Unit: Eukaryotic and Viral Translational Control - CNRS URA 1966

Director: KEAN, Katherine M.

Regulation of gene expression at the level of protein synthesis (translation) represents an important phenomenon. Both global modulation, depending on environmental conditions, and specific regulation, relying on the use of variant and alternative mechanisms for the translation of sub-sets of mRNAs, can be defined. The general focus of our research is directed towards the initial encounter between the 40S ribosomal subunit and the mRNA. We apply a combination of virology, specifically molecular genetics, biochemistry, functional genomics and cell biology to attempt to understand molecular mechanisms of translation initiation by the eukaryotic ribosome, and the regulation that can be brought to play by the structure of the mRNA and during infection by RNA viruses.

1) Development of in vitro model study systems that recapitulate translation properties/physiological conditions in the mammal

The aim is to simplify studies of phenomena/mechanisms by using cell culture rather than whole organisms, or test tube translation systems charged with a single population of mRNAs rather than the complex cell environment of some 20,000 mRNAs. Basically, starting from a model that gives discordant results in a simplified system to those seen in vivo, the simplified model system is modified successively, to gain understanding of the requirements and/or constraints affecting the phenomenon under study. Our main objective this year has been to study hepatitis C virus (HCV) translation. The RNA genome of this virus has long, structured 5' and 3' nontranslated regions that are highly conserved between different strains. In particular, in contrast to classical cellular mRNAs, the 5' region encompasses an IRES (Internal Ribosome Entry Segment) and instead of a poly(A) tail the 3' terminal sequence is composed of 98 nucleotides that form a cloverleaf structure (the X region). Three model systems are currently in use/under development :

i) study of hepatitis C virus (HCV) translation in cell culture

The translation system itself is currently being studied, with particular reference to the effects of changes in the oxygen concentration during cell culture on translation. Effectively, most research involving cell culture is carried out at atmospheric oxygen pressure (21% or 159 mmHg). However, it is known that most mammalian organs are at 3-5% oxygen (35-40 mmHg), and it has been shown that hepatocytes, the principal target of HCV infection, are subject to an even lower oxygen pressure. When cells are cultivated under experimental normoxia (1-5% oxygen) a net reduction in classical translation is observed, but IRES-dependent translation is maintained, particularly the expression of unusual transcription factors. Thus, the notion of a specific cellular protein context is invoked, and it is possible that these conditions are particularly favourable to the translation of the HCV genome.

Our studies of translation in cultured cells will be greatly facilitated by adapting the DNA microarray technology for the analysis specifically of human polysomal mRNA collaboration with the DNA microarray platform, Genopole, Institut Pasteur). We have developed a protocol that gives satisfactory quality and quantity of total RNA isolated from experimental normoxic Huh7 cells. In contrast, the purification protocol designed to isolate polysomal mRNAs is not quantitatively adequate to accommodate the drop in translation expected under experimental normoxia. To increase the yield of polysomal RNA, we have included a quantitative amplification step at the stage of labelled cDNA synthesis. The innovative nature of our approach is that a new RNA amplification protocol has been elaborated, designed to accommodate all types of noncoding sequences, irrespective of the length of the RNA.

ii) study of mRNA 5'-3' cross-talk, or synergy

We have previously reported the development of a partially ribosome-depleted rabbit reticulocyte translation system that promotes the 5'-cap/3'-poly(A) synergy observed in vivo that is the basis of the closed-loop RNA model of eukaryotic translation initiation. This translation system has allowed us to show that efficient translation of HCV RNA also relies on a closed-loop model, bringing into play the 5' IRES and the 3'X region. After having shown that the X region modulates translation depending on the exact sequence of the IRES, we have examined the effect of mutations in the X region on these same variant IRESes. We have found that mutations in the X region have variable consequences on translation efficiency depending on the sequence of the IRES. We are currently determining which changes in RNA structure could explain these translational modulations. For the same reasons, we have undertaken the measurement of the binding affinity of PTB (Polypyrimidine Tract Binding protein) to the mutant X regions. Our hypothesis is that PTB normally fixes both the IRES and the X region, forming a crucial bridge to favour RNA pseudocirularisation and increased translation efficiency.

Moreover, we have found this year that the synergy observed in Huh7 cells can reach a level of some 100-fold higher than that seen in vitro in the ribosome-depleted rabbit reticulocyte lysate system. Thus, we are currently identifying factors that intervene in the response of the IRES to the X region that would be limiting in rabbit reticulocyte lysates.

iii) study of the production of the HCV F protein (coll. P. Mavromara, Institut Pasteur, Athens, Greece)

The beginning of the HCV coding region that encodes the structural core protein encompasses a second open reading frame shifted to the +1 position. The F protein that could be encoded in this alternative reading frame is effectively expressed during natural human infection by HCV, as witnessed by the presence of specific antibodies in patients. Several lines of evidence prove that the synthesis of this protein in vivo does not occur as in vitro, where a simple controlled reading-frame shift during translation elongation requires only codons 8 to 14 of the HCV coding sequence. However, the mechanism of F protein synthesis in vivo remains unclear. We have tested F protein synthesis in the partially ribosome-depleted rabbit reticulocyte lysate and found that this was not adequate to reproduce results obtained in vivo. Thus, we are in the process of establishing an appropriate in vitro translation system to study this phenomenon.

2) Study of the Rabies virus M protein synthesis (coll. Y.Jacob, Virology Department)

Matrix protein (M) of Rabies virus (a Rhabdovirus) is known to down-regulate viral transcription activity and at the same time stimulate viral replication. Using a differential yeast two-hybrid screen of a human brain cDNA library we identified the p40 subunit of the mammalian eIF3 complex as a cellular partner of the rabies virus matrix protein (M), moreover the M-p40 interaction was confirmed by co-immunoprecipitation experiments. Since we previously found that the translation of mRNAs encoding M was 5-fold less efficient in vitro than that of control mRNAs with identical 5' and 3' nontranslated regions, we proposed an autoregulatory feedback inhibition effect of M protein on M mRNA translation. However, we now find that cap-poly(A) synergy can be reproduced on this mRNA in the basic rabbit reticulocyte lysate system, a system that, for the majority of mRNAs, exhibits synergy only in the presence of endogenous competitor mRNA. In addition, the translation of dicistronic mRNAs in which the poliovirus IRES served as the intercistronic spacer showed no negative positional effects of M on translation efficiency of the second cistron, nor even on its own cistron. Thus, the rules governing M mRNA translation in vitro are rather complex and further studies are necessary to provide insight into the regulation of M mRNA translation and the role of the M -eIF3 interaction.

3) Studies concerning enterovirus IRESes

Over the last 4 years, we have built up a solid understanding concerning the RNA structure-function relationship for domain E of the poliovirus IRES, and notably the correlation between a neuronal attenuation phenotype and mutations in this domain. We have now modelled the sequence of the related cardiotrophic coxackievirus B3 onto the poliovirus RNA structure, and begun to place the equivalent mutations into the coxackievirus B3 genome. The aim of this work is to determine whether IRES domain E structural modifications can serve in the conception of generalised enterovirus vaccines (collaboration with J.Gharbi, Monastir, Tunisia).

Another important aspect of our current work on enteroviruses concerns natural isolates of ECHOvirus 30 collected from an epidemic of meningitis (collaboration with J-L. Bailly, Clermont-Ferrand). The secondary structure of domain E of all enterovirus IRESes reported to date is characterised by a highly conserved 14 nucleotide lateral bulge. In one clade of these epidemic-derived ECHOviruses this bulge is replaced by a tightly closed branch-stem within domain E. We have found that this correlates with translation initiation from an upstream in-frame AUG codon as well as from the authentic initiation codon, but also with a change in the KCl optimum for in vitro translation between poly(A)(-) and poly(A)(+) RNA. This is indicative of a change in protein-RNA interactions or in the proteins required for translation, depending on the poly(A) status of the RNA.

Keywords: RNA virus, translation initiation, IRES, RNA structure-function, mRNA 5’-3’ cross-talk

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