Sequences 5' of the first upstream open reading frame in GCN4 mRNA are required for efficient translational reinitiation.

Translation of yeast GCN4 mRNA occurs by a reinitiation mechanism that is modulated by amino acid levels in the cell. Ribosomes which translate the first of four upstream open reading frames (uORFs) in the mRNA leader resume scanning and can reinitiate downstream. Under non-starvation conditions reinitiation occurs at one of the remaining three uORFs and GCN4 is repressed. Under starvation conditions, in contrast, ribosomes bypass the uORFs and reinitiate at GCN4 instead. The high frequency of reinitiation following uORF1 translation depends on an adequate distance to the next start codon and particular sequences surrounding the uORF1 stop codon. We present evidence that sequences 5' to uORF1 also strongly enhance reinitiation. First, reinitiation was severely inhibited when uORF1 was transplanted into the position of uORF4, even though the native sequence environment of the uORF1 stop codon was maintained, and this effect could not be accounted for by the decreased uORF1-GCN4 spacing. Second, insertions and deletions in the leader preceding uORF1 greatly reduced reinitiation at GCN4. Sequences 5' to uORF1 may influence the probability of ribosome release following peptide termination at uORF1. Alternatively, they may facilitate rebinding of an initiation factor required for reinitiation prior to resumption of the scanning process.     

The 5' UTR of HIV-1 full-length mRNA and the Tat viral protein modulate the programmed -1 ribosomal frameshift that generates HIV-1 enzymes

Translation of the full-length messenger RNA (mRNA) of the human immunodeficiency virus type 1 (HIV-1) generates the precursor of the viral enzymes via a programmed -1 ribosomal frameshift. Here, using dual-luciferase reporters, we investigated whether the highly structured 5' untranslated region (UTR) of this mRNA, which interferes with translation initiation, can modulate HIV-1 frameshift efficiency. We showed that, when the 5' UTR of HIV-1 mRNA occupies the 5' end of the reporter mRNA, HIV-1 frameshift efficiency is increased about fourfold in Jurkat T-cells, compared with a control dual-luciferase reporter with a short unstructured 5' UTR. This increase was related to an interference with cap-dependent translation initiation by the TAR-Poly(A) region at the 5' end of the messenger. HIV-1 mRNA 5' UTR also contains an internal ribosome entry site (IRES), but we showed that, when the cap-dependent initiation mode is available, the IRES is not used or is weakly used. However, when the ribosomes have to use the IRES to translate the dual-luciferase reporter, the frameshift efficiency is comparable to that of the control dual-luciferase reporter. The decrease in cap-dependent initiation and the accompanying increase in frameshift efficiency caused by the 5' UTR of HIV-1 mRNA is antagonized, in a dose-dependent way, by the Tat viral protein. Tat also stimulates the IRES-dependent initiation and decreases the corresponding frameshift efficiency. A model is presented that accounts for the variations in frameshift efficiency depending on the 5' UTR and the presence of Tat, and it is proposed that a range of frameshift efficiencies is compatible with the virus replication.     

L-Myc protein synthesis is initiated by internal ribosome entry

An internal ribosome entry segment (IRES) has been identified in the 5' untranslated region (5' UTR) of two members of the myc family of proto-oncogenes, c-myc and N-myc. Hence, the synthesis of c-Myc and N-Myc polypeptides can involve the alternative mechanism of internal initiation. Here, we show that the 5' UTR of L-myc, another myc family member, also contains an IRES. Previous studies have shown that the translation of mRNAs containing the c-myc and N-myc IRESs can involve both cap-dependent initiation and internal initiation. In contrast, the data presented here suggest that internal initiation can account for all of the translation initiation that occurs on an mRNA with the L-myc IRES in its 5' UTR. Like many other cellular IRESs, the L-myc IRES appears to be modular in nature and the entire 5' UTR is required for maximum IRES efficiency. The ribosome entry window within the L-myc IRES is located some distance upstream of the initiation codon, and thus, this IRES uses a "land and scan" mechanism to initiate translation. Finally, we have derived a secondary structural model for the IRES. The model confirms that the L-myc IRES is highly structured and predicts that a pseudoknot may form near the 5' end of the mRNA.     

Alternative splicing within the elk-1 5' untranslated region serves to modulate initiation events downstream of the highly conserved upstream open reading frame 2

The 5' untranslated region (UTR) plays a central role in the regulation of mammalian translation initiation. Key components include RNA structure, upstream AUGs (uAUGs), upstream open reading frames (uORFs), and internal ribosome entry site elements that can interact to modulate the readout. We previously reported the characterization of two alternatively spliced 5' UTR isoforms of the human elk-1 gene. Both contain two uAUGs and a stable RNA stem-loop, but the long form (5' UTR(L)) was more repressive than the short form (5' UTR(S)) for initiation at the ELK-1 AUG. We now demonstrate that ELK-1 expression arises by a combination of leaky scanning and reinitiation, with the latter mediated by the small uORF2 conserved in both spliced isoforms. In HEK293T cells, a considerable fraction of ribosomes scans beyond the ELK-1 AUG in a reinitiation mode. These are sequestered by a series of out-of-frame AUG codons that serve to prevent access to a second in-frame AUG start site used to express short ELK-1 (sELK-1), an N-terminally truncated form of ELK-1 that has been observed only in neuronal cells. We present evidence that all these events are fine-tuned by the nature of the 5' UTR and the activity of the α subunit of eukaryotic initiation factor 2 and provide insights into the neuronal specificity of sELK-1 expression.     

The zipper model of translational control: a small upstream ORF is the switch that controls structural remodeling of an mRNA leader

Transport of the essential amino acids arginine and lysine is critical for the survival of mammalian cells. The adaptive response to nutritional stress involves increased translation of the arginine/lysine transporter (cat-1) mRNA via an internal ribosome entry site (IRES) within the mRNA leader. Induction of cat-1 IRES activity requires both translation of a small upstream open reading frame (uORF) within the IRES and phosphorylation of the translation initiation factor eIF2alpha. We show here that translation of the upstream ORF unfolds an inhibitory structure in the mRNA leader, eliciting a conformational change that yields an active IRES. The IRES, whose activity is induced by amino acid starvation, is created by RNA-RNA interactions between the 5' end of the leader and downstream sequences. This study suggests that the structure of the IRES is dynamic and regulation of this RNA structure is a mechanism of translational control.     

Spurious splicing within the XIAP 5' UTR occurs in the Rluc/Fluc but not the betagal/CAT bicistronic reporter system

X-chromosome-linked inhibitor of apoptosis, XIAP, has been shown to contain a strong internal ribosome entry site (IRES) within its 5' untranslated region (UTR) that promotes translation of XIAP mRNA under conditions of cellular stress. This claim came under scrutiny in a recent report demonstrating that the XIAP 5' UTR undergoes splicing when inserted between the two reporter cistrons of the dual luciferase plasmid Rluc/Fluc. In this paper, we demonstrate that the splicing within the XIAP 5' UTR specifically occurs only in the context of mRNA produced from the Rluc/Fluc but not the pbetagal/CAT bicistronic reporter plasmid.     

Ribosomes stalling on uORF1 in the Xenopus Cx41 5' UTR inhibit downstream translation initiation

Translation of Xenopus laevis Connexin41 mRNA is strongly controlled by the three upstream open reading frames (uORFs) in its 5′ untranslated region. Mutation of uAUG1 into AAG induced a 100-fold increase in translation of a green fluorescent protein (GFP) reporter ORF. The termination codon of uORF1 was mutated and the uORF was linked in-frame with the GFP ORF, enabling visualisation of initiation at uAUG1 by synthesis of an elongated GFP form. Unexpectedly, hardly any elongated GFP was made, suggesting that translation of uORF1 in wild-type mRNA causes constraining of the entry of 40S ribosomal subunits upstream of uORF1. A rare leucine codon, the third codon of uORF1, contributed to the slow translation and thus to slow scanning. Replacement of the rare leucine codon in uORF1 with a common leucine codon stimulated GFP translation. Remarkably, the rare leucine codon, the termination codon of uORF1, uAUG2 and uAUG3 all improved recognition of uAUG1. Apparently, the block formed by a stalled ribosome on any element in uORF1 prevented the landing of new ribosomal subunits next to the cap and therefore downregulated GFP translation.     

The 5' untranslated region of protein kinase Cdelta directs translation by an internal ribosome entry segment that is most active in densely growing cells and during apoptosis

Protein kinase Cdelta (PKCdelta) is a member of the PKC family of phospholipid-dependent serine/threonine kinases and is involved in cell proliferation, apoptosis, and differentiation. Previous studies have suggested that different PKC isoforms might be translationally regulated. We report here that the 395-nt-long 5' untranslated region (5' UTR) of PKCdelta is predicted to form very stable secondary structures with free energies (deltaG values) of around -170 kcal/mol. The 5' UTR of PKCdelta can significantly repress luciferase translation in rabbit reticulocyte lysate but does not repress luciferase translation in a number of transiently transfected cell lines. By using a bicistronic luciferase reporter, we show that the 5' UTR of PKCdelta contains a functional internal ribosome entry segment (IRES). The activity of the PKCdelta IRES is greatest in densely growing cells and during apoptosis, when total protein synthesis and levels of full-length eukaryotic initiation factor 4G are reduced. However, the IRES activity of the 5' UTR of PKCdelta is not enhanced during serum starvation, another condition shown to inhibit cap-dependent translation, suggesting that its potency is dependent on specific cellular conditions. Accumulating data suggest that PKCdelta has a function as proliferating cells reach high density and in early and later events of apoptosis. Our studies suggest a mechanism whereby PKCdelta synthesis can be maintained under these conditions when cap-dependent translation is inhibited.     

The upstream open reading frame mediates constitutive effects on translation of cytochrome p-450c27 from the seventh in-frame AUG codon in rat liver

The 2.3-kb mRNA that codes for cytochrome P-450c27 (CYP27) has an unexpectedly long 5'-untranslated region (UTR) that holds six AUGs, leading to several upstream open reading frames (uORFs). The initiation of translation from the seventh AUG forms a putative 55-kDa precursor, which is processed in mitochondria to form a 52-kDa mature protein. The first three AUGs form fully overlapping uORF1, uORF2, and uORF3 that are in-frame with the seventh AUG and next two form fully overlapping uORF4 and uORF5 that are out-of-frame with the seventh AUG. Although not recognized by the scanning ribosomes under normal conditions, the sixth in-frame AUG forms a putative 57-kDa extension of the main open reading frame. The purpose of this study was to identify the elements in the 5'-UTR that direct CYP27 mRNA translation exclusively from the seventh AUG. Expression of 5' deletion mutants in COS cells reveal that the intact 5'-UTR not only directs the initiation of translation from the seventh AUG but also acts as a negative regulator. A 2-kb deletion mutant that lacks uORF1 initiates translation equally from the sixth and the seventh AUGs, forming both 57- and 55-kDa precursor proteins with a 2-fold increase in rate of translation. However, induction in translation does not affect the levels of the mature 52-kDa form in mitochondria but causes accumulation of the precursor form in cytosol not seen in COS cells transfected with wild-type cDNA. Mutation of the stop codon that terminates uORF1 completely shifts the initiation of translation from the seventh to the first AUG, forming a 67-kDa precursor that is processed into a 52-kDa mature protein in mitochondria. Confirmation of the bicistronic nature of CYP27 mRNA by epitope mapping of uORF1 suggests that translation of CYP27 mRNA from the seventh AUG is directed and regulated by uORF1 expression.     

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 O2-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 5' untranslated region of Rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems

Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5' UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5' UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and Drosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5' IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.     

The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5' secondary structure

Eukaryotic initiation factor (elF) 4A functions as a subunit of the initiation factor complex elF4F, which mediates the binding of mRNA to the ribosome. elF4A possesses ATPase and RNA helicase activities and is the prototype for a large family of putative RNA helicases (the DEAD box family). It is thought that the function of elF4A during translation initiation is to unwind the mRNA secondary structure in the 5' UTR to facilitate ribosome binding. However, the evidence to support this hypothesis is rather indirect, and it was reported that elF4A is also required for the translation of mRNAs possessing minimal 5' UTR secondary structure. Were this hypothesis correct, the requirement for elF4A should correlate with the degree of mRNA secondary structure. To test this hypothesis, the effect of a dominant-negative mutant of mammalian elF4A on translation of mRNAs with various degrees of secondary structure was studied in vitro. Here, we show that mRNAs containing stable secondary structure in the 5' untranslated region are more susceptible to inhibition by the elF4A mutant. The mutant protein also strongly inhibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents. UV crosslinking of elF4F subunits and elF4B to the mRNA cap structure is dramatically reduced by the elF4A mutant and RNA secondary structure. Finally, the elF4A mutant forms a more stable complex with elF4G, as compared to the wild-type elF4A, thus explaining the mechanism by which substoichiometric amounts of mutant elF4A inhibit translation.     

Identification of the TXNIP IRES and characterization of the impact of regulatory IRES trans-acting factors

Translation is a tightly regulated process and is predominantly controlled at the level of its initiation. Translation initiation mostly occurs in a cap-dependent manner. Under stress conditions when cap-dependent translation is hampered, internal ribosome entry sites (IRESes) allow for cap-independent translation of certain mRNAs. IRES-dependent translation is commonly regulated by RNA-interacting proteins, known as IRES trans-acting factors (ITAFs). In the present study, we found the 5′ untranslated region (UTR) of the thioredoxin-interacting protein (TXNIP) mRNA to be bound by the ITAF hnRNPA1. Upon verification of an IRES element within the 5′UTR of TXNIP, we determined additional interacting proteins, which predominantly appeared to interact with the IRES-regulatory second half of the 5′UTR. Amongst these PTB emerged as an inhibitory ITAF, whereas FBP3 and GEMIN5 appeared to contain TXNIP IRES-enhancing properties. In summary, we identified and characterized a novel IRES within the 5′UTR of TXNIP, which is regulated by the ITAFs PTB, FBP3, and GEMIN5.     

Pyrimidine tract binding protein and La autoantigen interact differently with the 5' untranslated regions of lentiviruses and oncoretrovirus mRNAs

Retrovirus genomic mRNA exhibits a several hundred nucleotides-long untranslated region (5' UTR) which encloses many control elements required for retrovirus replication. In addition, this 5' UTR contains translation regulatory elements, such as internal ribosome entry sites (IRESes) that have been described in oncoretroviruses, as well as in lentiviruses. UV cross-linking experiments suggested that the pyrimidine tract binding protein (PTB), a cellular protein known to regulate the activity of several picornaviral IRESes, binds to human T-cell leukemia virus (HTLV)-I RNA but not to lentiviral human immunodeficiency virus (HIV)-1, HIV-2 or simian immunodeficiency virus RNAs. To calculate the affinity of such RNA-protein interactions, we developed a new method based on the BIAcore technology. The absence of affinity of PTB for lentiviral RNAs was confirmed, whereas its affinity for HTLV-I RNAs was 1000-fold lower than for picornaviral RNAs. The BIAcore technology also revealed a significant affinity of the La autoantigen, previously described for its involvement in translational control of viral mRNAs, for HIV-1 and HTLV-I RNAs. Addition of recombinant PTB to in vitro translation experiments weakly enhanced translation initiation in the presence of HTLV-I IRES, suggesting that such an IRES requires additional trans-acting factors.     

Comparative analysis of translation efficiencies of hepatitis C virus 5' untranslated regions among intraindividual quasispecies present in chronic infection: opposite behaviors depending on cell type

Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5' untranslated region (5' UTR). While exhibiting the most highly conserved sequence within the genome, the 5' UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5' UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity. Sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell cultures lines, implying that interactions of RNA with related transacting factors may vary according to cell type. These data suggest that variants occasionally carried by the serum prior to reinfection could be selected toward different compartments of the same infected organism, thus favoring the hypothesis of HCV multiple tropism.