficiencies of candidate IRESs in most cases appeared to be very low and not sufficient to compensate for the reduction of cap-dependent initiation under stresses. The usually low efficiency of internal initiation of translation is inconsistent with postulated biological roles of IRESs.

The 5′ untranslated region of the maize alcohol dehydrogenase gene contains an internal ribosome entry site

Adh1, the maize gene encoding alcohol dehydrogenase ADH1, mRNA is efficiently translated in O 2-deprived roots of maize, whereas many normal cellular mRNAs are poorly translated. It has been shown that adh, the 5′ untranslated region of adh1 mRNA, provides effective translation of mRNA under hypoxia and heat shock conditions in Nicotiana benthamiana plants. We found that adh contains the internal ribosome entry site (IRES) active both in vivo, in N. benthamiana cells, and in vitro, in rabbit reticulocyte lysate translation system. It is widely supposed that cap-independent internal initiation may maintain efficient translation of particular cellular mRNAs under a variety of stresses and other special conditions when cap-dependent protein synthesis is impaired. We evaluated the level of IRES activity of adh and found that its contribution to the overall translation of adh-containing mRNA in plant cells is less than 1% both under normal conditions and under heat shock. The low efficiency of this IRES is inconsistent with its possible role as a main factor ensuring efficient translation of adh1 mRNA under stress conditions.

The Rhopalosiphum padi virus 5' internal ribosome entry site is functional in Spodoptera frugiperda 21 cells and in their cell-free lysates: implications for the baculovirus expression system.

Cap-independent internal initiation of translation occurs on a number of viral and cellular mRNAs and is directed by internal ribosome entry site (IRES) elements. Rhopalosiphum padi virus (RhPV) is a member of the Dicistroviridae. These viruses have single-stranded, positive-sense RNA genomes that contain two open reading frames, both preceded by IRES elements. Previously, the activity of the RhPV 5' UTR IRES has been demonstrated in mammalian, Drosophila and wheat germ in vitro translation systems. It is now shown that this IRES also functions within Spodoptera frugiperda (Sf21) cells which are widely used in the baculovirus expression system, and in a novel Sf21 cell-based lysate system. Inclusion of the RhPV IRES in a dicistronic reporter mRNA transcript increased translation of the second cistron 23-fold within Sf21 cells. In contrast, the encephalomyocarditis virus IRES was inactive in both systems. The RhPV IRES therefore has the potential to be utilized in insect cell expression systems.

The chicken c-Jun 5' untranslated region directs translation by internal initiation.

The 5' untranslated region (UTR) of the chicken c-jun message is exceptionally GC rich and has the potential to form a complex and extremely stable secondary structure. Because stable RNA secondary structures can serve as obstacles to scanning ribosomes, their presence suggests inefficient translation or initiation through alternate mechanisms. We have examined the role of the c-jun 5' UTR with respect to its ability to influence translation both in vitro and in vivo. We find, using rabbit reticulocyte lysates, that the presence of the c-jun 5' UTR severely inhibits translation of both homologous and heterologous genes in vitro. Furthermore, translational inhibition correlates with the degree of secondary structure exhibited by the 5' UTR. Thus, in the rabbit reticulocyte lysate system, the c-jun 5' UTR likely impedes ribosome scanning resulting in inefficient translation. In contrast to our results in vitro, the c-jun 5' UTR does not inhibit translation in a variety of different cell lines suggesting that it may direct an alternate mechanism of translational initiation in vivo. To distinguish among the alternate mechanisms, we generated a series of bicistronic expression plasmids. Our results demonstrate that the downstream cistron, in the bicistronic gene, is expressed to a much higher level when directly preceded by the c-jun 5' UTR. In addition, inhibition of ribosome scanning on the bicistronic message, through insertion of a synthetic stable hairpin, inhibits translation of the first cistron but does not inhibit translation of the cistron downstream of the c-jun 5' UTR. These results are consistent with a model by which the c-jun message is translated through cap independent internal initiation. Oncogene (2000) 19, 2836 - 2845

A selection system for functional internal ribosome entry site (IRES) elements: analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES.

Picornavirus internal ribosome entry site (IRES) elements direct cap-independent internal initiation of protein synthesis within mammalian cells. These RNA elements (about 450 nt) contain extensive secondary structure including a hairpin loop with a conserved GNRA motif. Such loops are important in RNA-RNA and RNA-protein interactions. Plasmids that express dicistronic mRNAs of the structure GUS/IRES/HOOK have been constructed. The HOOK sequence encodes a cell-surface-targeted protein (sFv); the translation of this open reading frame within mammalian cells from these dicistronic mRNAs requires a functional IRES element. Cells that express the sFv can be selected from nonexpressing cells. A pool of up to 256 mutant encephalomyocarditis virus IRES elements was generated by converting the wild-type hairpin loop sequence (GCGA) to NNNN. Following transfection of this pool of mutants into COS-7 cells, plasmids were recovered from selected sFv-expressing cells. These DNAs were amplified in Escherichia coli and transfected again into COS-7 cells for further cycles to enrich for plasmids encoding functional IRES elements. The sequence of individual selected IRES elements was determined. All functional IRES elements had a tetraloop with a 3' terminal A residue. Optimal IRES activity, assayed in vitro and within cells, was obtained from plasmids encoding an IRES with the hairpin loop sequence fitting a RNRA consensus. In contrast, IRES elements containing YCYA tetraloops were severely defective.

Attenuation of Theiler's murine encephalomyelitis virus by modifications of the oligopyrimidine/AUG tandem, a host-dependent translational cis element.

A set of Theiler's murine encephalomyelitis virus mutants with engineered alterations in the conserved oligopyrimidine/AUG tandem (E. V. Pilipenko, A. P. Gmyl, S. V. Maslova, G. A. Belov, A. N. Sinyakov, M. Huang, T. D. K. Brown, and V. I. Agol, J. Mol. Biol. 241:398-414, 1994) were assayed for their growth potential in BHK-21 cells (as reflected in plaque size) and for neurovirulence upon intracerebral inoculation of mice. Tandem-destroying mutations, which included substitutions in the oligopyrimidine moiety and extended insertions into the oligopyrimidine/AUG spacer, exerted relatively little effect on the plaque size but ensured a high level of attenuation. The attenuated mutants exhibited remarkable genetic stability upon growth in BHK-21 cells. However, the brains of rare animals that developed symptoms after the inoculation with high doses of these mutants invariably contained pseudorevertants with the oligopyrimidine/AUG tandem restored by diverse deletions or an AUG-generating point mutation. The AUG moiety of the tandem in the revertant genomes was represented by either a cryptic codon or initiator codon. The results demonstrate that the tandem, while dispensable for the Theiler's murine encephalomyelitis virus growth in BHK-21 cells, is essential for neurovirulence in mice. Thus, the oligopyrimidine/AUG tandem is a host-dependent cis-acting control element that may be essential for virus replication under certain conditions. The functional activity of the tandem was retained when its oligopyrimidine or AUG moieties were made double stranded. A possible role of the tandem in the cap-independent internal initiation of translation on the picornavirus RNA templates is discussed.

Starting Window, a Distinct Element in the Cap-independent Internal Initiation of Translation on Picornaviral RNA

Initiation of translation on picornaviral RNA templates occurs via cap-independent ribosome binding to a cis-acting element, internal ribosome entry site (IRES). Mapping of the starting point of translation relative to the IRES was attempted using Theiler's murine encephalomyelitis virus (TMEV) RNA as a model. The possibility that the starting point is determined by the conserved oligopyrimidine upstream of the initiator codon was studied. In contrast to poliovirus, neither the conserved oligopyrimidine nor an AUG at a fixed distance downstream of this oligopyrimidine are required for efficient translation of the TMEV RNA in Krebs-2 extracts or reticulocyte lysates or for viral infectivity; mutants lacking the oligopyrimidne/AUG tandem were stable upon passage in BHK-21 cells. A short template segment, the starting window, was defined, wherefrom ribosomes begin translation or downstream scanning depending, respectively, on the presence or absence of a good-context AUG within this window. Using a collection of the engineered TMEV mutant RNAs, the starting window was mapped to 16-17 nt downstream of the IRES and was found to be approximately a dozen nt long. The efficiency of translation initiation from an AUG linearly increased upon the 5′→3′ displacement of the initiator codon within the window. The competence of the starting window did not appear to depend markedly on its primary structure; however, it was completely inactivated ("closed") with concomitant dramatic inhibition of total protein synthesis upon conversion of the corresponding RNA segment into a double-stranded form.

Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation.

Eukaryotic translation initiation factor-4A (eIF-4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA-dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA-ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF-4F. To gain further understanding into the mechanism of action of eIF-4A in mRNA-ribosome binding, defective eIF-4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF-4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap-independent internal initiation mechanism. Addition of eIF-4A or eIF-4F relieved inhibition of translation, but eIF-4F was six times more effective than eIF-4A, whereas eIF-4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF-4A is defective in recycling through eIF-4F, thus explaining the dramatic inhibition of translation. Mutant eIF-4A proteins also inhibited eIF-4F-dependent, but not eIF-4A-dependent RNA helicase activity. Taken together these results suggest that eIF-4A functions primarily as a subunit of eIF-4F, and that singular eIF-4A is required to recycle through the complex during translation. Surprisingly, eIF-4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.

Conservation of RNA-protein interactions among picornaviruses.

Picornavirus genomes encode unique 5' noncoding regions (5' NCRs) which are approximately 600 to 1,300 nucleotides in length, contain multiple upstream AUG codons, and display the ability to form extensive secondary structures. A number of recent reports have shown that picornavirus 5' NCRs are able to facilitate cap-independent internal initiation of translation. This mechanism of translation occurs in the absence of viral gene products, suggesting that the host cell contains the necessary components for the cap-independent internal initiation of translation of picornavirus RNAs as well as cellular mRNAs. In an attempt to identify some of the perhaps novel cellular proteins involved in this newly discovered mechanism of translation, we utilized RNA mobility shifts assays to identify and characterize interactions that occur between the 5'NCR of poliovirus type 1 (PV1) and cellular proteins. In this report, we describe two separate interactions between RNA structures from the 5' NCR of PV1 and proteins present in extracts from HeLa cells as well as other cell types. We describe the interaction between nucleotides 186 to 220 (stem-loop D) and a cellular protein(s) present in HeLa cell extracts. Mutational analysis of this stem-loop structure suggests that maintenance of a base-paired structure in the lower stem is necessary to present the sequences which directly interact with the protein(s). We also describe the interaction between nucleotides 220 to 460 (stem-loop E) and a cellular protein present in HeLa cell extracts. This RNA binding activity fractionates to a specific ammonium sulfate fraction (A cut) of a ribosomal salt wash. Mutational analysis of the stem-loop E structure suggests that the preservation of an extensive RNA structure is necessary for a strong interaction with the cellular protein(s), although smaller RNAs derived from this region of the 5' NCR can interact to lesser extents. Finally, we show that both of these RNA-protein interactions are conserved among the closely related enteroviruses PV1 and coxsackievirus type B3, human rhinovirus type 14, and the more distantly related cardiovirus Theiler's murine encephalomyelitis virus, suggesting that such RNA-protein interactions serve basic functions which are conserved and utilized by each of these picornaviruses.

Prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus RNA

Initiation of translation on picornavirus RNAs is accomplished through internal binding of ribosomes to a complex cis-acting element. Here we show that efficient function of this element involves two appropriately spaced smaller elements: UUUCC and an AUG. This conclusion emerged from analysis of the genome structures of spontaneous revertants of mutant polioviruses with extended insertions between the UUUCC and AUG motifs. It was confirmed by the results obtained with specially designed constructs. A similarity to the prokaryotic translation initiation mechanism, which involves the Shine-Dalgarno sequence, is emphasized, but in the picornavirus system the position of the UUUCC must be strictly fixed relative to upstream cis-acting elements, and the AUG may not necessarily serve as an initiation codon.

In vitro mutational analysis of cis-acting RNA translational elements within the poliovirus type 2 5' untranslated region.

Initiation of translation on poliovirus RNA occurs by internal binding of ribosomes to a region within the 5' untranslated region (UTR) of the mRNA. This region has been previously roughly mapped between nucleotides 140 and 631 of the 5' UTR and termed the ribosome landing pad. To identify cis-acting elements in the 5' UTR of poliovirus type 2 (Lansing strain) RNA that confer cap-independent internal initiation, we determined the in vitro translational efficiencies of a series of deletion and point mutations within the 5' UTR of the mRNA. The results demonstrate that the 3' border of the core poliovirus ribosome landing pad is located between nucleotides 556 and 585, whereas a region extending between nucleotides 585 and 612 confers enhanced translation. We studied two cis-acting elements within this region of the 5' UTR: a pyrimidine stretch which is critical for translation and an AUG (number 7 from the 5' end) that is located approximately 20 nucleotides downstream from the pyrimidine stretch and augments translation. We also show that the stem-loop structure which contains this AUG is not required for translation.