京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21654号 / 医博第4460号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 朝長 啓造, 教授 妹尾 浩, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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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...

Translation is the process by which proteins are synthesized from the instructions in the genetic code. Translation is mediated by the ribosome, a large ribonucleoprotein complex, in concert with messenger RNA (mRNA), transfer RNA (tRNA), and a variety of proteins. The canonical mechanism of translation, introduced in Part I of my thesis, is divided into four distinct phases: initiation, elongation, termination, and recycling. Under unusual circumstances, each phase of translation can also proceed via a number of noncanonical mechanisms, many of which are vitally important for cellular growth or viral infectivity. My thesis describes structural insights into two such noncanonical mechanisms. The aim of the first project, described in Part II, was to structurally characterize a noncanonical mechanism of translational termination in bacteria. In the absence of a stop codon, ribosomes arrest at the 3′ end of an mRNA and are unable to terminate. In bacteria, the primary mechanism for rescuing such nonstop complexes is known as trans-translation. In the absence of a functional trans-translation system, however, the small protein ArfA recognizes the empty mRNA channel and recruits the release factor RF2 to the ribosome, enabling termination to occur. Using single-particle electron cryomicroscopy (cryo-EM), I obtained four high-resolution structures of nonstop complexes that reveal the mechanism of ArfA-mediated ribosome rescue and have wider implications for understanding canonical termination in bacteria. The aim of the second project, described in Part III, was to gain structural insights into a noncanonical mechanism of translational initiation in eukaryotes known as internal ribosome entry. Instead of a 5′ cap, many viruses contain intricately structured, cis-acting internal-ribosome-entry sites (IRESs) within their genomes that direct end-independent initiation. The IRES of hepatitis-C virus (HCV), for example, interacts directly with the mammalian ribosome and functionally replaces many of the canonical initiation factors. However, the mechanism by which the HCV IRES coordinates assembly of an initiation complex and progresses through the initiation phase remains poorly understood. I developed a method for purifying native ribosomal complexes from cell lysate that enabled me to obtain multiple cryo-EM maps of the HCV IRES in complex with the 80S ribosome, including a previously unseen conformation of the IRES induced by rotation of the ribosomal small subunit, and to make progress towards capturing earlier steps in the initiation pathway.

Protein synthesis is a complex process performed by sophisticated cellular particles known as ribosomes. Although RNA constitutes the major structural and functional component, ribosomes from all kingdoms contain an extensive array of proteins with largely undefined functional roles. The work presented in this thesis addresses ribosomal complexity using mutants of Salmonella typhimurium to examine the physiological effects of ribosomal protein (r-protein) removal and orthologous replacement on bacterial fitness and ribosome function. The results of paper I demonstrate that removal of small subunit protein S20 conferred two independent translation initiation defects: (i) a significant reduction in the rate and extent of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in subunit association. The topographical location of S20 in mature 30S subunits suggests that these perturbations are the result of improper orientation of helix 44 of the 16S rRNA when S20 is absent. In paper II we show that the major functional impairment associated with loss of large subunit protein L1 manifested as an increase in free ribosomal subunits at the expense of translationally active 70S particles. Furthermore, the formation of free ribosomal subunits was imbalanced suggesting that L1 is required to suppress degradation or promote formation of 30S subunits. Compensatory evolution revealed that mutations in other large subunit proteins mitigate the cost of L1 removal, in one case seemingly via an increase in 70S complex formation. As shown in paper III, the large fitness costs associated with complete removal of r-proteins is in contrast to the generally mild costs of orthologous protein replacement, even in the absence of a high degree of homology to the native protein. This clearly demonstrates the robustness and plasticity of the ribosome and protein synthesis in general and it also implies that functional constraints are highly conserved between these proteins. The findings of paper III also allowed us to examine the barriers that constrain horizontal gene transfer and we find that increased gene dosage of the sub-optimal heterologous protein may be an initial response to stabilize deleterious transfer events. Overall the results highlight the requirement of r-proteins for the maintenance of ribosomal structural integrity.

Title from second page of PDF document. Includes bibliographical references (p. Xx-Xx).

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-10T16:16:48Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-LarissaNascimento.pdf: 5009861 bytes, checksum: e41e4e6a4cc83034688c263b000766c1 (MD5) / Made available in DSpace on 2017-04-10T16:16:48Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-LarissaNascimento.pdf: 5009861 bytes, checksum: e41e4e6a4cc83034688c263b000766c1 (MD5) Previous issue date: 2012-03-13 / O gênero Leishmania compreende 30 espécies de protozoários flagelados pertencentes a famíla Trypanosomatidae, donde 20 são patogênicas ao homem. Esses organismos apresentam ciclos de vida complexos e peculiaridades moleculares frente à maioria dos eucariontes, como a ausência de regulação transcricional. Desse modo, a regulação da expressão gênica nesses parasitas é efetuada em etapas pós-transcricionais, dentre essas a mais importante é o processo de iniciação da tradução dos mRNAs, onde diferentes fatores denominados eIFs (eukariotic initiation factors) estão envolvidos. Dentre esses fatores se destaca o complexo eIF4F com função de promover o reconhecimento e ligação de RNAs maduros aos ribossomos. Tal complexo é composto de três sub-unidades: eIF4A (RNA helicase); eIF4E (proteína de ligação ao cap); e eIF4G (proteína multidomínio estruturadora do complexo eIF4F). Em tripanossomatídeos se sabe da existência de cinco homólogos da sub-unidade eIF4G distintos (EIF4G1 ao G5), contudo, pouco se sabe sobre a ocorrência e funções celulares desses homólogos. Portanto o objetio do presente trabalho foi avaliar a expressão dos diferentes homólogos do eIF4G durante o ciclo de vida de Leishmania amazonensis, caracterizando as possíveis modificações pós-traducionais por fosforilação que possam estar agindo sobre tais fatores, uma vez que em eucariotos superiores mecanismos de regulação global da tradução por fosforilação dos eIFs via MAP quinases já são conhecidos. Para tal, foram realizadas culturas de L. amazonensis nas formas promastigota e amastigota-axênicas, e os extratos protéicos provenientes de diferentes fases do crescimento foram analisados através de Western blot. Foi observado que os homólogos de eIF4G estão presentes durante todo o ciclo de vida de L. amazonensis. Podendo ser observado que os EIF4G1, G4 e G5 apresentaram mais de uma isoforma proteica sugestiva de possíveis modificações pós-traducionais desses homólogos. Em conseguinte, a expressão de EIF4G3 e EIF4G4 foi analisada em condições especiais de cultivo na presença de seis inibidores diferentes, contudo nenhuma dessas condições alterou a expressão desses fatores, revelando que essas proteínas são bastante estáveis e possuem tempo de meia-vida prolongado. Posteriormente, o mapeamento in silico de sítios de fosforilação por MAP quinases nos EIF4Gs de Leishmania spp. demonstra a existência de sítios de fosforilação específicos em todos os homólogos E a purificação de fosfoproteínas confirma a existência de mecanismos de fosforilação agindo nos EIF4G3 e EIF4G4. Esses resultados auxiliam no esclarecimento dos mecanismos moleculares, até então obscuros, envolvidos na regulação da expressão gênica pós-transcricional característica desses organismos. / The genus Leishmania comprises 30 species of flagellated protozoa from the family Tripanosomatidae, of which 20 are pathogenic to humans. These organisms have complex life cycles and molecular particularities not observed in most eukaryotes, like absence of transcriptional regulation. Thus, the regulation of gene expression occurs in post-transcriptional steps, with the initiation of mRNA translation representing the most important event. Different factors called eIFs (eukariotic initiation factors) are involved, with emphasis in eIF4Fcomplex, which promotes mRNA recognition and its ribosome interaction. This eIF4F complex consists of three subunits: eIF4A (RNA helicase), eIF4E (cap binding protein) and eIF4G (scaffold protein, for eIF4F complex maintenance). In trypanosomatids, five eIF4G homologous (EIF4G1 to G5) was described, however, little is known about the specific cellular functions of these homologous. In this manner, we evaluated the expression and characterized post-translational modifications of the eIF4G homologous during the Leishmania amazonensis life cycle, especially phosphorylation, considering the translational regulation by phosphorylation of eIF ́s via MAP kinases observed in higher eukaryote. For this reason, cultures were performed with the L. amazonensis promastigote and amastigote axenic forms, and the protein extract, collected from different growth phases, were analyzed by Western blotting. These results demonstrated the detection of all eIF4G homologues during the life cycle of L. amazonensis, while more than one protein isoform were observed for the EIF4G1, G4 and G5 homologues, suggesting possible post-translational modifications. Posteriorly, the EIF4G3 and EIF4G4 gene expression were investigated under differential growth conditions using six distinct inhibitors, however no change was observed in the expression pattern, which suggests that these proteins are quite stable and have long half-life extending. Finally, In silico analysis shows specifics MAP kinase-dependent phosphorylation sites presents in all eIFG homologues and further analysis with EIF4G3 and EIF4G4 confirmed the existence of phosphorylation mechanisms acting on these factors.These present data help to clarify the molecular mechanisms involved in post-transcriptional regulation of gene expression characteristic of these organisms.