Protein synthesis is a fundamental and ultimate process in living cells. Cells possess sophisticated machineries and continuously carry out complex processes. Monitoring protein synthesis in living cells not only inform us about the mechanism of translation but also deepen our insights about all aspects of life. Understanding the structure and mechanism of the ribosome and its associated factors helped us enlarge our knowledge on protein synthesis. Recently, with the dramatic advances of high-throughput sequencing and bioinformatics, a new technique called ribosome profiling emerged. By retrieving mRNA fragments protected by translating ribosomes, ribosome profiling reveals global ribosome occupancy along mRNAs in living cells, which can inform us with the identity and quantity of proteins being made. Easily adapted to other organisms, ribosome profiling technique is expanding its application in revealing various cellular activities as well as the knowledge on protein synthesis. Here, we report the mechanism of translating mRNA cleavage by endoribonuclease RelE in vivo. RelE is an endoribonuclease that is induced during nutrient deficiency stress and specifically cleaves translating mRNAs upon binding to the ribosomal A site. Overexpression of RelE in living cells causes growth arrest by inhibiting global translation. We monitored RelE activity in vivo upon overexpression using ribosome profiling. The data show that RelE actively cuts translating mRNAs whenever the ribosomal A site is accessible, resulting in truncated mRNAs. RelE causes the ribosome complexes to accumulate near the 5' end of genes as the process of ribosome rescue, translation, and cleavage by RelE repeats. RelE cleavage specific sub-codon level ribosome profiling data also represent reading frame in Escherichia coli and sequence specificity of RelE cleavage in vivo. We report another ribosome profiling study on a methyltransferase TrmD in E. coli. TrmD is known to methylate G37 (the residue at 3' side of anticodon) of some tRNAs and be responsible for codon-anticodon interaction. We constructed a TrmD depletion E. coli strain, whose deletion results in lethality of cells. Resulting depletion of m1G37 in the strain leads to growth arrest. Lack of m1G37 of some tRNAs whose codons start with C showed frequent frameshift when translating the gene message in vitro. By using ribosome profiling, we successfully observed significant difference on translation process when codons interact with anticodons of tRNAs lacking m1G37. The data reveal slow translation rate or pauses on the tRNAs when missing the appropriate methylation, which corresponds to the previous biochemical data in vitro.

La traduction est considérée comme une étape clé de l'expression des gènes permettant à la cellule de s'adapter aux variations de son environnement en réponse aux signaux internes ou externes. Des études bioinformatiques ont montrés que la moitié des ARN messagers chez l'homme portent, en amont de leur phase codante, des éléments régulateurs appelés uORF. Le laboratoire a montré qu'un défaut de terminaison de la traduction par déplétion du facteur de terminaison eRF3 modifie l'expression de gènes dont l'ARNm contient des uORF comme le gène ATF4. Cette modification se fait soit par un mécanisme de réinitiation après traduction de l'uORF soit par une augmentation de la stabilité de l'ARNm résultant d'un défaut de sa dégradation par la voie du "Nonsense-mediated mRNA Decay" (NMD). A travers leur association dans le même complexe et leur implication dans la terminaison de la traduction et la NMD, eRF3 et Upf1 contribuent à la régulation fine de l'expression des gènes. Cependant, on ne sait pas dans quelle mesure ces deux facteurs affectent la traduction et la stabilité des ARNm. Nous avons évalué la traduction par ribosome profiling et le taux de transcrits par RNA-seq dans les cellules humaines déplétées en eRF3 ou en Upf1. Ces analyses nous ont permis de dresser une carte des uORF traduites dans le transcriptome des cellules humaines HCT116. Nous avons également observé que peu de gènes cibles sont communs entre la déplétion en eRF3 ou en Upf1. Nos résultats appuient fortement l'hypothèse qu'il y a au moins deux classes de transcrits portant des uORF, l'une dont la régulation implique la terminaison de la traduction et l'autre dont la régulation implique la NMD. / Regulation of gene expression at the translational level is increasingly being recognized as a key mechanism by which cells can rapidly change their gene expression pattern in response to internal or external stimuli. Bioinformatic studies revealed that half of human transcripts present at least one expression regulatory element uORF in the 5’ leader sequence preceding the main ORF. We have previously shown that translation termination disruption caused by eRF3a depletion induces upregulation of the transcriptional activator ATF4 and its targeted genes partly by a translational control at uORFs, and partly in relation to a defect in Nonsense-mediated mRNA Decay activation, increasing ATF4 mRNA stability. Through their physical association and their involvement in translation termination and NMD, eRF3 and Upf1 are regulating the protein and mRNA levels of a significant number of genes and thus contribute to the fine-tuning of their expression. It is not known yet, in what extent both of these factors affect translational control and what is the subset of genes that are regulated by these factors. In this study, we evaluated translation by ribosome profiling and mRNA level by RNA-seq in human cells subjected to either eRF3a or Upf1 depletion. These analyses allowed us to draw a transcriptome-wide map of uORFs and obtained a list of functional uORFs in our reference HCT116 transcriptome. We also observe that only a small fraction of these are common targets for both eRF3a and Upf1. Our results provide strong support for the notion that different classes of transcripts bearing uORFs are regulated either by translational processes involving translation termination or by NMD.

Submitted by Karin Goebel (karing@fiocruz.br) on 2014-11-25T18:05:56Z No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) / Approved for entry into archive by Karin Goebel (karing@fiocruz.br) on 2014-11-25T18:06:29Z (GMT) No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) / Made available in DSpace on 2014-11-25T18:06:29Z (GMT). No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / As células-tronco (CTs) caracterizam-se por possuírem a capacidade de se autorrenovar e de dar origem a um ou mais tipos celulares diferenciados. Nos últimos anos, diversos trabalhos mostraram a existência de CTs em tecidos adultos, tornando-as uma alternativa interessante para uso em terapias celulares. Contudo, para melhor utilizar as CTs, é preciso primeiramente compreender como ocorre a diferenciação em um tipo celular específico e, principalmente, como é regulada a expressão gênica durante este processo. Em 2009, Ingolia e colaboradores apresentaram uma nova técnica conhecida como ribosome profiling, a qual consiste no isolamento e sequenciamento em larga escala dos fragmentos de RNA associados e protegidos pelos ribossomos, os quais têm um tamanho aproximado de 30 nucleotídeos (conhecido com footprint ribossomal). Ao mapear as sequências obtidas, é possível obter informações não apenas sobre quais sequências estão sendo traduzidas, mas também sobre a cinética da tradução e sua extensiva rede de regulação. Assim, o objetivo deste trabalho foi aplicar a técnica de ribosome profiling ao estudo do processo de diferenciação de CTs adultas. Como modelo de estudo, foram utilizadas CTs obtidas de tecido adiposo antes (t=0) e após a indução para diferenciação adipogênica por 3 dias (t=72h). O primeiro passo do trabalho foi a adaptação do protocolo de ribosome profiling para o estudo de CTs adultas, o qual consiste na lise celular, digestão do lisado com uma RNA nuclease (a qual irá degradar o RNA exposto, preservando os fragmentos protegidos pelo ribossomo), ultracentifrugação do homogenato sobre colchão de sacarose 1 M para sedimentação dos ribossomos, extração de RNA e isolamento dos fragmentos de 30 nucleotídeos. Também foi feita extração do RNA poliA. As amostras foram sequenciadas (SOLiD™) e os dados obtidos foram triados e mapeados contra um banco de dados de RNAm, utilizando-se a ferramenta CLC Genomics Workbench. Foram identificados mais de 8.000 transcritos para as amostras de ribosome profiling e mais de 17.000 para as de poliA. Ao calcular o fold change entre as condições t=0 e t=72h, foi possível verificar que mais de 50% dos genes foram detectados como diferencialmente expressos apenas por ribosome profiling. Observou-se que genes relacionados com vias de diferenciação adipogênica e de metabolismo de lipídeos encontravam-se regulados positivamente em ambas as amostras de RNA. Por outro lado, observou-se que vias de regulação do citoesqueleto de actina e de adesão focal estavam reguladas negativamente apenas nas amostras de ribosome profiling. Isso é interessante, uma vez que a inibição destas vias já foi descrita como importante para o processo de adipogênese. Além disso, foi observada uma forte redução na eficiência de tradução de genes relacionados com a tradução após 72 horas de indução para diferenciação. Os resultados obtidos no presente trabalho reforçam as evidências de que os mecanismos de regulação pós-transcricionais e traducionais têm um papel muito importante na regulação da diferenciação celular de CTs, sendo que a técnica de ribosome profiling permitiu obter informações mais detalhadas de como este processo pode estar acontecendo. / Stem cells (SC) are characterized by their capacity of both self-renewing and giving rise to new differentiated cells. SC are found in adult tissues, which are considered a putative source for cell therapy. However, little is known about the mechanisms involved in the trigger of SCs differentiation into a specific cell type. Understanding adult SCs differentiation process is a fundamental step to better use and to take advantage of their potential. In 2009, Ingolia and collaborators presented a new methodology of transcriptome analysis named ribosome profiling, which consists on the isolation and deep-sequencing of the mRNA fragments enclosed by ribosomes. When lysed cells are submitted to nuclease digestion, unprotected mRNA is degraded, while fragments within ribosomes are preserved and have a known footprint of 30 nucleotides. Sequencing these ribosome-protected fragments results in a high-precision measurement of in vivo translation, providing precise information about translation kinetics and its extensive regulation. The objective of this work was to apply the ribosome profiling methodology to the study of adipogenic differentiation in adult SCs. SCs were isolated from human adipose tissue from three donors and were cultured in a control medium (t=0) and induced to adipogenic differentiation for 72 hours (t=72h). The first step was to adapt and optimize the ribosome profiling protocol to the SC model, which consists in cell lysis, cell lysate digestion by nuclease (to degrade unprotected RNA, preserving ribosome-protected fragments), ultracentrifugation over a 1M sucrose cushion to pellet ribosomes, RNA extraction and 30 nucleotides fragments isolation. poliA RNA was also isolated. Samples were submitted to deep-sequencing (SOLiD™) and the reads obtained were trimmed and mapped onto the reference mRNA database using the CLC Genomics Workbench. Over 8000 transcripts were identified in ribosome profiling samples and over 17000 in poliA samples. Fold change analysis between t=0 and t=72h of both RNA samples showed that differential expression of more than 50% of the genes was identified only by ribosome profiling. Pathways related to adipogenesis and lipid metabolism were upregulated in both RNA samples. However, regulation of the actin cytoskeleton and focal adhesion proteins were downregulated only in ribosome profiling samples. Interestingly, downregulation of these pathways was already described as an important phenomenon to cell adipogenesis. Besides, we observed a strong reduction of translational efficiency of genes involved in translation at t=72h. Our results reinforce previous data, suggesting that posttranscriptional and translational regulation play a fundamental role in the regulation of SC differentiation process and that ribosome profiling is an important tool to better understand this process.

Covalent nucleotide modifications in RNAs affect numerous biological processes, and novel functions are continually being revealed even for well-known modifications. Among all RNA species, transfer RNAs (tRNAs) are highly enriched with diverse modifications, which are known to play roles in decoding and tRNA stability, charging, and cellular trafficking. However, studies of tRNA modifications have been limited in a small scale and performed by groups with different methodologies. To systematically compare the functions of a large set of noncoding RNA modifications in translational regulation, I carried out ribosome profiling in 57 budding yeast mutants lacking nonessential genes involved in tRNA modifications. Deletion mutants with enzymes known to modify the anticodon loop or non-tRNA substrates such as rRNA exhibited the most dramatic translational perturbations, including altered dwell time of ribosomes on relevant codons, and altered ribosome density in protein-coding regions or untranslated regions of specific genes. Several mutants that result in loss of tRNA modifications in locations away from the anticodon loop also exhibited altered dwell time of ribosomes on relevant codons. Translational upregulation of the nutrient-responsive transcription factor Gcn4 was observed in roughly half of the mutants, consistent with the previous studies of Gcn4 in response to numerous tRNA perturbations. This work also discovered unexpected roles for tRNA modifying enzymes in rRNA 2’-O-methylation, and in transcriptional regulation of TY retroelements. Taken together, this work revealed the importance and novel functions of tRNA modifications, and provides a rich resource for discovery of additional links between tRNA modifications and gene regulation.

Ribosome profiling (RP) is a novel technique that exploits RNA sequencing and ribosome immobilization to quantify transcription and translation at different cell growth stages. Therefore, RP provides invaluable information for expression dynamics studies. Quantitative –omics studies are of crucial importance for identification of potential biomarkers of infection. An ideal parasite detection system should definitely establish the presence or absence of infection; determine the species involved; be detectable even in low concentrations; be proportional to parasite density; and determine the presence of antibiotic resistance. Here, we propose a simple workflow that attempts to identify a set of biomarkers that fulfill some of the above criteria for the ideal detection system. RP expression profiles were ranked for abundance, crosschecked with PlasmoDB for homogeneity along infection cycles and probed for availability of structural stability. The latter is of fundamental importance for the development of molecular biosensors to be give birth to rapid diagnostic kits. In addition, a simple biochemistry workflow was developed for easy production of the selected biomarkers in E. coli. Altogether, the present work provides two complementary and novel workflows that shall aid researchers to rapidly produce molecular biomarkers and develop biosensors based on antibodies or aptamers. / Tesis

Mutations in TSC2 cause the disorder tuberous sclerosis (TSC), which has a high incidence of autism and intellectual disability. TSC2 regulates mRNA translation required for group 1 metabotropic glutamate receptor-dependent synaptic long-term depression (mGluR-LTD), but the identity of mRNAs responsive to mGluR-LTD signaling in the normal and TSC brain is largely unknown. We generated Tsc2+/- mice to model TSC autism and performed ribosome profiling to identify differentially expressed genes following mGluR-LTD in the normal and Tsc2+/- hippocampus. Ribosome profiling reveals that in Tsc2+/-mice, RNA-binding targets of Fragile X Mental Retardation Protein (FMRP) are increased. In wild-type hippocampus, induction of mGluR-LTD caused rapid changes in the steady state levels of hundreds of mRNAs, many of which are FMRP targets. Moreover, mGluR-LTD signaling failed to promote phosphorylation of eukaryotic elongation factor 2 (eEF2) in Tsc2+/- mice, and chemically mimicking phospho-eEF2 with low cycloheximide enhances mGluR-LTD in the Tsc2+/- brain. These results suggest a molecular basis for bidirectional regulation of synaptic plasticity by TSC2 and FMRP. Furthermore, deficient mGluR-regulated translation elongation contributes to impaired synaptic plasticity in Tsc2+/- mice.

Translation control is crucial during virus-host interaction, in which the host relies on the translation machinery to mount an antiviral response or induce the inflammation response to reduce virus spread, while the virus aims to take control of this system to thwart the host defense while producing viral progeny. The field of oncolytic virus (OV) therapy relies on replicating, engineered viruses that preferentially infect tumor cells to induce direct oncolysis or promote an antitumor immune response. Despite the importance of translation control in virus-host interaction, not much has been described on the interaction at the translation level between OV and cancer cells. I propose that this knowledge gap could reveal significant improvements in OV efficacy in treating cancer. In my first study, I set out to characterize the translatome of an infection-resistant breast cancer cell line infected by three clinically advanced OVs to identify residual antiviral activity in cancer cells regulated by translation control. I found the inositol phosphatase Inpp5e to be a novel antiviral gene that is translationally induced during infection via a transcript variant shift. Mechanistically, I showed that the majority of Inpp5e transcripts in uninfected cells contain a long 5’ UTR that harbor four translationally inhibitory upstream reading frames (uORF). Yet, OV infection induced the expression of a shorter 5’ UTR with a spliced intron that removes three uORFs, derepressing the translation of Inpp5e mRNA. CRISPR-Cas9 knockout of Inpp5e also enhanced the infectivity of oncolytic HSV1 and VSV. My study suggests the existence of a class of translationally regulated antiviral genes in cancer cells. In my second study, I sought to adapt the translation of transgenes to the unique translation condition imposed by the infecting virus via the incorporation of a viral 5’UTR. I identified HSV1 5’UTRs by locating the transcription start site of most HSV1 genes using RNA-seq data, then determined the 5’UTR of US11 as a potent translation enhancer during HSV1 infection. Incorporation of this 5’UTR into the transgene expression cassette inserted into the HSV1 genome enhanced transgene expression significantly at the translation level. In my third study, I set out to explore the mechanism of miR-223 mediated inflammation inhibition. miR-223 is a protective miRNA in the context of atherogenesis via suppressing inflammatory signaling. Using transcriptome and translatome profiling (RNA-seq and Ribo-seq), I found that the inhibitory effect of miR-223 on inflammation occurs primarily at the translation level. Overall, my work highlights the importance of translation control in OV-cancer cells interaction, as well as in inflammation-related diseases.

Sex differences are widespread in both mouse and human liver, and are associated with sex differences in drug metabolism and liver pathophysiology. The secretory patterns of growth hormone (GH) is one of the major drivers of liver sex specificity, where intermittent and continuous secretion in male and female respectively lead to sex bias in the expression of more than 1000 genes in mouse liver, via a complex interplay of GH-responsive transcription factors and epigenetic mechanisms. This thesis explores three themes of molecular control in the regulation of liver sex differences: microRNAs, DNA methylation, and translational control. Studies herein identified two microRNAs, miR-1948-5p and miR-802-5p, whose expression is sex biased and regulated by GH and the transcription factor STAT5b. Small RNA sequencing confirmed the sex specificity of these two microRNAs and identified an additional 18 sex-biased microRNAs. Computational and experimental characterization of miR-1948-5p and miR-802-5p confirmed their authenticity. In vivo inhibition of these microRNAs by locked nucleic acids indicated that miR-1948-5p and miR-802-5p played a functional role in repressing female-biased genes and male-biased genes, respectively. This thesis also investigated the impact of GH and STAT5b on liver DNA methylation profiles. Reduced representation bisulfite sequencing was performed on liver tissues from four mouse models that perturbed the GH and STAT5b axis. In the wildtype liver, sex biased demethylation was positively associated with sex biased chromatin opening and gene expression. Global hypermethylation was observed in livers of mice with lit/lit mutation resulting in GH deficiency or with hepatocyte-specific deletion of the STAT5ab locus. Strikingly, these hypermethylated loci were enriched for enhancer elements and STAT5b binding sites found in wild-type mouse liver. Hypophysectomy followed by GH replacement mouse models identified differentially methylated regions that were sex-biased and rapidly methylated and demethylated in response to GH stimulation. Finally, we used ribosome profiling to validate sex-biased protein translation and identify mechanisms of translational control. In sum, this body of work provides novel insights and broadens our understanding of the diverse molecular mechanisms underlying sexual dimorphism in the liver. / 2020-10-08T00:00:00Z

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京都大学 / 新制・課程博士 / 博士(理学) / 甲第24462号 / 理博第4961号 / 新制||理||1708(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 青山 卓史, 教授 高田 彰二, 准教授 遠藤 寿 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM