eIF3 and eIF4E are very important eukaryotic translation initiation factors. eIF3 is practically involved in every step of translation initiation, eIF4E is important mainly for its ability to bind the cap. Mammalian factor eIF3 consists of thirteen subunits, many subunits have a function apart from translation, such as in apoptosis and mitosis. It was proved that upregulated or downregulated expression of some subunits as well as upregulated expression of eIF4E is linked with different types of tumours and malignancies in human. In the first part of my work, I was examining the amount of transcripts of subunits eIF3a, b, d, e, f, g, h, i and j in cell lines which are used for study of acute lymphoblastic leukaemia. I tried to find if there is a difference in the amount of trancripts between lines or between lines and control line in these subunits. According to experiments and statistical analysis, I proved increased amount of mRNA for eIF3b subunit in control cell line NC-NC in comparison with other used leukaemic cell line except from line NALM6. Other differences were not statistically important. In the second part of my work, I was analysing 3' UTR region of transcripts of eIF4E1 and utilising of polyadenylation signals in this trancript. I used the leukeamic cell lines again. The experiments clearly...

Les microARN (miARN) sont de petits ARN non-codants qui contrôlent l’expression génique, en s’hybridant, le plus souvent, de manière imparfaite à des séquences spécifiques qui se trouvent généralement dans la région non traduite en 3' (3’UTR) de transcrits cibles. Les miARN guident sur l’ARN messager (ARNm) un complexe protéique appelé RNA-induced Silencing Complex (RISC), composé des protéines Argonaute et TNRC6, qui perturbe l’initiation de la traduction et provoque la déadénylation et la dégradation du transcrit. C'est l’interaction entre le RISC et le complexe de pré-initiation de la traduction 43S (composé de la petite sous-unité ribosomique 40S et des facteurs d’initiation associés) qui entraîne la répression traductionnelle de l’ARNm ciblé. Des résultats récents ont démontré que le RISC perturbe le balayage de la région non traduite en 5’ (5’UTR) par le ribosome, étape qui requiert la présence de 2 facteurs d’initiation qui sont eIF4F qui reconnaît et lie la coiffe ainsi que la protéine PABP, fixée le long de la queue poly(A). Toutefois, les miARN peuvent également induire la stimulation de la traduction des transcrits cibles dans les cellules quiescentes, dans un lysat d’embryons de drosophiles ou encore dans les ovocytes de Xénope. Le mécanisme moléculaire de stimulation de l’expression par les miARN est encore mal connu mais requiert l’absence de queue poly(A) en 3’ des ARN cible et de TNRC6 au sein du complexe RISC. Le Virus de l’Hépatite C (VHC) possède en 5’ de son ARNg un site d’entrée interne du ribosome (IRES) qui recrute la petite sous-unité ribosomique 40S, sans nécessiter la reconnaissance de la coiffe par eIF4F, ni la protéine PABP, ni le balayage de la 5’UTR par le ribosome. Ces caractéristiques singulières nous ont conduits à rechercher l'impact du complexe RISC fixé en 3’ de l’ARNm sur l’initiation de la traduction du VHC. Pour cela, nous avons utilisé des transcrits contenant l'IRES du VHC en 5' et des sites d’hybridation du miARN let-7 en 3’. Ces ARNm ont ensuite été transfectés dans des lignées cellulaires hépatocytaires, ou non. A notre grande surprise, nous avons observé que la fixation du miARN let-7 sur la région 3' du transcrit stimulait fortement l’expression dirigée par l’IRES de VHC. Toutefois, l’augmentation de l’expression n’est pas due à la stabilisation du transcrit mais bien à une hausse significative de la synthèse protéique indépendamment d’un quelconque effet de miR-122. En utilisant d’autres IRES dites 'HCV-like', nous avons pu confirmer ces résultats et démontrer que, l’ajout d’une queue poly(A) en 3’ du transcrit, capable de fixer la PABP, annule cet effet stimulateur suggérant que l’absence de cette protéine est nécessaire pour que le complexe RISC stimule la traduction du VHC. / MicroRNAs (miRNAs) are small non coding RNAs which control gene expression by recognizing and hybridizing to a specific sequence generally located in the 3’UTR of targeted messenger RNA (mRNA). miRNAs serve as a guide for the RNA-Induced Silencing Complex (RISC) that is composed by, at least, the Argonaute proteins and TNRC6. Recent studies have suggested that translation inhibition occurs first and is then followed by deadenylation and degradation of the targeted transcript. The miRNA-induced inhibition of protein synthesis occurs at the level of translation initiation during the ribosomal scanning step and it requires the presence of both the initiation factor eIF4G and the poly(A) Binding Protein (PABP). In this process, the RISC interacts with both PABP and 43S pre-initiation complex (composed by initiation factors and ribosome) and it results in the disruption of linear scanning of the ribosome along the 5’ Untranslated Region (5’UTR). In some specific cases, the binding of miRNAs to their target sequences can upregulate translation initiation. This has notably been demonstrated in G0 quiescent cells, drosophila embryos and Xenopus oocytes. Although the molecular mechanism by which upregulation occurs remains to be precisely determined, it appears that the absence of a poly(A) tail and the lack of availability of the TNRC6 proteins are amongst the major determinants. In the particular case of the Hepatitis C Virus (HCV), the genomic RNA is uncapped and non polyadenylated and harbors an Internal Ribosome Entry Site (IRES) which directly binds to the ribosome with no need for cap-recognition, PABP binding and ribosome scanning. These peculiar features of the HCV IRES prompted us to investigate how viral translation can be regulated by the miRNA machinery. In order to do that, we have used a mRNA that contains the HCV IRES in 5’ and 4 let-7 binding sites in its 3’ extremity. To most of our surprise, we have observed a strong stimulation of the expression of the HCV IRES when the construct is bearing the let-7 sites. This effect is not due to any interference with the miR-122 binding sites although the magnitude of stimulation reached the same level. Our data show that it is the presence of the RISC on the 3' end of the transcript that can stimulate internal ribosome entry at the 5' end. By using other HCV-like IRESes, we could confirm these data and further showed that the absence of a poly(A) tail was an absolute requirement for the stimulation to occur. These effects are not due to an increase of mRNA stability and are rather exerted at the level of translation.

Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs) together with the small ribosomal subunit to locate the mRNA's translational start and to properly decode the genetic message that it carries. The largest of these factors, eIF3, forms the scaffold for other initiation factors to promote their spatially coordinated placement on the ribosomal surface. It is our long-standing pursuit to map the 40S-binding site of the yeast multisubunit eIF3 and here we present three new mutual interactions between these two macromolecules (i) The C-terminal region of the eIF3c/NIP1 subunit is comprised of the conserved bipartite PCI domain and we show that a short C-terminal truncation and two clustered mutations directly disturbing the PCI domain produce lethal or slow growth phenotypes and significantly reduce amounts of 40S-bound eIF3 in vivo. The extreme C-terminus directly interacts with small subunit ribosomal protein RACK1/ASC1, which is a part of the 40S head, and, consistently, deletion of ASC1 impairs eIF3 association with ribosomes. The PCI domain per se shows strong but unspecific binding to RNA, for the first time implicating this protein fold in protein-RNA interactions. We conclude that the c/NIP1...

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Translation initiation is a complex process which results in the assembly of the elongation competent 80S ribosome from the 40S and 60S ribosomal subunits, the initiator tRNA and mRNA, and is orchestrated by numerous eukaryotic initiation factors (eIFs). Although it represents one of the most regulated processes of gene expression, the exact mechanism of one of the key steps of translation initiation - mRNA recruitment to the 43S pre-initiation complex (PIC) - is still only poorly understood. Recent studies indicated that besides eIF4F and poly(A)-binding protein, also eIF3 might play an important, if not crucial, role in this step. In our laboratory, we recently identified a 10 Ala substitution (Box37) in the a/TIF32 subunit of Saccharomyces cerevisiae eIF3, which interfered with translation initiation rates. Detailed analysis showed that this mutation significantly reduces the amounts of model mRNA in the gradient fractions containing 48S PICs as the only detectable effect in vivo. Moreover, a recently solved crystal structure of the N-terminal part of a/TIF32 pointed to two Box37 residues, Arg363 and Lys364, both proposed to contribute to one of the positive, potentially RNA-binding areas on the a/TIF32 surface. The fact that also their substitutions with alanines severely impaired the mRNA recruitment...

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.

In eukaryotic translation, eukaryotic initiation factors (eIFs) are at least as important as the ribosome itself. Some of these factors play different roles throughout the entire process to ensure proper assembly of the preinitiation complex on mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most important factor integrating signals from others and coordinating their functions on the ribosome is eIF3. In Saccharomyces cerevisiae, eIF3 is formed by five subunits. All these subunits contain structural motifs responsible for contact with ribosomal proteins and RNAs. In addition to these highly structured parts, the rest of eIF3 is unstructured and very flexible. Therefore, despite the recent progress thanks to the use of a cryo-electron microscopy, a precise structure and position of eIF3 on the 40S ribosomal subunit are still not known. Also, the presence of eIF3 on 80S during early elongation and its role in reinitiation and readthrough are not fully understood. In order to crack mysteries of yeast eIF3, we used x-ray crystallography, chemical cross- linking coupled to mass spectrometry, and various biochemical and genetic assays. We demonstrated that eIF3 is very compactly packed when free in solution. This...

Eukaryotic translation initiation factor eIF4E1 (eIF4E1) plays a pivotal role in the control of cap-dependent translation initiation, occurs in P- bodies and is important for the formation of stress granules (SG). Human cells encompass two other non-canonical translation initiation factors capable of cap binding although with a lower affinity for the cap: eIF4E2 and eIF4E3. Here, I investigated the ability of individual eIF4E family members and their variants to localize to SGs and P-bodies in stress-free, arsenite and heat shock conditions. Under all tested conditions, both eIF4E1 and eIF4E2 proteins and all their variants localized to P-bodies unlike eIF4E3 protein variants. Under both arsenite and heat stress conditions all tested variants of eIF4E1 and the variant eIF4E3-A localized to SGs albeit with different abilities. Protein eIF4E2 and all its investigated variants localized specifically to a major part of heat stress-induced stress granules. Further analysis showed that approximately 75% of heat stress-induced stress granules contain all three eIF4Es, while in 25% of them eIF4E2 is missing. Large ribosomal subunit protein L22 was found specifically enriched in arsenite induced SGs. Heat stress-induced re- localization of several proteins typical for P-bodies such as eIF4E2, DCP-1, AGO-2...

From RNA-to-protein, translation initiation and protein synthesis is mediated by trans-acting factors that recognize mRNA features common to almost all eukaryotes. Eukaryotic translation initiation factor 3 complex (eIF3) is a highly conserved protein complex that recognizes 5'-CAP elements of the mRNA to initiate translation. eIF3 consists of nine subunits, three of them having two isoforms: eIF3A, eIF2B1, eIF3B2, eIF3C1, eIF3C2, eIF3D, eIF3E, eIF3F, eIF3G1, eIF3G2, eIF3H and eIF3K. This work deals with functional characterization, expression and subcellular localization of eIF3B1, eIF3B2 and eIF3E in Arabidopsis thaliana male gametophyte and interaction of eIF3E with the Constitutive photomorphogenesis 9 (COP9) complex as a regulatory complex of eIF3E post-translational control. Here we show that depletion of eif3b1 or eif3b2 is not gametophytic lethal and that the two protein might function redundantly, whereas, knockout of eIF3E causes male gametophyte lethality. Interestingly, eif3b1 show post-fertilization defects during embryogenesis, suggesting that its redundancy with eIF3B2 is restricted to the gametophyte. Gene expression studies revealed high expression of eIF3 subunits in actively dividing zones of leaf primordia, root meristem and root elongation zones as well as in the vegetative...