Olivier Jean-Jean

Expression mechanism of the hepatitis B virus (HBV) C gene and biosynthesis of HBe antigen

The C gene of the hepatitis B virus, which contains two in-phase initiation codons delimiting the pre-C sequence and the C region, directs the synthesis of the major protein of the capsid (HBcAg) and of a precore protein which upon processing results in the secretion of the HBeAg. We used an adenovirus-based vector to study in the human 293 cell line the C gene products, the intermediates of the precore protein processing and the kind of protease involved in this processing. The synthesis of the 21-kDa HBcAg polypeptide was dependent on the deletion of the pre-C sequence suggesting that a pre-C mRNA is not used for the synthesis of the major capsid protein. With the construct containing the complete C gene, two proteins of 25 and 22 kDa were detected intracellularly, corresponding to the unprocessed and partially processed precore protein, respectively. In addition, a 15-kDa protein (HBeAg) was secreted in the culture medium. Using pepstatin, an inhibitor specific for aspartyl proteinases, reduction of HBeAg secretion and accumulation of the 22-kDa processing intermediate were observed, suggesting the involvement of an aspartyl proteinase in the conversion of the 22-kDa protein into HBeAg.

Involvement of Human Release Factors eRF3a and eRF3b in Translation Termination and Regulation of the Termination Complex Formation

ABSTRACT eRF3 is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. Both bind eRF1 and stimulate its release activity in vitro. However, whether both proteins can function as termination factors in vivo has not been determined. In this study, we used short interfering RNAs to examine the effect of eRF3a and eRF3b depletion on translation termination efficiency in human cells. By measuring the readthrough at a premature nonsense codon in a reporter mRNA, we found that eRF3a silencing induced an important increase in readthrough whereas eRF3b silencing had no significant effect. We also found that eRF3a depletion reduced the intracellular level of eRF1 protein by affecting its stability. In addition, we showed that eRF3b overexpression alleviated the effect of eRF3a silencing on readthrough and on eRF1 cellular levels. These results suggest that eRF3a is the major factor acting in translation termination in mammals and clearly demonstrate that eRF3b can substitute for eRF3a in this function. Finally, our data indicate that the expression level of eRF3a controls the formation of the termination complex by modulating eRF1 protein stability.

Stop codon recognition in ciliates: Euplotes release factor does not respond to reassigned UGA codon

In eukaryotes, the polypeptide release factor 1 (eRF1) is involved in translation termination at all three stop codons. However, the mechanism for decoding stop codons remains unknown. A direct interaction of eRF1 with the stop codons has been postulated. Recent studies focus on eRF1 from ciliates in which some stop codons are reassigned to sense codons. Using an in vitro assay based on mammalian ribosomes, we show that eRF1 from the ciliate Euplotes aediculatus responds to UAA and UAG as stop codons and lacks the capacity to decipher the UGA codon, which encodes cysteine in this organism. This result strongly suggests that in ciliates with variant genetic codes eRF1 does not recognize the reassigned codons. Recent hypotheses describing stop codon discrimination by eRF1 are not fully consistent with the set of eRF1 sequences available so far and require direct experimental testing.

Hepatitis B virus (HBV) X gene expression in human cells and anti-HBx antibodies detection in chronic HBV infection

All mammalian hepatitis B virus genomes contain an open reading frame X (X-ORF) of unknown function which could encode a protein of 17 kDa. Using a plasmid containing the entire X-ORF preceded by the adenovirus type 2 major late promoter and its tripartite leader sequence efficient expression of the HBV X-gene was achieved. The X protein of 17 kDa was characterized by immunoblotting and immunoprecipitated with an antiserum prepared against a X fusion protein produced in E. coli. By cell fractionation and indirect immunofluorescence the X-protein was found at least in part associated with nuclei. Human cell extracts containing the X protein have been used to screen human sera for anti-HBx antibodies. Such antibodies were detected in sera from patients with active chronic hepatitis with ongoing viral replication. The efficient expression of the HBV X protein obtained will facilitate its functional analysis.

Stop codon selection in eukaryotic translation termination: comparison of the discriminating potential between human and ciliate eRF1s

During eukaryotic translation termination, eRF1 responds to three stop codons. However, in ciliates with variant genetic codes, only one or two codons function as a stop signal. To localize the region of ciliate eRF1 implicated in stop codon discrimination, we have constructed ciliate–human hybrid eRF1s by swapping regions of human eRF1 for the equivalent region of ciliate Euplotes eRF1. We have examined the formation of a cross‐link between recombinant eRF1s and mRNA analogs containing the photoactivable 4‐thiouridine (s4U) at the first position of stop and control sense codons. With human eRF1, this cross‐link can be detected only when either stop or UGG codons are located in the ribosomal A site. Here we show that the cross‐link of the Euplotes–human hybrid eRF1 is restricted to mRNAs containing UAG and UAA codons, and that the entire N‐terminal domain of Euplotes eRF1 is involved in discriminating against UGA and UGG. On the basis of these results, we discuss the steps of the selection process that determine the accuracy of stop codon recognition in eukaryotes.

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly

Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation.

The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element

Selenocysteine insertion into selenoproteins involves the translational recoding of UGA stop codons. In mammals, selenoprotein expression further depends on selenium availability, which has been particularly described for glutathione peroxidase 1 and 4 (Gpx1 and Gpx4). The SECIS element located in the 3′UTR of the selenoprotein mRNAs is a modulator of UGA recoding efficiency in adequate selenium conditions. One of the current models for the UGA recoding mechanism proposes that the SECIS binds SECIS-binding protein 2 (SBP2), which then recruits a selenocysteine-specific elongation factor (EFsec) and tRNASec to the ribosome, where L30 acts as an anchor. The involvement of the SECIS in modulation of UGA recoding activity was investigated, together with SBP2 and EFsec, in Hek293 cells cultured with various selenium levels. Luciferase reporter constructs, in transiently or stably expressing cell lines, were used to analyze the differential expression of Gpx1 and Gpx4. We showed that, upon selenium fluctuation, the modulation of UGA recoding efficiency depends on the nature of the SECIS, with Gpx1 being more sensitive than Gpx4. Attenuation of SBP2 and EFsec levels by shRNAs confirmed that both factors are essential for efficient selenocysteine insertion. Strikingly, in a context of either EFsec or SBP2 attenuation, the decrease in UGA recoding efficiency is dependent on the nature of the SECIS, GPx1 being more sensitive. Finally, the profusion of selenium of the culture medium exacerbates the lack of factors involved in selenocysteine insertion.

Human eukaryotic release factor 3a depletion causes cell cycle arrest at G1 phase through inhibition of the mTOR pathway.

ABSTRACT Eukaryotic release factor 3 (eRF3) is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. Studies have related eRF3 with cell cycle regulation, cytoskeleton organization, and tumorigenesis. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. eRF3a is the major factor acting in translation termination, and its expression level controls termination complex formation. Here, we investigate the role of eRF3a in cell cycle progression using short interfering RNAs and flow cytometry. We show that eRF3a depletion induces a G1 arrest and that eRF3a GTP-binding activity, but not the eRF3a N-terminal domain, is required to restore G1-to-S-phase progression. We also show that eRF3a depletion decreases the global translation rate and reduces the polysome charge of mRNA. Finally, we show that two substrates of the mammalian TOR (mTOR) kinase, 4E-BP1 and protein kinase S6K1, are hypophosphorylated in eRF3a-depleted cells. These results strongly suggest that the G1 arrest and the decrease in translation induced by eRF3a depletion are due to the inhibition of mTOR activity and hence that eRF3a belongs to the regulatory pathway of mTOR activity.

Translation termination efficiency modulates ATF4 response by regulating ATF4 mRNA translation at 5′ short ORFs

The activating transcription factor 4 (ATF4) promotes transcriptional upregulation of specific target genes in response to cellular stress. ATF4 expression is regulated at the translational level by two short open reading frames (uORFs) in its 5′-untranslated region (5′-UTR). Here, we describe a mechanism regulating ATF4 expression in translation termination-deficient human cells. Using microarray analysis of total RNA and polysome-associated mRNAs, we show that depletion of the eucaryotic release factor 3a (eRF3a) induces upregulation of ATF4 and of ATF4 target genes. We show that eRF3a depletion modifies ATF4 translational control at regulatory uORFs increasing ATF4 ORF translation. Finally, we show that the increase of REDD1 expression, one of the upregulated targets of ATF4, is responsible for the mTOR pathway inhibition in eRF3a-depleted cells. Our results shed light on the molecular mechanisms connecting eRF3a depletion to mammalian target of rapamycin (mTOR) pathway inhibition and give an example of ATF4 activation that bypasses the signal transduction cascade leading to the phosphorylation of eIF2α. We propose that in mammals, in which the 5′-UTR regulatory elements of ATF4 mRNA are strictly conserved, variations in translation termination efficiency allow the modulation of the ATF4 response.

Importance of the C terminus of the hepatitis B virus precore protein in secretion of HBe antigen

The hepatitis B virus (HBV) e antigen (HBeAg) is a 15 kDa soluble antigen derived from a precursor protein (precore protein) by two processing events, cleavage of the N-terminal signal peptide and cleavage of the C-terminal 34 amino acids. So far, the role of the C-terminal sequences in secretion has not been analysed in full. In this study deletion of the last 60 amino acids was found to abrogate HBeAg secretion whereas deletions of the last 10, 25 or 39 amino acids decreased its secretion rate. These data demonstrate that C-terminal precore protein sequences are crucial for HBe secretion and determine its secretion rate.