Molecular functions of eukaryotic initiation factor 3 (eIF3) in translation are manifold, encompassing events from initiation complex assembly to translation termination. The contribution of the individual subunits of eIF3 to its multiple activities is quite unclear. It has been hypothesized that several of its 13 subunits contribute to mRNA specific regulation. Prior research had established that the h subunit of eIF3 in Arabidopsis was required for translation of specific mRNAs as well as for organ formation and meristem development. This study aims towards understanding the functions of individual subunits of eIF3 in the context of plant development and to further define the role of eIF3h at the molecular level. This dissertation describes an effort to identify mutations affecting each of the 13 eIF3 subunits. Using a panel of pollen-specific fluorescent marker genes, eIF3 subunits e, h and i1 were demonstrated to be essential for normal male gametophyte development. Furthermore, subunits b and c proved to be essential for embryo development. In contrast, a mutation in eIF3k revealed no phenotypic abnormalities. This work represents a systematic effort to attribute functions to many of the eIF3 subunits in growth and development in a multicellular eukaryote. The h subunit of eIF3 is necessary for the efficient translation of specific mRNAs in Arabidopsis. In particular, eIF3h fosters the translation of those mRNAs that harbor multiple upstream open reading frames (uORFs) in their 5’ leader. The specific molecular activity of eIF3h was investigated by structure-function analysis of the 5' leader of the Arabidopsis AtbZip11 mRNA, which harbors a set of four uORFs that is evolutionarily conserved. By pairing extensive mutagenesis of the AtbZip11 5' leader with gene expression analysis in Arabidopsis seedlings, it was revealed that eIF3h helps the ribosome to retain its reinitiation competence during uORF translation. These data establish a function for the h subunit of eIF3 in a special case of translation initiation, reinitiation. Finally, the molecular events during translation reinitiation were investigated further for a functional cooperation between eIF3h and the large subunit of the ribosome, given that the large ribosomal subunit had been implicated in reinitiation in other biological contexts. Reinitiation profiling using the AtbZip11 leader demonstrated that a protein of the large ribosomal subunit, RPL24B, bolsters reinitiation similar to eIF3h. Taken together, there exists a functional cooperation between the large ribosomal subunit and eIF3 that helps ribosomes to reinitiate after translation of uORFs.

Translation initiation is a multi-step process culminating in formation of the elongation- competent 80S ribosome. It requires accurate assembly of small and large ribosomal subunits, mRNA, initiation Met-tRNAi Met and at least 12 eukaryotic initiation factors (eIFs). This phase of protein synthesis is also one of the key points of regulation of gene expression. One of the main aims of our laboratory is a complex characterization of the multiprotein eIF3 complex that has been implicated in most of the steps of translation initiation. For example, we revealed and described its novel role in translation reinitiation (REI), a gene-specific translational control mechanism that among others governs expression of an important yeast transcriptional activator GCN4. Here I present a detailed characterization of the multi-functional N-terminal domain of Tif32 (subunit eIF3a). We demonstrated that the Tif32-NTD functionally interacts with the 5' sequences of short upstream ORF (uORF1) in the GCN4 mRNA leader and thus allows efficient reinitiation downstream of this critical reinitiation-permissive uORF. Four REI- promoting elements (RPEs) were identified in the 5' sequences of uORF1, two of which were shown to work in the Tif32-NTD-dependent manner. The structure of the 5' sequences was determined...

Double-stranded RNA fungal virus Helminthosporium victoriae virus 190S (genus Victorivirus, family Totiviridae) contains two large open reading frames (ORFs) that overlap in the tetranucleotide AUGA. Translation of the downstream ORF, which encodes the RNA-dependent RNA polymerase (RdRp), was previously proposed to depend on ribosomal reinitiation following termination of the upstream ORF, which encodes the capsid protein. In this study, I provided evidence to confirm that coupled termination-reinitiation (stop-restart) is indeed used. A dual-fluorescence method was established to define the RNA sequence determinants for RdRp translation. Stop-restart depends on a 32-nt stretch of RNA sequence immediately upstream of the AUGA motif, including a predicted pseudoknot structure. The presence of similar sequence motifs and predicted RNA structures in other victoriviruses suggest that they all share a related stop–restart strategy for RdRp translation. The close proximity of the secondary structure to the AUGA motif appears to be especially important for promoting translation of the downstream ORF. Normal strong preferences for AUG start codons and canonical sequence context for translation initiation of the downstream ORF appear somewhat relaxed. With dual-fluorescence system, reinitiation efficiency of the downstream ORF was determined to be ~3.9%. Pseudoknot swapping between the one in HvV190S and those predicted from other victoriviruses showed that reinitiation from the downstream ORF of HvV190S is quite tolerant to varying primary sequences of the various pseudoknots. Mutational analysis by introducing different combinations of nucleotide mutations into pseudoknot stems reproducibly confirmed the determinant role of pseudoknot on reinitiation using two different experimental systems. Together, these results provide the first example of coupled termination-reinitiation regulated by a simple pseudoknot stucture. These data expanded the understanding of coupled termination-reinitiation mechanism employed by RNA viruses and refined a new model for genus victorivirus, the largest genus in the family Totiviridae. The dual fluorescence system used in this study represented the first application of an efficient in vivo assay for recording low-frequency events in filamentous fungi.

La réinitiation de la traduction est un mécanisme permettant de traduire des ORF qui sont présents dans la région leader de différents ARNm cellulaires (uORF). La majorité des cas de réinitiation de la traduction chez les eucaryotes concerne des uORF de petite taille. Des stratégies alternatives ont été développées, entre autres par les virus, afin de réinitier la traduction après un long uORF. Le virus de la mosaïque du chou-fleur (CaMV) exprime un ARNm polycistronique codant la totalité des protéines virales. L’une d’entre elle, la protéine TAV (TransActivateur/Viroplasmine) est un facteur essentiel qui rend possible la réinitiation de la traduction après de longs ORF et qui, de plus, active la protéine kinase TOR. La sous-unité h du facteur d’initiation de la traduction eIF3, requise pour promouvoir la reinitiation après un petit ORF chez les plantes, a été identifiée comme étant une nouvelle cible de phosphorylation de la voie de signalisation de TOR. L’objectif principal de ma thèse a été d’élucider la fonction de la protéine eIF3h dans la réinitiation après un petit ORF ainsi que dans la réinitiation de la traduction, assurée par TAV, après un long ORF. Nous avons exploité les lignées transgéniques eif3h-1 d’Arabidopsis exprimant la protéine eif3h tronquée de son extrémité C-terminale, qui sont déficientes pour la réinitiation mais pas pour l’initiation de la traduction. Nous avons montré que la phosphorylation de eIF3h est essentielle pour stabiliser eIF3 au niveau des ribosomes durant l’élongation, ce qui favorise la ré-acquisition par le ribosome de facteurs nécessaires à la réinitiation de la traduction, et que la délétion de sa région Ct abolit son intégration dans le complexe eIF3. De plus, nous avons montré que eIF3h, la cible de la voie de signalisation de TOR, interagit avec S6K1. Des protoplastes préparés à partir des plantes mutantes eif3h-1 sont incapables de promouvoir la réinitiation après de longs ORF en présence de TAV. La surexpression de eIF3h, indifféremment de son état de phosphorylation, est indispensable pour restaurer la reinitiation assurée par TAV dans les protoplastes eif3h-1. Par ailleurs, les plantes eif3h-1 déficientes dans la réinitiation, sont résistantes à l’infection par le CaMV démontrant l’importance de eIF3h pour la réplication du CaMV. En revanche, ces plantes eif3h-1 peuvent être infectées par d’autres virus dont la traduction de l’ARN génomique est coiffe- ou IRES-dépendante. Ainsi, nos résultats suggèrent que eIF3h est un facteur de reinitiation important aussi bien pour la reinitiation après un petit qu’après un long ORF (controlée par TAV), et que TAV exploite cette machinerie cellulaire, et plus particulièrement TOR et eIF3h, pour exprimer ses propres protéines par réinitiation de la traduction. / Translation of mRNAs that harbor upstream open reading frames (uORFs) within their leader regions operates via a reinitiation mechanism. In plants, reinitiation is up regulated by the target of rapamycin (TOR) signaling via phosphorylation of the subunit h of initiation factor 3 (eIF3). The eif3h-1 mutant expressing the C-terminally truncated eIF3h while maintaining high translation initiation efficiency is not active in reinitiation. Cauliflower mosaic virus (CaMV) pregenomic polycistronic RNA is translated via an exceptional mechanism of reinitiation after long ORF translation under control of CaMV protein TAV, which ensures activation of TOR. To find the link between underlying mechanisms, we examined eIF3h function in cellular and viral context. Here we show that eIF3h, if phosphorylated, has a role in recruitment of eIF3 into actively translating ribosomes that is a prerequisite for formation of reinitiation-competent ribosomal complexes. C-terminal truncation of eIF3h abolished its integration into the eIF3 complex and eIF3 loading on polysomes as manifested by the eIF3 core subunit c. We also show that eIF3h as a putative target of TOR/S6K1 binds S6K1 in vitro. eIF3h phosphorylation is not required for eIF3 complex formation. We demonstrated that eIF3h is essential for TAV to activate reinitiation after long ORF translation. Protoplasts derived from eif3h-1 mutant failed to support TAV function in reinitiation, which is restored only upon overexpression of recombinant eIF3h indifferent to its phosphorylation status. eif3h-1 mutant defective in reinitiation was found resistant to CaMV infection suggesting that eIF3h is critical for virus amplification. In contrast, viruses that evolve translation initiation dependent on either cap or the internal ribosome entry site infect reinitiation deficient mutant. Thus, we conclude that TAV exploits the basic cell reinitiation machinery, particularly TOR and eIF3h, to overcome cellular barriers to reinitiation after long ORF translation.

Chez Arabidopsis, la protéine RISP est détournée par le virus CaMV pour assurer, ensemble avec la protéine virale TAV, la traduction de son ARN polycistronique. RISP a été identifiée comme une cible de la voie de signalisation de TOR et il a été montré que sa phosphorylation est requise pour promouvoir la réinitiation de la traduction activée par TAV. Les résultats que j’ai obtenus suggèrent que RISP, lorsqu’elle n’est pas phosphorylée, intervient ensemble avec eIF3, au niveau du complexe de pré-initiation 43S pour recruter le complexe ternaire grâce à l’interaction entre RISP et la sous-unité b du facteur eIF2. Il s’est avéré que RISP a la capacité, lorsqu’elle est phosphorylée, d’interagir non seulement avec la protéine ribosomique eL24 mais également avec eS6. Nos résultats indiquent que la liaison entre les sous-unités ribosomiques 60S et 40S sous l’effet de RISP, est régulée par la voie de TOR et qu’elle joue un rôle important dans le contrôle de la réinitiation de la traduction. / Many factors are required to recruit the tRNAi and a 60S ribosomal subunit to the 40S ribosomal subunit preinitiation complex. This recruitment is normally strictly limited during reinitiation of translation if factors recruited during the primary translation event are shed from 40S. However, factor retention can occur during long ORF translation if the CaMV viral factor TAV is present. RISP is a downstream target of TOR and found either within the 43S preinitiation complex, if bound to eIF3, and/or attached to 60S, if phosphorylated by TOR. We show here that RISP interacts with subunit b of eIF2 before phosphorylation. Critically, TOR activation up-regulates phosphorylation of both RISP and eS6 as well as the binding of both factors. Importantly, eS6-deficient plants are less active in TAV-mediated reinitiation and are thus less susceptible to CaMV infection. It is attractive to propose that eS6 phosphorylation contributes to retention and re-use of 60S during 40S scanning.

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