Translational errors: from yeast to new therapeutic targets

Errors occur randomly and at low frequency during the translation of mRNA. However, such errors may also be programmed by the sequence and structure of the mRNA. These programmed events are called 'recoding' and are found mostly in viruses, in which they are usually essential for viral replication. Translational errors at a stop codon may also be induced by drugs, raising the possibility of developing new treatment protocols for genetic diseases on the basis of nonsense mutations. Many studies have been carried out, but the molecular mechanisms governing these events remain largely unknown. Studies on the yeast Saccharomyces cerevisiae have contributed to characterization of the HIV-1 frameshifting site and have demonstrated that frameshifting is conserved from yeast to humans. Yeast has also proved a particularly useful model organism for deciphering the mechanisms of translation termination in eukaryotes and identifying the factors required to obtain a high level of natural suppression. These findings open up new possibilities for large-scale screening in yeast to identify new drugs for blocking HIV replication by inhibiting frameshifting or restoring production of the full-length protein from a gene inactivated by a premature termination codon. We explore these two aspects of the contribution of yeast studies to human medicine in this review.     

Two yeast PUF proteins negatively regulate a single mRNA

mRNA stability and translation are regulated by protein repressors that bind 3'-untranslated regions. PUF proteins provide a paradigm for these regulatory molecules: like other repressors, they inhibit translation, enhance mRNA decay, and promote poly(A) removal. Here we show that a single mRNA in Saccharomyces cerevisiae, encoding the HO endonuclease, is regulated by two distinct PUF proteins, Puf4p and Mpt5p. These proteins bind to adjacent sites and can co-occupy the mRNA. Both proteins are required for full repression and deadenylation in vivo; their removal dramatically stabilizes the mRNA. The two proteins act through overlapping but non-identical mechanisms: repression by Puf4p is dependent on deadenylation, whereas repression by Mpt5p can occur through additional mechanisms. Combinatorial action of the two regulatory proteins may allow responses to specific environmental cues and be common in 3'-untranslated region-mediated control.     

Nonsense-mediated mRNA decay: terminating erroneous gene expression

Nonsense-mediated mRNA decay is a surveillance pathway that reduces errors in gene expression by eliminating aberrant mRNAs that encode incomplete polypeptides. Recent experiments suggest a working model whereby premature and normal translation termination events are distinct as a consequence of the spatial relationship between the termination codon and mRNA binding proteins, a relationship partially established by nuclear pre-mRNA processing. Aberrant termination then leads to both translational repression and an increased susceptibility of the mRNA to multiple ribonucleases.     

The yeast nuclear cap binding complex can interact with translation factor eIF4G and mediate translation initiation

The mRNA cap structure is bound by either the nuclear (CBC) or the cytoplasmic (eIF4F) cap binding complex. Following mRNA export, CBC must be exchanged for eIF4F in the cytoplasm. It is not known how this exchange occurs or how this RNP remodeling event is integrated with mRNA function. Here we report genetic and biochemical evidence that the yeast translation initiation factor eIF4G associates with CBC, and that eIF4E, the eIF4F component that binds both the cap and eIF4G, antagonizes this interaction. Furthermore, we find that CBC can stimulate translation in extracts containing an eIF4G protein deficient for eIF4E binding. These data suggest that eIF4E binding to the eIF4G-CBC complex on newly exported mRNA displaces CBC, and that the first round of translation on mRNA may occur via a different mechanism than subsequent rounds.     

mRNA turnover

Nuclear RNA-binding proteins can record pre-mRNA processing events in the structure of messenger ribonucleoprotein particles (mRNPs). During initial rounds of translation, the mature mRNP structure is established and is monitored by mRNA surveillance systems. Competition for the cap structure links translation and subsequent mRNA degradation, which may also involve multiple deadenylases.     

Translation of non-capped mRNAs in a eukaryotic cell-free system: acceleration of initiation rate in the course of polysome formation

Real-time monitoring of the translation of non-capped luciferase mRNA in a wheat germ cell-free system has been performed by continuous in situ measurement of the luminescence increase in the translation mixture. The phenomenon of acceleration of translation has been revealed. It has been shown that the acceleration is accompanied by the loading of translating polysomes with additional ribosomes, and thus is caused mainly by a rise in the initiation rate, rather than the stimulation of elongation or the involvement of additional mRNA molecules in translation. The acceleration requires a sufficient concentration of mRNA and depends on the sequence of the 5′ untranslated region (UTR). It can be abolished by the addition of excess cap analog (m⁷GpppGm). As the acceleration does not depend on the preliminary translation of other mRNAs in the same extract, the conclusion has been made that the effect is not due to activation of the ribosome population or other components of the system during translation, but rather it is the consequence of intra-polysomal events. The acceleration observed is discussed in terms of the model of two overlapping initiation pathways in eukaryotic polysomes: translation of non-capped mRNAs starts with eIF4F-independent initiation at 5′ UTR, and after the formation of sufficiently loaded polysomes, they rearrange in such a way that a mechanism of re-initiation of terminating ribosomes switches on. The eIF4F-mediated circularization of polysomes may be considered as a possible event that leads to the re-initiation switch and the resultant acceleration effect.     

Conducting the initiation of protein synthesis: the role of eIF4G

The eukaryotic initiation factor eIF4G is a large modular protein which serves as a docking site for initiation factors and proteins involved in RNA translation. Together with eIF4E and eIF4A, eIF4G constitutes the eIF4F complex which is a key component in promoting ribosome binding to the mRNA. Thus, the central role of eIF4G in initiation makes it a valid target for events aimed at modulating translation. Such events occur during viral infection by picornaviruses and lentiviruses and result in the hijack of the translational machinery through cleavage of eIF4G. Proteolysis of eIF4G is also mediated by caspases during the onset of apoptosis causing inhibition of protein synthesis. We will review the role of eIF4G and protein partners as well as the cellular and viral events that modulate eIF4G activity in the initiation of translation.     

AUG_hairpin: prediction of a downstream secondary structure influencing the recognition of a translation start site

Background  

The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. The recognition of the start AUG codon by eukaryotic ribosomes is considered to depend on its nucleotide context. However, the fraction of eukaryotic mRNAs with the start codon in a suboptimal context is relatively large. It may be expected that mRNA should possess some features providing efficient translation, including the proper recognition of a translation start site. It has been experimentally shown that a downstream hairpin located in certain positions with respect to start codon can compensate in part for the suboptimal AUG context and also increases translation from non-AUG initiation codons. Prediction of such a compensatory hairpin may be useful in the evaluation of eukaryotic mRNA translation properties.     

A cis-Acting Element Present within the gag Open Reading Frame Negatively Impacts on the Activity of the HIV-1 IRES

Translation initiation from the human immunodeficiency virus type-1 (HIV-1) mRNA can occur through a cap or an IRES dependent mechanism. Cap-dependent translation initiation of the HIV-1 mRNA can be inhibited by the instability element (INS)-1, a cis-acting regulatory element present within the gag open reading frame (ORF). In this study we evaluated the impact of the INS-1 on HIV-1 IRES-mediated translation initiation. Using heterologous bicistronic mRNAs, we show that the INS-1 negatively impact on HIV-1 IRES-driven translation in in vitro and in cell-based experiments. Additionally, our results show that the inhibitory effect of the INS-1 is not general to all IRESes since it does not hinder translation driven by the HCV IRES. The inhibition by the INS-1 was partially rescued in cells by the overexpression of the viral Rev protein or hnRNPA1.     

Translation of nonSTOP mRNA is repressed post-initiation in mammalian cells

We investigated the fate of aberrant mRNAs lacking in-frame termination codons (called nonSTOP mRNA) in mammalian cells. We found that translation of nonSTOP mRNA was considerably repressed although a corresponding reduction of mRNA was not observed. The repression appears to be post-initiation since (i) repressed nonSTOP mRNAs were associated with polysomes, (ii) translation of IRES-initiated and uncapped nonSTOP mRNA were repressed, and (iii) protein production from nonSTOP mRNA associating with polysomes was significantly reduced when used to program an in vitro run-off translation assay. NonSTOP mRNAs distributed into lighter polysome fractions compared to control mRNAs encoding a stop codon, and a significant amount of heterogeneous polypeptides were produced during in vitro translation of nonSTOP RNAs, suggesting premature termination of ribosomes translating nonSTOP mRNA. Moreover, a run-off translation assay using hippuristanol and RNAse protection assays suggested the presence of a ribosome stalled at the 3' end of nonSTOP mRNAs. Taken together, these data indicate that ribosome stalling at the 3' end of nonSTOP mRNAs can block translation by preventing upstream translation events.     

Poly(A)-binding proteins: Structure, domain organization, and activity regulation

RNA-binding proteins are of vital importance for mRNA functioning. Among these, poly(A)-binding proteins (PABPs) are of special interest due to their participation in virtually all mRNA-dependent events that is caused by their high affinity for A-rich mRNA sequences. Apart from mRNAs, PABPs interact with many proteins, thus promoting their involvement in cellular events. In the nucleus, PABPs play a role in polyadenylation, determine the length of the poly(A) tail, and may be involved in mRNA export. In the cytoplasm, they participate in regulation of translation initiation and either protect mRNAs from decay through binding to their poly(A) tails or stimulate this decay by promoting mRNA inter-actions with deadenylase complex proteins. This review presents modern notions of the role of PABPs in mRNA-dependent events; peculiarities of regulation of PABP amount in the cell and activities are also discussed.     

Zinc and the initiation of myoblast differentiation

Previous studies have indicated that a lack of available zinc inhibited myoblast differentiation as shown by a failure of the cells to fuse and low expression of creatine kinase mRNA and activity. However, the nature of the requirement for zinc and its relationship to the events leading to differentiation have been unclear. The current studies with C₂C₁₂ cells indicated that the muscle-specific enhancer present in the 5′-flanking region of the creatine kinase gene contributed to the zinc sensitivity of this enzyme. Because this enhancer can be activated by expression of the myogenic factors MyoD and myogenin, their sensitivity to zinc was investigated. The concentrations of both MyoD and, particularly, myogenin mRNA, were decreased by zinc deficiency. In vitro translation experiments suggested that these changes closely corresponded with alterations in their rates of synthesis. Further experiments failed to indicate a major effect of zinc on the stabilities of these mRNAs. Because an induction of myogenin mRNA is one of the earliest known events in myoblast differentiation, its particular sensitivity to lack of zinc suggests that zinc may be required before or during the initiation of myoblast differentiation.     

A simplified reconstitution of mRNA-directed peptide synthesis: activity of the epsilon enhancer and an unnatural amino acid

The study of the early events in translation would be greatly facilitated by reconstitution with easily purified components. Here, Escherichia coli oligopeptide synthesis has been reconstituted using five purified recombinant His-tagged E. coli initiation and elongation factors. Highly purified ribosomes are required to yield products with strong dependencies on the translation factors. Based on HPLC separation of radiolabeled translation products from an mRNA encoding a tetrapeptide, approximately 80% of peptide products are full length, and the remaining 20% are the dipeptide and tripeptide products resulting from pausing or premature termination. Oligopeptide synthesis is enhanced when a commonly used epsilon (enhancer of protein synthesis initiation) sequence is included in the mRNA. The system incorporates a selectable, large, unnatural amino acid and may ultimately form the basis of a pure translation display technology for the directed evolution of peptidomimetic ligands and drug candidates. The recombinant clones can be exploited to prepare initiation factors and initiation complexes for structural studies, to study initiation and elongation in ribosomal peptide synthesis, and to screen for eubacterial-specific drugs.     

Predicted structures of apolipoprotein II mRNA constrained by nuclease and dimethyl sulfate reactivity: stable secondary structures occur predominantly in local domains via intraexonic base pairing

Analyses of apolipoprotein II mRNA with chemical and enzymatic probes showed that double- and single-stranded regions were distributed uniformly along the mRNA except for a large (72 nucleotides) single-stranded region containing the translation stop codon. Secondary structure models constrained by the experimental data were made by varying the distance (along the mRNA) over which base pairing was allowed. Four prominent secondary structures were seen with restrictions of 165, 330, or 659 nucleotides suggesting that such structures from via local interactions over distances of 50-120 nucleotides. Predicted long range interactions involve only 2-3 base pairs while local interactions involve helices of 4-10 base pairs. Predicted helices of greater than or equal to 4 base pairs occur primarily within exons, raising the possibility that prominent secondary structures in mRNAs may be largely due to intraexonic base pairing. Tests of single- and double-stranded domains by oligonucleotide-directed RNase H cleavage and primer extension were in accord with the structure model and with nuclease and chemical modification data. The model predicting base pairing between the coding and the 3' noncoding regions was tested by RNase H cleavage followed by oligo(dT)-cellulose chromatography to separate 5' and 3' mRNA fragments. Most (82%) of the 5' fragment remained associated with the 3' noncoding region in a structure with a tm = 50 degrees C in 0.2 M Na+ suggesting that this stem could be stable in vivo. This stem may be stable in the isolated mRNA, but would likely occur transiently in polyribosomal apolipoprotein II mRNA due to ribosome transit through the 5' side of the stem. Alternate structures may occur in this region during ribosome transit and play a role in translation termination or in determining the susceptibility of the mRNA to degradation.