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  Director : KEAN, Katherine M. (kathiemb@pasteur.fr)



This year's main aim was to consolidate our preliminary results and hypotheses concerning mechanisms of initiation of viral protein synthesis (translation). Several RNA viruses have been studied (picornaviruses, hepatitis C virus, rabies virus, rotavirus). Concerning cellular protein synthesis, our work has focused primarily on FGF6, a member of the fibroblast growth factor family - mitogenic peptides implicated in physiological processes including oncogenesis, angiogenesis, morphogenesis, tissue regeneration and survival.



Structure-function analysis of the poliovirus (PV) IRES

Picornavirus mRNAs are amongst those translated by an alternative mechanism of initiation, from an IRES (for Internal Ribosome Entry Segment). Picornavirus IRESes are approximately 450 nucleotides long, and are structured into several domains. Domain E is accepted to be important in PV neurovirulence, as it contains residues that are mutated in the three vaccine strains which have played a crucial role in the control of poliomyelitis since the 1950s and which have been a keystone of the imminent world-wide poliovirus eradication goal. These mutations have been implicated as strong determinants of attenuation and to understand how they act, we have been comparing their effects in a single genomic background, that of wild-type PV type 1. We found last year that only one of the three mutations resulted in a significantly defective IRES. Previously, it had been proposed that the structure of a stem within domain E could be destabilised upon introduction of this mutation. This year we have examined this question biophysically, using RNA melting curve techniques (in collaboration with E Westhof and A Werner, IBMC, Strasbourg). Instead of a structural destabilisation, our experimental results indicate that the mutation strengthens the stem structure at low ionic strength. This goes against the current dogma concerning the correlation between IRES structure and function. Thus we are currently confirming our results by biochemical and enzymatic probing analyses.

Communication between the IRES and the 3' poly(A) tail of picornavirus mRNAs

Picornavirus IRESes have been classified into 3 groups on the basis of primary sequence and secondary structure conservation, as well as their functional properties. We previously showed that the presence of the 3' poly(A) tail stimulated translation initiation efficiency from representatives of all three types of picornavirus IRES. This is mediated by interactions between the proteins that bind the 5' IRES and the poly(A) tail, which are analagous to those that allow physical circularisation of cellular capped, polyadenylated mRNAs and are required for their efficient translation. However, we found last year that trans stimulation of standard capped mRNAs by the presence of exogenous poly(A) chains could mechanistically substitute for 3' poly(A) tail co-operation with the 5' cap. This year we examined the question of the effects of exogenous poly(A) chains on IRES-driven translation. Not only stimulation but also translation inhibition was evidenced according to the IRES examined and most interestingly, members of one of the groups of IRES split into the two opposing subsets. The repartition observed correlates with biological properties of the viruses which up until now had been believed to intervene later in the viral life-cycle than poly(A) tail-mediated stimulation of translation.

Communication between the 5' and 3' ends of rotavirus and hepatitis C virus (HCV) mRNAs

Rotavirus mRNAs are capped at the 5' end, but are not polyadenylated at their 3' ends, carrying instead a 7 nucleotide consensus sequence which binds a viral protein (NSP3) that could perform the equivalent role to that of the cellular poly(A) binding protein (PABP) in terms of mRNA circularisation. This year we have shown that the functional analogies between these proteins extend to include a dramatic NSP3-induced stimulation of rotavirus mRNA translation efficiency, i.e. once again implicating RNA-protein and protein-protein interactions that would ensure circularisation of the mRNA in efficient translation initiation. As seen for poly(A) effects on 5' cap mediated translation, we found that the rotavirus 3' end sequence could exert its stimulatory effect equally well when supplied in trans as when present in cis. The most "atypical" viral mRNA sequence we have studied is that of HCV, which has an IRES at the 5' end and a 98 nucleotide highly structured cloverleaf (the X region) at the 3' end rather than a poly(A) tail. Nevertheless, we previously showed that translation from the HCV IRES is stimulated by the presence of the X region at the 3' end of the RNA, and proposed that HCV RNA would be circularised via a completely different protein complex from other mRNAs analysed. We are currently testing this hypothesis using electron microscopy techniques (in collaboration with G. Pehau-Arnaudet, Plate-forme de cryomicroscopie moléculaire, Institut Pasteur — see relevant activity report). In the meantime, we have shown that despite the significant differences between this mRNA and other mRNAs studied, once again translation stimulation can be ensured by the cognate 3' end sequence supplied in trans.

The different cases seen of trans stimulation by mRNA 3' end sequences refute the hypothesis of continuous ribosome recycling at the post-termination stage of translation. Such results suggest either stimulation by increased de novo ribosome recruitment, or by direct transfer of the 40S ribosomal subunit from the stop codon to the 5' end of the mRNA. Furthermore, they open the possibility to the fact that competent mRNAs could be altruistic and lend their 3' ends to disabled neighbours.

Characterisation of the quasi-species distribution of the HCV IRES in infected individuals

We have extended a functional study concerning patients infected with HCV genotype 1b or 3a that we began last year (in collaboration with J-M. Pawlotsky and M. Soler, hôpital Henri Mondor, Créteil), looking at the intrinsic activity of variant IRESes and their stimulation by the X region. The HCV X region is a most remarkable modulator of variant IRES activity. Its stimulatory capacities vary between 3 and 1000-fold depending on the exact sequence and intrinsic activity of the variant IRES, but it can equally well be neutral or exert an inhibitory effect on IRES activity. These modulatory effects are not specific for a single IRES genotype. Functional differences between variant IRESes correlate with structural differences as assessed by biochemical and enzymatic probing of the RNA.

Regulation of rabies virus (RV) translation (in collaboration with Y. Jacob and E. Real, Virology Department, Institut Pasteur)

RV, a Rhabdovirus, induces a partial inhibition of host cell macromolecular synthesis. For VSV, the prototype of this family, the M2 viral protein has been implicated in this process. Using the yeast two-hybrid system, we have shown that the p40 subunit of the cellular eIF3 complex (responsible for the delivery of the 40S ribosomal subunit to the 5' end of mRNAs via its interaction with both this subunit and eIF4G) interacts with the RV M2 protein. We have confirmed the M2-p40 interaction by co-immunoprecipitation of these two proteins synthesised in vitro. In addition, we have found that the translation efficiency of an artificial mRNA coding for M2 is 10 times less efficient than that of a control mRNA in vitro. The mutation of a PPxY motif strongly conserved throughout rhabdovirus M2 proteins reduced the affinity of the M2-p40 interaction and partially rescued the in vitro translation efficiency. Our global results led us to propose an original model of translation control whereby the neo-synthesised M2 protein interacts immediately with the eIF3 complex fixed on the viral RNA, blocking successive cycles of translation initiation. This hypothesis is supported by recent results whereby an insect virus IRES that does not require eIF3 for translation initiation was used to drive M2 protein synthesis. In this case, the translation of the RNA coding for M2 was as efficient as that of the control mRNA..

Regulation of murine FGF6 translation

We chose to examine translation of this protein because of its physiological properties, but also because it is known that the mRNA encompasses three potential translation initiation sites. In addition, gene expression is extremely tightly controlled both spatially and temporally, being restricted to skeletal muscle precursors during 7 days of embryonic development. We found that two FGF6 mRNAs exist, which differ in the length of their 5' noncoding regions. Furthermore, we have shown that the endogenous protein profile in different mouse cell lines is dramatically different, i.e. it seems that the initiation codon usage profile varies according to the cell type and differentiation state. For the moment we have been unable to determine whether differential use of the two mRNAs contributes to FGF6 translation regulation.

Keywords: translation initiation, IRES, RNA structure-function, mRNA 5’-3’ cross-talk, eIF3


puce Publications of the unit on Pasteur's references database


  Office staff Researchers Scientific trainees Other personnel
  BALLET, Saskya, IP, (secretary,sballet@pasteur.fr) KEAN, Katherine M., CNRS, (Research Director, Head of Laboratory,kathiemb@pasteur.fr)

BORMAN, Andrew M., IP, (Researcher)

MICHEL, Yanne M., PhD student

MALNOU, Cécile E., PhD student

QUESNOIT, Mélanie, Diploma student

DEVEAUX, Vanessa, undergraduate rotation student

JUFFROY, Olivier, undergraduate rotation student

LHUILLIER, Pierre, undergraduate rotation student

PAULOUS, Sylvie, IP (technician)

Activity Reports 2002 - Institut Pasteur

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