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The role of translation initiation factor 3 (eIF3) in translation termination.
Beznosková, Petra ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Staněk, David (referee)
Protein synthesis is a tightly regulated process of gene expression. Each gene has its start and its stop, which is determined by one of the three stop codons. Many recent articles describe ribosomes that purposely bypass stops on specific mRNAs to extend the nascent polypeptide to alter its properties. It is called programmed stop codon readthrough. Since over 15% of human genetic diseases are caused by so called premature termination codons (PTC) that halt translation and produce truncated proteins, this mechanism has a great potential implication in medical research. Numerous labs search for non-toxic drugs specifically increasing readthrough at PTCs; however, the success of this effort requires identification and understanding of all factors that are involved in this process. Here, we present one such factor eukaryotic initiation factor 3 (eIF3) and describe its ability to induce readthrough on stop codons in termination non-favorable context during programmed readthrough and also the consequences of its action on translation regulation. We additionally analyzed which near-cognate (nc) tRNAs are incorporated at UGA stop codons depending on the nucleotide that immediately follows them (so called +4 base). This way we established new rules for stop codon decoding and identified so called...
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Characterization of the molecular mechanism of translation reinitiation in yeast.
Pondělíčková, Vanda ; Valášek, Leoš (advisor) ; Hašek, Jiří (referee) ; Vopálenský, Václav (referee)
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...
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Mapping the contact points between eukaryotic translation initiation factor eIF3 and the 40S ribosomal subunit.
Kouba, Tomáš ; Valášek, Leoš (advisor) ; Pospíšek, Martin (referee) ; Staněk, David (referee)
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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The role of the N-terminal domain of the a/TIF32 subunit of eIF3 in mRNA recruitment to the 43S pre-initiation complexes.
Vlčková, Vladislava ; Valášek, Leoš (advisor) ; Mašek, Tomáš (referee)
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...
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Study of translation initiation factors eIF3 and eIF4E in leukemic cell lines
Mrvová, Silvia ; Mašek, Tomáš (advisor) ; Haškovec, Cedrick (referee)
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...
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Dissection of eIF3 functional domains promoting the 48S pre-initiation complex assembly
Beznosková, Petra ; Valášek, Leoš (advisor) ; Novotný, Marian (referee)
In eukaryotes, translation initiation is guided by up to twelve protein initiation factors (eIFs) and begins with the formation of the 43S pre-initiation complex (PIC) composed of the small ribosomal subunit (40S), eIF2.GTP/Met-tRNAi Met ternary complex, and eIFs 1, 1A, 3 and 5. The 43S PIC subsequently interacts with the 5'end of an mRNA (an mRNA recruitment step) and thus formed 48S PIC travels in 5' to 3' direction along the mRNA leader sequence to locate the AUG start codon (this presumably linear movement is generally known as scanning). Start site selection results in the dissociation of the initiation factors and joining of the large (60S) ribosomal subunit to form the 80S initiation complex poised for elongation. Eukaryotic initiation factor 3 (eIF3) plays a critical role in most of these events; however, the molecular details of most of its contributions are still unknown to us. Previous in vivo studies generated numerous mutations in all eIF3 subunits with specific defects either in the PICs assembly or in the following steps such as scanning, AUG recognition, etc. To understand the exact role of eIF3 in this intriguing process at the molecular level, we have embarked on a study that aims to dissect the individual functions of each eIF3 subunit in translation initiation using the purified...
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RNAi of the a subunit of human translation initiation factor 3 (eIF3).
Peclinovská, Lucie ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Translation initiation is the first step of protein synthesis that captures the flow of gene expression pathway in all living organisms. The advantage of regulation of gene expression at the level of translation initiation is that it allows for more rapid changes in the proteome and serves as the rate limiting step under certain conditions such as stress. This process is masterminded by many initiation factors. One of them, a multisubunit eukaryotic initiation factor 3 (eIF3), is a very efficient player in this field taking a part in the most of the initiation steps. The largest subunit of the eIF3 complex is called eIF3a p170 and TIF32 in mammals and yeast, respectively, and at least in yeast, it was shown to represent an essential constituent of the translational machinery. This work is based on all that has been learned about the eIF3a roles in translation initiation in the model organism of yeast Saccharomyces cerevisiae in effort to examine the degree of the functional conservation with its human ortholog. This is achieved by the RNAi-mediated knock-down of eIF3a in HeLa and HEK cell lines followed by variety of well established assays to monitor translational status of eIF3a depleted cells. In the first part, I describe optimization of the RNA interference protocol with respect to the choice...
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Translation reinitiation mechanism on mRNA of trascriptional activator GCN4.
Vlčková, Vladislava ; Valášek, Leoš (advisor) ; Strachotová, Dita (referee)
Translation reinitiation is a gene-specific translational control mechanism exploiting the ability of some short upstream open reading frames (uORFs) to retain post-termination 40S ribosomal subunit on the mRNA. Reinitiation efficiency depends on cis-acting sequences surrounding the uORF, translation elongation rates on the uORF, selected initiation factors, and the intercistronic distance of the short uORF from the main ORF. Although the precise mechanism of reinitiation is still not known, great progress in elucidating some of its details has been recently made with help of the GCN4 translational control model system. Among them, involvement of eIF3 was shown to play a critical role for efficiency of this process. In particular, it was proposed that eIF3 specifically interacts with sequences located upstream of a reinitiation-permissive uORF upon termination, and that this step is instrumental in stabilizing the 40S ribosomal subunit on the mRNA to allow subsequent resumption of scanning for reinitiation downstream. In this thesis, the current knowledge of the translation reinitiation mechanism is summarized. As a typical example, the yeast transcriptional activator GCN4 has been chosen, the mRNA of which is subjected to a tight translational control via the very reinitiation mechanism.
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The role of eIF3 in mRNA recruitment to the 43S pre-initiation complex
Beznosková, Petra ; Mašek, Tomáš (referee) ; Valášek, Leoš (advisor)
Translation initiation in eukaryotes plays an important role in gene expression and relies on interactions between many eukaryotic initiation factors, small and large ribosomal subunits, mRNA and initiator tRNA. The largest of these initiation factors, the eukaryotic initiation factor 3 (eIF3), participates in the most of translation initiation reactions. In yeast, eIF3 occurs together with eIF1, eIF5 and ternary complex (TC, eIF2-GTP-Met-tRNAi Met complex) in the multifactor complex (MFC) and promotes formation of the 43S preinitiation complexes. eIF3 also stimulates mRNA recruitment to the 43S preinitiation complexes, scanning the 5' untranslated region of mRNA for AUG recognition and has an important role in the gene-specific translational control mechanism called reinitiation involving short upstream open reading frames (uORFs).
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C-konc. segment NIP1/eIF3c a HCR1 subjednotky eIF3 umožňují jeho vazbu na 40S ribozóm přes RPS33/ASC1 a RPS22
Rutkai, Edit ; Herrmannová, Anna ; Szamecz, Bela ; Valášek, Leoš
One of the rst critical steps of protein synthesis is the recruitment of the eIF2.GTP.Met-tRNAiMet ternary complex (TC) to the 40S ribosome. In yeast, the TC occurs together with eIFs 1, 3 and 5 in a Multifactor complex (MFC) that was shown to function as an important intermediate of the initiation pathway. Our previous study determined several critical domains of the TIF32 and NIP1 subunits of yeast eIF3, the deletion of which, in the background of a wild type gene, signifcantly aected binding of the mutant MFC to the 40S ribosomes. Subsequent identifcation of the two contact points between TIF32 and components of the 40S ribosome enabled us to propose that the major body of the eIF3 complex resides under the head region on the solvent side of the small ribosomal subunit facing down towards its left foot
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