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. Specific aims are 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) Study of hepatitis C virus translation

The RNA (+) genome of hepatitis C virus (HCV) 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). Our working hypothesis was that PTB (Polypyrimidine Tract Binding protein) normally fixes both the IRES and the X region, forming a crucial bridge to favour RNA pseudocirularisation and increased translation efficiency. This would be analogous to the model proposed for classical capped, polyadenlyated mRNAs, in which an mRNA5'-eIF4E-eIF4G-PABP-mRNA3' bridge is implicated in efficient translation initiation under competitive conditions. The hypothesis supposes X-mediated stimulation of efficiency of translation from the HCV IRES.

Effectively, we have found such X-mediated translational stimulation for several years, but this remains a controversial subject. Reports from different groups published concerning effects of the X region on translation efficiency are distinctly contradictory, ranging from stimulation, through no effect, to inhibition. Therefore, we have constructed a translation plasmid in which the X sequence was replaced by X in the antisense orientation. These RNAs showed functional properties that were indistinguishable from negative control RNAs without the X region at the 3' end of the RNA. Thus, under our experimental conditions, translational stimulation by the 3' X region is a specific effect, and it seems that the effect is modulated according to the exact sequence of the IRES. We have also now set-up a specific semi-quantitative RT-PCR assay, that enabled us to verify that differential RNA stabilities were not intervening in the observed X-effect. We have previously described the study of variant IRESes that are distinguished from the prototype stimulation by the X region at the 3' end of the RNA, being refractory to X or inhibited by this sequence. One of the genotype 3a variants that is insensitive to the 3' X region differs from the prototype by a G to A substitution at nt. 243. Interestingly, this is one of the 4 differences between the extremely closely related genotype 1a and 1b sequences. We thus thought that such minor sequence differences in the strains studied by different groups could explain discordant results reported concerning the effects of the X region on translation efficacy. Therefore, we constructed by site-directed mutagenesis a number of chimeric type 1a/1b IRESes covering the sum of the discriminatory residues. No position could be clearly identified as being responsible for the contradictory effects of X described in the literature to date.

By site-directed mutagenesis, we have re-constructed mutants in the loops of the 3' X region of the RNA that should be defective for PTB binding. EMSA (or band shift assay), showed that an altered loop 2 mutant fixed recombinant PTB slightly less well than the prototype X sequence, whereas a loop 3 mutant was severely handicapped for binding of PTB. However, when we assessed RNAs carrying these mutant X regions by in vitro translation, it was the loop 2 mutant that was severely handicapped functionally, behaving like an RNA that had no X sequence at the 3' end. In contrast, the loop 3 mutant was able to stimulate translation efficiency from the HCV IRES equally well as the wild-type X sequence. Thus it seems unlikely that PTB binding to the X region intervenes in X-mediated translational stimulation. To confirm this conclusion, we wished to carry out translations in extracts that had been depleted for PTB by passage over an Encephalomyocarditis Virus IRES domain I affinity column as has been described. This technique presented major inconveniences. Firstly, relatively little extract could be prepared from a reasonable amount of starting material, and the preparation was expensive, and secondly the depletion was not entirely specific. Thus, we find that results to date using this translation system are not completely satisfactory, and we are carrying out additional experiments to confirm preliminary data.

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 h subunit of the mammalian eIF3 complex as a cellular partner of the rabies virus matrix protein (M). Moreover the M-eIF3h interaction was confirmed by co-immunoprecipitation experiments. We have pursued our studies to provide insight into the regulation of M mRNA translation and the role of the M-eIF3h interaction. We purified M protein from eukaryotic cells and showed that the M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5'-UTRs. It is remarquable that inhibition could be observed in some cases at approximately equimolar ratios of protein to mRNA. In contrast, translation of hepatitis C virus IRES-containing mRNA, which does not require eIF3 for 40S ribosomal subunit recruitment, was unaffected by the M protein. After transfection of cultured eukaryotic cells, M protein could be found associated with the polysome fraction of cell lysates, and this seemed to inhibit the polysome-association of a control protein co-transfected in parallel.

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

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