Dissertations / Theses on the topic 'IRES – Internal Ribosome Entry Site'

Le complexe Scc2-Scc4 est essentiel pour l’association du complexe cohésine sur l’ADN. Les proteines Cohésine génèrent la cohésion entre les chromatides sœurs, ce qui est essentiel pour la ségrégation des chromosomes. Scc2 (également connu sous le nom NIPBL) est muté chez les patients atteints du syndrome de Cornelia de Lange, une maladie multi-organique caractérisée par des anomalies du développement du visage, de la developpement mental cardiaque et du tractus gastro-intestinal. Comment les mutations localisées au niveau du gène codant pour la proteine Scc2 conduisent à des anomalies du développement chez les patients n’a pas encore été élucidé. Une des hypothèses est que la liaison de Scc2 / cohésine à différentes régions du génome a une incidence sur la transcription. Chez la levure de bière, il a été montre que Scc2 se lie aux genes transcrits par l'ARN Pol III (les ARNt et spliceosomals) , ainsi qu‘aux gènes transcrits par l'ARN Pol II codant pour des petits ARN nucléolaires et nucléaires (snARN et snoARNs ) et des gènes de protéines ribosomiques. Nous rapportons ici que Scc2 est important pour l'expression de ces gènes. Scc2 et le régulateur transcriptionnel Paf1 collaborent pour promouvoir la production de Box H / ACA snoARNs qui guident la pseudouridylation des ARN y compris l'ARN ribosomal. Une mutation de Scc2 a été associée à des défauts dans la production d'ARN ribosomal, la biogenèse des ribosomes, et del’épissage. Alors que le mutant Scc2 n'a pas de défaut général de la synthèse protéique, il montre un déphasage accrue et une réduction de l’utilisation du site interne d'entrée ribosomale (IRES)/ coiffe-indépendante. Ces résultats suggèrent que Scc2 favorise normalement un programme d'expression génétique qui prend en charge la fidélité de la traduction. Nous émettons l'hypothèse que le dysfonctionnement de traduction peut contribuer au syndrome de Cornelia de Lange, qui est causé par des mutations dans Scc2
The Scc2-Scc4 complex is essential for loading the cohesin complex onto DNA. Cohesin generates cohesion between sister chromatids, which is critical for chromosome segregation. Scc2 (also known as NIPBL) is mutated in patients with Cornelia de Lange syndrome, a multi-organ disease characterized by developmental defects in head, limb, cognition, heart, and the gastrointestinal tract. How mutations in Scc2 lead to developmental defects in patients is yet to be elucidated. One hypothesis is that the binding of Scc2/cohesin to different regions of the genome will affect transcription. In budding yeast, Scc2 has been shown to bind to RNA Pol III transcribed genes (tRNAs, and spliceosomal), as well as RNA Pol II-transcribed genes encoding small nuclear and nucleolar RNAs (snRNAs and snoRNAs) and ribosomal protein genes. Here, we report that Scc2 is important for gene expression. Scc2 and the transcriptional regulator Paf1 collaborate to promote the production of Box H/ACA snoRNAs which guide pseudouridylation of RNAs including ribosomal RNA. Mutation of Scc2 was associated with defects in the production of ribosomal RNA, ribosome biogenesis, and splicing. While the scc2 mutant does not have a general defect in protein synthesis, it shows increased frameshifting and reduced internal ribosomal entry site (IRES) usage/cap-independent translation. These findings suggest Scc2 normally promotes a gene expression program that supports translational fidelity. We hypothesize that translational dysfunction may contribute to the human disorder Cornelia de Lange syndrome, which is caused by mutations in Scc2

Le virus de l’hépatite C pose un grave problème de santé du fait de sa prévalence élevé (3%) dans la population mondiale. Il est l’agent viral responsable de la survenue d’hépatites chroniques évoluant progressivement vers une cirrhose et/ou un carcinome hépatocellulaire. Son génome est composé d’un ARN monocaténaire de polarité positive d’environ 9600 nucléotides, flanqué à ses deux extrémités des régions 5’ (5’NC) et 3’ (3’NC) non codantes. Sa traduction est initiée par l’entrée directe des ribosomes au niveau d’une séquence hautement structurée et complexe, localisée dans la région 5’NC : l’IRES (Internal Ribosome Entry Site). La conservation de cette région entre les différents génotypes et sous-types viraux et son rôle fondamental dans l’initiation de la traduction, première étape du cycle viral, font de l’IRES une cible thérapeutique considérable en l’absence d’un traitement efficace et d’un vaccin. Les mécanismes d’initiation de la traduction au niveau de cette structure sont relativement bien connus, cependant la modulation de son activité reste mal comprise et demeure un sujet de controverse. Nous avons mis en évidence, in vitro, à l’aide d’un système de gène rapporteur un rôle modulateur dose-dépendant de la protéine structurale de capside (C-VHC) ajoutée en trans, ainsi qu’un rôle activateur en cis de la région 3’NC sur l’activité de l’IRES. Afin d’étudier et de valider ces observations ex vivo, nous avons mis au point un système cellulaire d’expression basé sur l’emploi de vecteurs lentiviraux. Ces derniers expriment un transcrit bicistronique comportant l’IRES du VHC et ont permis de quantifier l’activité traductionnelle de l’IRES dans les conditions proches de celles de l’infection virale au sein de différentes lignées cellulaires, et de valider notre système d’étude en testant l’inhibition de l’IRES par des agents thérapeutiques utilisés dans le traitement contre l’infection par le VHC.

Le Virus de l'Hépatite C (VHC) est un virus à génome ARN positif, membre de la famille des Flaviviridae, dont la traduction est guidée par un Site d'Entrée Interne du Ribosome (IRES) situé dans la région 5' non traduite du génome. Il est connu que l'initiation de la traduction médiée par l'IRES du VHC nécessite peu de facteurs classiques de la traduction et alors que de nombreux facteurs cellulaires sont capables d'interagir avec cet IRES, aucun n'a été reconnu capable d'une interaction fonctionnelle de type ITAF. Nous avons démontré d'une part dans ce travail que la protéine PTB n'était pas un cofacteur de ce mécanisme. D'autre part ce travail a abouti à l'idée qu'une activité ATP-dépendante non classique est nécessaire à la formation du complexe d'initiation sur l'IRES du VHC. Ce dernier résultat est particulièrement intéressant car pourrait conduire à la détermination d'une nouvelle cible thérapeutique anti-virale.

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

Le virus de l’hépatite C (VHC) est actuellement responsable d’un problème de santé mondiale infectant environ 3% de la population. Le VHC possède une molécule d’ARN simple brin et de polarité positive ainsi que deux régions non codantes (NC) 5’ et 3’. La traduction virale a lieu via une séquence interne d’entrée des ribosomes (IRES) située dans la région 5’NC. L’objectif de ce travail de thèse a été de clarifier le rôle de facteurs viraux en cis (3’NC) et en trans (C, NS5A, NS5B) dans la régulation de l’activité traductionnelle de l’IRES. Nous avons utilisé un système double rapporteur ARN, ciblant ainsi la traduction. D’après l’activité relative de l’IRES mesurée (rapport RLuc/FLuc) différents points ont été observés : 1) la région 3’NC stimule fortement l’activité IRES en cis, ce qui dépend de la structure secondaire globale; 2) une modulation dose et génotype dépendante de la traduction a été démontrée en présence de C et NS5B ; 3) aucun effet synergique n’a été observé entre les protéines virales ou entre les différents facteurs viraux. D’après ces résultats, les facteurs viraux étudiés agiraient de façon séquentielle pour moduler la traduction virale. Nous nous sommes ensuite focalisés sur le développement de nouveaux outils. Nous avons établi un système lentiviral bicistronique, qui s’est révélé efficace pour le criblage de drogues. Les expériences actuellement en cours visent l’établissement d’une lignée exprimant constitutivement l’ARN polymérase du phage T7 (T7 RNAp), permettant à la fois une analyse fine de la fonctionnalité de l’IRES, le criblage de drogues ainsi que l’étude d’autres virus, remplaçant le système vaccine couramment utilisé
Hepatitis C virus (HCV) is responsible of a major health problem, infecting 3% of world population. Hepatitis C Virus (HCV) possesses a positive single-stranded RNA genome with highly structured non coding (NC) regions at its extremities: 5’NC and 3’NC. Translation initiation of HCV RNA occurs via an Internal Ribosome Entry Site (IRES) located at its 5’end. Our aim was to clarify the role of cis (3’NCR) and trans (C, NS5A, NS5B) viral factors on the regulation of IRES activity. By the use of a dual RNA reporter system, targeting the translation step and avoiding the cryptic IRES promoter activity, relative IRES activities measured in luminometry (= RLuc/FLuc activities ratio) revealed the following features : 1) all the HCV 3’ non coding (NC) sequences tested highly stimulate in cis the IRES efficiency; 2) a dose and genotype dependent modulation of the translation in trans was shown with the capsid and NS5B ; and 3) not any cooperative effect could be obtained either between viral proteins, or in the presence of both cis and trans factors. Taking together these results encouraged us to propose a model in which the viral factors tested act sequentially to modulate viral translation and the switch to replication. We then focus on the development of novel tools for evaluating the IRES activity analysis. We established a bicistronic lentiviral system, which revealed efficient for drugs screening, however not adequate for a precise IRES activity analysis. Experiments actually in progress aim the precise analysis of IRES activity, drugs screening and in addition the study of other viruses, replacing the vaccine system currently used

The IRES found in the intergenic (IGR) region of viruses belonging to the Dicistroviridae family is remarkable for its ability to bind directly to the ribosome with high affinity and initiate translation without the requirement for any initiation factors by mimicking a P/E hybrid tRNA. Here, we have conducted an in-depth biochemical characterization of the CrPV IGR IRES. We have found that the L1.1 region of the IRES is responsible for 80S assembly and reading frame maintenance, and may play an additional role downstream of ribosome binding. Additional studies on the modularity of the IRES showed that the two domains of the IGR IRES work independently, but in concert with one another to manipulate the ribosome. We then addressed the question of how the IGR IRES recruits ribosomes during periods of cellular stress, when inactive 80S couples accumulate in the cell. Here, we found that the IRES is able to bind directly to eEF2-associated 80S couples, providing a rationale as to how the IRES remains translated during these periods. Finally, we developed a new in vitro translation system to assess the functionality of specialized ribosomes, and used this system and the IGR IRES in order to ask questions about the pathology of dyskeratosis congenita. Though divergent from other viral IRESs, the simplicity of this tRNA-like IRES serves as a powerful model for understanding IRES functions in general, the role of tRNA/ribosome interactions that occur normally during translation, and how these processes are linked to the greater context of the cell.

The picornaviruses are a family of positive sense, single-stranded RNA viruses that initiate translation of their genome using an internal ribosome entry site (IRES), which is typically around 450 nt in length. Currently, three classes of picornavirus IRES element exist, based on structural similarities and biological properties. A novel picornavirus IRES element was identified within the 5' untranslated regions of porcine teschovirus-1 (PTV-1), porcine enterovirus-8 (PEV-8) and simian virus-2 (SV2). These related elements are considerably shorter than other picornavirus IRES elements and surprisingly formation of 48S pre-initiation complexes on the PTV-1 IRES is achieved in the absence of the translation initiation complex eIF4F. Indeed, toeprinting analysis with the PTV-1 IRES indicated that it is able to directly interact with the 40S ribosomal subunit. These properties are unusual for picornavirus IRES elements but are remarkably similar to those of the Flaviviridae IRES elements. Secondary structure models for the PTV-1, PEV-8 and SV2 IRES elements have been derived and key structural features verified through mutagenesis and functional assays. These studies have suggested that a fourth type of IRES element exists within the Picornaviridae, which is structurally and functionally related to the hepatitis C virus (HCV) and pestivirus IRES elements. Picornavirus IRES elements require accessory proteins for their activity. The availability of these proteins may provide insight into the tissue tropism of the viruses. To assess the binding of accessory proteins to the FMDV IRES in cells, a system was designed that could allow the capture of RNA transcripts containing the FMDV IRES and attached proteins from cells. This system made use of a specific protein-RNA interaction between the iron response protein and iron response element, which was placed at the 3' end of the FMDV IRES. Functional assays indicated that key features of this system were operational as expected, but currently no proteins have been identified as FMDV IRES binding proteins using this system.

Picornaviruses have a single-stranded, positive-sense RNA genome. An internal ribosome entry site (IRES) situated within the 5' untranslated region of the genome mediates cap-independent translation of the picornavirus RNA. In an attempt to further understand the mechanism of IRES-mediated translation, it was decided to investigate the role of several nucleotides within the encephalomyocarditis virus IRES which are absolutely conserved among all cardiovirus and aphthovirus IRES elements and in close proximity to the binding site of the translation initiation factor, eIF4GI. Four nucleotides within the J domain were randomised to generate a pool of mutant IRES elements containing up to 256 different sequences. Analysis of this pool, using a cell selection system to isolate functional IRES elements, revealed that the four nucleotides were essential for IRES activity. Furthermore, it was shown that mutations at these nucleotides severely affected the binding of eIF4GI and eIF4A to the IRES. A clear correlation was seen between the activity of the mutant IRES element and its ability to bind eIF4GI/eIF4A. This strongly suggests that the binding of eIF4GI to the IRES is functionally relevant in vivo. It was also shown that the IRES elements from several different hepatitis C virus genotypes could be used within the cell selection system. This is particularly useful since the analysis of HCV within cells is currently restricted. Finally, the role of residue 20 within the swine vesicular disease virus 2A protease was investigated. This residue is known to affect translation of the picornavirus RNA and virus virulence. To analyse the role of residue 20 to the function of 2A, this residue was substituted for each of the 20 amino acids. This revealed that amino acids substitutions were reasonably well tolerated with the exception of proline.

Dicistroviruses possess a positive-sense, monopartite single-strand RNA genome that encodes two open reading frames containing the nonstructural and structural polyproteins (ORF1 and ORF2) separated by the intergenic region (IGR) internal ribosome entry site (IRES). Translation of each ORF is directed by distinct IRESs, a 5’ untranslated region (UTR) and an IGR IRES. Previous bioinformatic studies have shown that a subset of dicistroviruses contain an overlapping gene in the +1 translational reading frame within the structural polyprotein gene near the IGR IRES region. We hypothesize that the IGR IRES directs translation of two overlapping ORFs, a novel +1 frame ORFx and the 0 frame ORF which encodes the viral structural polyprotein. In this thesis, using Israeli acute paralysis virus (IAPV) as a model, the existence and start site of ORFx were identified using mutagenesis and Mass Spectrometry analyses. In addition, the structural elements within the IAPV IGR IRES that determine alternative reading frame translation initiation were explored. Lastly, the localization of overexpressed tagged-ORFx in Drosophila S2 cells was examined to gain insights of its function. Summarizing, we have discovered a novel mechanism that increases the coding capacity of a virus through an IGR IRES. These studies of IAPV IGR-IRES will further our understanding of IRESs mediated translation initiation and reading frame decoding.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

Avian encephalomyelitis virus (AEV) is a picornavirus that affects young chickens, quails, pheasants and turkeys. Translation initiation on picornavirus mRNA is cap independent and occurs through a mechanism known as internal initiation, which depends on Internal Ribosome Entry Site (IRES) element within the 5' untranslated region (UTR) of the viral RNA. AEV has been assigned within the Hepatovirus genus and shares protein sequence similarity with hepatitis A virus (HAV). I have demonstrated that the 494 nucleotide 5' UTR of the AEV genome contains an internal ribosome entry site (IRES) element. However, in contrast to the HAV IRES, the AEV IRES functions efficiently in the presence of cleaved eEF4G, suggesting functional differences exist. Characterization of the AEV IRES element revealed that there are remarkable structural and functional similarities between the AEV, flavivirus [especially hepatitis C virus (HCV)] and newly discovered type 4 picornaviras IRES elements, including porcine teschovirus-1 (PTV-1), porcine enterovirus-8 (PEV-8) and simian virus-2. These related IRES elements are generally shorter than other picornaviras IRES elements and are able to directly interact with 40S ribosomal subunits. These results indicate that despite classification of AEV as a Hepatovirus, the AEV IRES element should be grouped into the picornavirus type four IRES elements. Seneca Valley virus-001 (SVV) has recently been isolated from pigs and identified as a member of the Picornaviridae capable of killing tumor cells. Initial analysis of the SVV- 001 5' UTR revealed that it may contain an IRES element highly reminiscent of those of the Flaviviridae, especially classical swine fever virus (CSFV). However, further research is required to fully characterize the SVV-001 IRES element.

Bag-1 is an anti-apoptotic protein involved in the regulation of a number of cellular processes, notably as a co-chaperone for the 70kDa heat shock proteins. At least four protein products of Bag-1 have been isolated, p50, p46, p36 and a minor isoform, p29. The 5' UTR of the p36 isoform of Bag-1 has been shown to contain an internal ribosome entry segment (IRES). The internal ribosome entry mediated mechanism of translation has been shown to maintain Bag-1 expression when cap- dependent translation is compromised during heat shock. Many IRESes require trans-acting protein factors for optimal IRES activity. Bag-1 IRES activity is cell-type specific and is inefficient in cell lines with low endogenous levels of the /r < ms-acting factors poly (rC) binding protein 1 (PCBP1) and polypyrimidine tract binding protein 1 (PTB1). Activity of the Bag-1 IRES can be stimulated in vitro and in vivo by overexpression of PTB and PCBP1. PTB and PCBP1 bind specifically to the minimal active Bag-1 IRES element. A secondary structural model of the minimal Bag-1 IRES was obtained by chemical and enzymatic probing of IRES RNA in vitro. Addition of PTB and PCBP1 modulates the secondary structure of the Bag-1 IRES in the ribosome-landing region. Overexpression of Bag-1 proteins in cells subjected to genotoxic stress has been shown to protect cells from stress-induced growth inhibition and cell death. The Bag-1 IRES is functional in heat-shocked cells and cells treated with chemotherapeutic agents and this correlates with a redistribution of PTB and PCBP1 from the nucleus of the cell to the cytoplasm. A model for the mechanism of action of the Bag-1 IRES and the influence of PTB and PCBP1 is proposed.

The Dicistroviridae intergenic region internal ribosome entry site (IGR IRES) exhibits the remarkable ability to bind the conserved core of the ribosome with high affinity. By mimicking the conformation of a tRNA, the IGR IRES can bypass the requirement for canonical initiation factors and Met-tRNAi, and initiate translation from a non-AUG start codon in the ribosomal A site. The pseudoknot (PKI) domain of the IRES engages the decoding center upon initial ribosome binding, and subsequently translocates into the P site to allow delivery of the incoming aminoacyl-tRNA. Within the P site, the IRES adopts a conformation that is reminiscent of a P/E hybrid state tRNA to effectively co-opt the canonical elongation cycle. How the IGR IRES establishes the translational reading frame in the absence of initiation factors remains an outstanding question. Here, we elucidate the mechanism of reading frame selection by performing mutagenesis and biochemical assays to explore the function of specific IRES structural elements. We demonstrate that constituents of the Cricket paralysis virus PKI domain, including the helical stem, anticodon:codon-like base-pairing, and the variable loop region are optimized for IRES-mediated translation. Additionally, we reveal through extensive structural and biochemical studies that stem-loop III of the Israeli acute paralysis virus (IAPV) IRES mimics the acceptor stem of tRNA and functions in supporting efficient 0 frame translation. Finally, we established an infectious chimeric clone to investigate how translational regulation by the IAPV IRES affects the viral life cycle. Studies using this chimera demonstrate that formation of stem-loop VI upstream of the IAPV IRES contributes to optimal IRES activity and viral yield. Our findings suggest that extensive and complete tRNA-mimicry by the IAPV IGR IRES facilitates IRES-mediated translation and reading frame selection.
Medicine, Faculty of
Graduate

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus, the etiological agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). One of the key viral proteins that contribute to tumorigenesis is vFLIP, a viral homolog of the FLICE inhibitory protein. This KSHV protein interacts with the NFκB pathway to trigger the expression of antiapoptotic and proinflammatory genes and ultimately leads to tumor formation. The expression of vFLIP is regulated at the translational level by an internal ribosomal entry site (IRES) element. However, the precise mechanism by which ribosomes are recruited internally and the exact location of the IRES has remained elusive. The aims of this study were to confirm the previously identified 252-nt fragment directly upstream of vFLIP as the location of the vFLIP IRES in cellulo and to determine the structure and mechanism of action of the vFLIP IRES. Here we show that a 252-nt, within a coding region, directs translation in HEK293 cells. We have also established its RNA structure using chemical and enzymatic probing of RNA structure in solution and mutational analysis studies revealed that the domain If of the vFLIP IRES is crucial for its activity. Also, we demonstrate that IRES activity requires the presence of eIF4A and the eIF4E-eIF4G interaction. These interactions may define a new paradigm for IRES-mediated translation. Finally, we attempted to identify cellular proteins that may interact with the vFLIP IRES using several types of protein affinity chromatography, but we could detect a protein interacting with vFLIP IRES but yet to be confirmed.

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.

Translation of mRNA into protein represents the final step in the gene-expression pathway, driving the formation of the proteome from genomic information. The regulation of this process is a mechanism that is used to modulate gene expression in a wide range of biological situations. Protein synthesis is principally regulated at the initiation stage, allowing for rapid, reversible control of gene expression. Progress over recent years in determining the structures and activities of regulatory factors, and in mapping their interactions, have advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studying the regulation of translation initiation by mechanisms that include the modulation of initiation factor activity, internal ribosome initiation and through sequence-specific RNA-binding proteins. This thesis focused on translational control during viral infection, where we investigated the role of Severe Acute Respiratory Syndrome non-structural protein 1 and Enterovirus 71 Internal Ribosome Entry Site in this process. To establish the function of SARS NSP1 protein in translation regulation we attempted the identification of NSP1 protein partners using several types of protein affinity chromatography. Using a wide range of approaches, we could not detect nor confirm the association of NSP1 with any cellular proteins. To dissect the role of FBP2, we engineered a wide range of recombinant FBP2 proteins of different lengths and analysed their interactions with IRES elements using biochemical techniques. This allowed us to characterize the interaction of EV71 IRES with initiation factors eIF4A, eIF4E, eIF4G and FBP2. Finally, we used chemical probing of RNA structure in solution to establish the secondary structure of the BiP IRES. We identified the formation of a structured RNA scaffold of 220 nucleotides comprising 3 major domains.

CoxsackievirusB3 (CVB3), a member of the Picornaviridae family is the causative agent of Virus-induced Myocarditis and Dilated Cardiomyopathy. The 5’UTR contains an Internal Ribosome Entry Site or IRES element that recruits ribosomes in a cap-independent manner. The ribosomes are recruited upstream of the AUG triplet at 591 (AUG591), also called as the cryptic AUG, after which they scan downstream for about 150 nucleotide, before initiating at the initiator AUG or AUG741. The 3’UTR of CVB3 is 99 nts long, highly structured RNA containing conserved domains, and is followed by a poly (A) tail of variable lengths. We have investigated possible involvement of host proteins which may interact with CVB3 IRES and influence its activity. We have demonstrated the role of Poly-pyrimidine tract binding protein (PTB) and established PTB as a bona-fide ITAF for CVB3, by characterizing the effect of partial silencing of PTB ex-vivo in HeLa cells. The IRES activity in BSC-1 cells, reported to have very low level of endogenous PTB, is found to be significantly low compared to that in HeLa cells. PTB is observed to interact with both the 5’ and 3’ UTR of CVB3, although with different affinities. Finer mapping of the interaction between PTB and the UTRs showed that the protein interacts with multiple regions of both UTRs. We have also shown the cis-acting effect of the CVB3-3’UTR on IRES mediated translation. The PTB contact points on the 3’UTRwas found to map to conserved regions, the deletion of which abrogates the 3’UTR mediated enhancement of the IRES activity. The possible role played by PTB in enhancing IRES activity by CVB3 3’UTR suggests that PTB protein might help in circularization of the CVB3 RNA by bridging the ends necessary for efficient translation of the viral RNA. In the second part, we have investigated possible role of some of the cis-acting element present in the CVB3 5’UTR RNA particularly the cryptic AUG. We have shown that mutation in cryptic AUG reduces the efficiency of translation mediated by the CVB3 IRES. Mutation in cryptic AUG moiety also reduces the interaction of mutant RNA with La protein. We have demonstrated that binding of 48S ribosomal complex with mutant IRES RNA was weaker compared to wt IRES RNA. We have investigated the possible alteration in secondary structure in the mutant RNA by chemical and enzymatic modification, which suggests that there is marginal alteration in the local structure due to mutation. It appears that integrity of cryptic AUG is important for efficient translation initiation by the CVB3 IRES. Results suggest that cryptic AUG plays a significant role in mediating internal initiation of translation of CVB3 RNA by mediating precise La binding and correct positioning of the 48S ribosomal complex. Finally, we have investigated the importance of a conserved hexa-nucleotide stretch in the apical loop within stem loop C (SLC, nt104-180), upstream of the ribosome landing site, on CVB3 IRES function. It has been already shown from our laboratory that the deletion at this apical loop resulted in significant decrease in IRES activity. This deletion mutant was shown to alter the secondary structure of the CVB3 5’UTR RNA. Here we have investigated the effect of point mutation in the apical loop SLC/c on CVB3 IRES activity by generating substitution mutation in the apical loop SLC/c in order to avoid possible alteration in secondary structure. Both the deletion or substitution mutation at this apical loop resulted in significant decrease in IRES activity. Both the mutant IRES RNAs (deletion and substitution mutant) failed to interact with certain trans-acting factors. Furthermore, expression of CVB3 2A protease significantly enhanced IRES activity of the wild type, but the effect was not so pronounced on the mutant IRESs. It is possible that the mutant RNAs were unable to interact with some trans-acting factors critical for enhanced IRES function. We have short-listed three proteins of approximate molecular mass of 56, 64 and 90 kDa, which showed reduced binding with mutant IRESs. By using RNA affinity column with biotinylated UTP labeled RNA we have purified couple of proteins and identified p64 as Cyto Keratin 1 protein by performing in-gel trypsin digestion followed by MALDI analysis. Overall, the results characterize the CVB3 IRES structurally and functionally, which could be useful in targeting critical RNA-protein interactions to develop candidate antiviral agent against Coxsackievirus infection.

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018
Em eucariotas, a informação genética está codificada na molécula de ácido desoxirribonucleico (DNA, do inglês deoxyribonucleic acid), sendo transcrita para ácido ribonucleico mensageiro prematuro (pre-mRNA, do inglês premature ribonucleic acid), num processo denominado transcrição, e posteriormente traduzida para proteína, num processo designado tradução. Durante a transcrição, a molécula de pre-mRNA sofre um processo de maturação durante o qual lhe são adicionadas uma estrutura cap (guanina metilada, m7G) na extremidade 5’, e uma cadeia de poliadenosinas [cauda poli(A)] na extremidade 3’, e num fenómeno designado por splicing, se dá a remoção das regiões não codificantes (intrões) que intercalam com regiões codificantes/regulatórias (exões), e a junção das últimas. Seguidamente, o mRNA maduro é translocado para o citoplasma onde é traduzido para proteína nos ribossomas. A tradução implica, normalmente, o reconhecimento da estrutura cap por factores de iniciação da tradução (eIF, do inglês eukaryotic translation initiation factor). Após este reconhecimento, o complexo de preiniciação da tradução 43 S (43S PIC, do inglês 43S preinitiation complex), que depende da formação prévia do complexo ternário [factor eucariótico de iniciação da tradução 2 (eIF2, do inglês eukaryotic translation initiation factor 2) ligado a uma guanosina trifosfato (GTP, do inglês guanosine triphosphate) e à molécula de RNA de transferência (tRNA, do inglês transfer ribonucleic acid) que transporta a primeira metionina da cadeia peptídica (Met-tRNAi, do inglês initiator tRNA methionine complex)], é recrutado para a extremidade 5’ do mRNA, de onde iniciará o rastreamento da região 5’ transcrita mas não traduzida do mRNA (5’UTR, do inglês 5’untranslated region), até que este reconheça um codão de iniciação num contexto favorável. Quando um codão de iniciação é reconhecido, inicia-se a fase de alogamento da tradução, que consiste na síntese de uma cadeia peptídica. A terminação da tradução ocorre quando um codão stop é reconhecido pelo ribossoma, o que conduz à dissociação da recentemente formada cadeia peptídica do ribossoma e à reciclagem deste. Em condições de stresse, as células reduzem globalmente a síntese proteica sobretudo através da inibição da iniciação canónica da tradução. Esta inibição pode ser mediada, entre outras vias, pela fosforilação da subunidade alfa (α) do eIF2 (eIF2α), impedindo a sua reciclagem que é necessária para a formação de um novo complexo ternário, e consequentemente, do 43S PIC. A fosforilação do eIF2α é mediada por diferentes cinases em resposta a diferentes estímulos, designadamente stresse do retículo endoplasmático (RE), escassez de nutrientes e danos no DNA. Contudo, proteínas associadas à resposta ao stresse podem continuar a ser traduzidas usando mecanismos alternativos, permitindo às células redireccionar os seus esforços para combater o stresse. Algumas dessas proteínas são codificadas por mRNA que contêm regiões estruturadas designadas por locais de entrada internos do ribossoma (IRES, do inglês internal ribosome entry sites), que recrutam o ribossoma internamente para a vizinhança de codões de iniciação. A iniciação da tradução através destas estruturas requer muitas vezes a interacção com proteínas específicas, ITAF (do inglês, IRES trans-acting factor), e elimina a necessidade de reconhecimento da estrutura cap. A transformação de células normais em células tumorais ocorre devido a um acumular de mutações que conduz à inactivação ou à ativação de proteínas, ou ainda à alteração da sua actividade biológica. Muitas das proteínas que se encontram alteradas em cancro regulam vias essenciais ao crescimento e desenvolvimento de uma célula, e estão também associadas a programas de resposta ao stresse. Deste modo, e como seria expectável, são muitas as proteínas associadas ao cancro, cujos mRNA contêm IRES, permitindo, deste modo, a sua expressão em condições em que a tradução canónica está inibida. Este mecanismo de protecção celular, é explorado por células tumorais, que estão muitas vezes sujeitas a condições de stresse (escassez de oxigénio, escassez de nutrientes, ou danos no DNA), de forma a aumentar a sua capacidade de sobrevivência e proliferação. Este projecto teve por objectivo o estudo da expressão mediada por IRES de isoformas de proteínas que se encontram alteradas em diversos tipos de cancro: a isoforma Δ160p53 do supressor de tumores p53, que parece apresentar funções oncogénicas previamente associadas a mutações missense no gene TP53; e uma isoforma ainda não descrita do GTPase H-Ras, p14H-Ras, cuja expressão parece ser induzida em condições de stresse do RE a um nível bastante superior em relação à expressão da isoforma canónica do H-Ras (p21H-Ras). Recorrendo a uma análise in silico da estabilidade da região com possível actividade de IRES para cada um dos alvos, e de acordo com o conteúdo em GC e a energia mínima livre de Gibbs previstos, concluímos que ambos se tratavam de bons candidatos. Adicionalmente, pretendíamos avaliar o efeito de mutações associadas ao desenvolvimento de cancro no funcionamento deste mecanismo alternativo. Desta forma, usámos um vector bicistrónico que contém, como cistrão a 5’, a região codificante da luciferase da medusa Renilla reniformis (Rluc, do inglês Renilla luciferase), e, como cistrão a 3’, a região codificante da luciferase do pirilampo Photynus pyralis (Fluc, do inglês firefly luciferase). Deste modo, analisámos a expressão de cada uma das proteínas através da respectiva actividade de luciferase por medição directa da luminescência resultante de cada uma das reacções com o respectivo substrato. Este vector bicistrónico contém um hairpin (estrutura em grampo) estável a jusante do codão stop da Rluc, para impedir que o ribossoma reinicie a tradução após terminação da tradução canónica da Rluc, de maneira que a tradução da Fluc ocorrerá de forma independente da estrutura cap, e apenas na presença de estruturas no mRNA localizadas na vizinhança do respectivo codão de iniciação que permitam o recrutamento interno do ribossoma. As sequências em estudo para a actividade de IRES foram inseridas imediatamente a montante do codão de iniciação da Fluc e as mutações estudadas foram inseridas nesses mesmos constructos por mutagénese dirigida. A actividade de IRES foi estudada na ausência e na presença de thapsigargina, uma droga inibidora da bomba de cálcio do RE, que induz o stresse deste organelo, promovendo assim a inibição da tradução canónica por fosforilação do eIF2α. Relativamente ao estudo da expressão mediada por IRES do Δ160p53, observações anteriores indicaram que Δ160p53 contém um IRES nos primeiros 432 nucleótidos (nt) codificantes da isoforma Δ160p53, e que parte da região codificante de outra isoforma do p53, Δ133p53, localizada a montante do correspondente codão de iniciação do Δ160p53 (5’UTR do Δ160p53), inibe a actividade de IRES. Usando o sistema bicistrónico descrito anteriormente, analisámos a actividade de IRES dos 432 nt do Δ160p53 e a sua inibição por parte da respectiva 5’UTR. No constructo bicistrónico que contém os 432 nt do Δ160p53 observámos um aumento (não estatisticamente significativo) na actividade de luciferase da Fluc, e que, na presença da 5’UTR, esta é inibida. Além disso, analisámos o efeito de três das mutações missense mais comuns do TP53 (R175H, R248Q e R273H) na actividade de IRES. De acordo com os nossos resultados, as mutações R248Q e R273H parecem induzir a actividade do IRES em condições de stresse do RE. Portanto, a função oncogénica destas mutações poderá estar relacionada com o aumento da expressão dependente de IRES do Δ160p53 em tecidos tumorais, promovendo a capacidade de proliferação, sobrevivência e invasão das células. Além de identificar IRES, pretendíamos caracterizá-los a nível da estrutura e da regulação. Deste modo, iniciámos o processo de optimização da caracterização in vivo da estrutura secundária do IRES do Δ160p53 através do método de modificação química de ácidos nucleicos usando o dimetilsulfato (DMS), bem como das condições de imunoprecipitação do Hdm2 (do inglês murine double minute 2 human homolog)⸺foi descrito como ITAF capaz de regular a expressão dependente de IRES do XIAP (do inglês X-linked inhibitor of apoptosis protein) e cuja interação com um IRES presente no mRNA do p53 foi observada⸺para identificar novos IRES regulados por esta ITAF através da sequenciação de RNA previamente co-immunoprecipitados usando anticorpos anti-Hdm2. Em diferentes condições de stresse, foi observado recentemente no nosso laboratório o aumento da expressão de uma isoforma ainda não descrita do GTPase H-Ras, p14H-Ras, indicando que um mecanismo de tradução alternativo poderá regular a sua expressão. Além disso, observou-se também que a presença da mutação silenciosa H27H (T81>C), associada a um maior risco de desenvolvimento de cancro, promovia o aumento da expressão de p14H-Ras em condições de stresse. Usando o mesmo sistema bicistrónico, analisámos a actividade de IRES de 195 nt da região codificante do p21H-Ras (região codificante limitada pelo codão de iniciação do p21H-Ras e pelo hipotético codão de initiação do p14H-Ras). Além disso, analisámos o efeito na actividade deste hipotético IRES da mutação silenciosa T81>C. Os nossos resultados sugerem que, em condições de stresse, esta região é capaz de mediar a tradução de forma independente da estrutura cap e que a mutação estimula a actividade de IRES. Um possível papel oncogénico desta mutação será promover a expressão desta isoforma, que, tal como Δ160p53, poderá apresentar funções oncogénicas. No futuro, pretendemos realizar um rastreio de drogas capazes de inibir a actividade dos IRES aqui estudados, e avaliar se estas poderão reverter o processo de tumorigénese. Além disso, pretendemos caracterizar a estrutura secundária de cada um destes IRES, identificar novos IRES e novas ITAF. Pretendemos, assim, identificar proteínas cuja expressão através de IRES possa estar implicada no desenvolvimento de cancro, e assim fornecer novas abordagens para a terapia desta doença.
In eukaryotes, most proteins are translated through a canonical translation initiation mechanism that involves recognition of the cap structure at the messenger ribonucleic acid (mRNA) 5’end in order to recruit the ribosome. Yet, during certain physiological and pathological conditions, canonical translation is impaired and protein synthesis is globally decreased, in part due to eIF2α phosphorylation. However, some mRNA that encode, among others, proteins associated with stress-response are translated through alternative cap-independent translation initiation mechanisms. Internal ribosome entry sites (IRES) consist of structures within the mRNA that can recruit the ribosome to the vicinities of, or directly to, the initiation codon, in a cap-independent manner. Overall, IRES-dependent translation initiation does not require the complete set of eukaryotic translation initiation factors (eIF) for ribosomal recruitment but additional factors named IRES trans-acting factors (ITAF) are required to modulate the IRES activity. Several cellular mRNA-containing IRES are related to stress-response, programmed cell death, cell proliferation, cell growth and angiogenesis, and their deregulation has been associated with tumor development. Nonetheless, IRES-mediated translation mechanisms are not well understood in eukaryotic cells nor is it their role in cancer. Therefore, the main goal of this work was to understand the role of IRES-dependent translation in cancer development with possible implications for cancer treatment. Here, we studied the putative IRES-mediated translation of two isoforms of proteins that were shown to be upregulated in several cancers, and whose expression was shown to be promoted during cap-dependent translation inhibition: the tumor suppressor p53 isoform, Δ160p53, and a yet-to-be described GTPase H-Ras isoform, p14H-Ras. Additionally, we evaluated the effect of cancer-related mutations in the activity of each putative IRES. Therefore, we used a bicistronic construct, which contained as the 5’ cistron the coding sequence of Renilla luciferase (Rluc)⸺cap-dependently translated⸺and as the 3’ cistron the coding sequence of firefly luciferase (Fluc)⸺cap-independently translated⸺, and immediately upstream Fluc’s initiation codon the putative IRES’ sequence. The expression of each protein was assessed by quantifying their respective luciferase activity by measuring the resulting bioluminescence from each reaction with the corresponding substrate. We studied the activity of both putative IRES in the absence and in the presence of thapsigargin, an inhibitory drug of a calcium pump from the endoplasmic reticulum (ER), which leads to ER stress, and, consequently to eIF2α phosphorylation. In previous reports, Δ160p53 was shown to be expressed in an IRES-dependent way from an IRES located within the first 432 nucleotides (nt) from Δ160p53 coding sequence. Throughout this work, we performed an in silico analysis of Δ160p53’s 432-nt sequence, which indicated that this region might be, indeed, a good IRES candidate. Although not statistically significant, our bioluminescence assays’ results suggest a putative wild-type Δ160p53 IRES activity and that Δ160p53 5’UTR represses its putative IRES activity. Regarding the effect of p53’s cancer-related missense mutations (R175H, R248Q and R273H) in the putative IRES activity, our results indicate that both R248Q and R273H are capable of inducing Δ160p53 putative IRES activity in the presence of Δ160p53 5’UTR during thapsigargin-induced ER stress, whereas R175H seems to have no effect in the IRES activity. This suggests that R248Q and R273H p53 cancer-related mutations may drive tumorigenesis by promoting IRES-dependent expression of Δ160p53, which has been shown to harbor oncogenic functions. Furthermore, according to the in silico analysis, these two mutations are located within the same loop, which corresponds to the most stable one, thus suggesting that this loop may be more important for IRES activity. Additionally, we performed initial experiments to characterize the secondary structure of Δ160p53 putative IRES by chemical probing using dimethyl sulfate (DMS) as well as to detect new IRES regulated by murine double minute 2 human homolog (Hdm2), a known ITAF of X-linked Inhibitor of Apoptosis Protein (XIAP) IRES that is also known to bind to Δ40p53 IRES and to regulate p53 expression, by RNA deep sequencing of Hdm2-bound RNA previously co-immunoprecipitated (co-IP) using anti-Hdm2 antibodies⸺we started by optimizing Hdm2 immunoprecipitation (IP). Regarding H-Ras putative IRES, preliminary experiments from our lab, showed that the expression of a yet-to-be described H-Ras short isoform, p14H-Ras, was upregulated during stress conditions, and that an H-Ras cancer-related silent mutation (T81>C), which is associated with higher risk for developing cancer, promoted its expression. Therefore, we hypothesized that H-Ras mRNA might contain an IRES within a 195-nt sequence, which corresponds to the putative sequence between the initiation codons of p21H-Ras and p14H-Ras. We started by performing an in silico analysis regarding the stability of possible structures located within the 195-nt sequence, which indicated that this region might be a good candidate, as well. Our results from the bioluminescence assays suggest that wild-type H-Ras putative IRES sequence is able to drive IRES-dependent expression under ER stress conditions, as well as the T81>C-mutated H-Ras putative IRES sequence. This suggests that T81>C mutation may induce the IRES-dependent expression of H-Ras, which may contribute for cancer development. In the future, we aim to perform a drug screening for drugs targeting both putative IRES and evaluate if we can possibly revert tumor progression using the most promising screened drugs. Additionally, we are expecting to characterize the IRES structure of both putative IRES studied throughout this work and to identify new IRES through RNA deep sequencing of samples obtained by Hdm2-bound RNA co-IP. We intend to identify proteins, whose IRES-mediated translation may be implicated in tumorigenesis, thus allowing the development of new cancer therapies.

Tese de doutoramento em Biociências, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
A regulação ao nível da iniciação da tradução de mRNAs é fundamental no processo de controlo de expressão génica uma vez que permite uma resposta celular rápida face a estímulos externos. Este controlo pode ocorrer de forma específica de transcrito, através de elementos reguladores em cis, tais como internal ribosome entry sites (IRESs), que medeiam a tradução de forma independente de alguns factores de iniciação canónicos que são inibidos em condições de stress celular, ou em algumas condições fisiológicas ou patológicas. Desta forma, a tradução dependente de IRES é refractária a condições que inibem a síntese proteica global. Estes elementos encontram-se em transcritos que codificam proteínas responsivas a stress, oncogenes ou supressores de tumor. O trabalho apresentado nesta dissertação mostra que os transcritos que codificam o mammalian (or mechanistic) target of rapamycin (MTOR) e a isoforma proteica de P53, Δ160P53, possuem elementos IRESs a regular a sua expressão. O MTOR é uma serina/treonina quinase conservada que integra sinais provenientes da estimulação por factores de crescimento, assim como dos estados nutricional e energético da célula actuando, nomeadamente, na maquinaria de tradução. Apesar da crescente compreensão acerca dos mecanismos de regulação e efeitos da via de sinalização do MTOR, o controlo da sua própria expressão, nomeadamente ao nível da tradução, permanece largamente desconhecido. Os resultados descritos nesta tese demonstram que a região 5´ transcrita e não traduzida (5’UTR) do mRNA MTOR humano contém um elemento IRES que permite a sua tradução de forma independente da estrutura cap. Adicionalmente, demonstra-se que a tradução de MTOR mediada por IRES é estimulada em hipoxia com associado aumento da fosforilação de EIF2α e que esta estimulação é independente da indução de hypoxia-inducible factor 1α (HIF1α) per se.
A fase anti-apoptótica da unfolded protein response induzida por stress do retículoendoplasmático (RE) estimula a tradução de MTOR mediada por IRES, contudo um efeito mais pronunciado é observado na fase pró-apoptótica com associado aumento da fosforilação de EIF2α. Mostra-se ainda que a inactivação de MTORC1 é acompanhada por estimulação do IRES do MTOR, sugerindo um circuito de auto-regulação com o intuito de manter os níveis proteicos de MTOR constantes. Estes resultados demonstram um novo mecanismo regulador da expressão génica de MTOR, que integra o rearranjo de perfil proteico observado em condições que inibem globalmente a tradução. Para além disso, os resultados aqui apresentados podem explicar o facto da via de sinalização do MTOR não ser perdida em condições que inibem a síntese proteica. A proteína P53 possui papel fundamental no impedimento de desenvolvimento tumoral. Em condições de stress, P53 desenvolve um programa protector que, dependendo da severidade do stress e/ou dano causado, poderá promover a sobrevivência celular através da indução de uma paragem temporária do ciclo celular e da reparação dos danos ou promover a inviabilização da célula através da indução de senescência celular ou morte por apoptose ou autofagia. O gene tumor protein p53 (TP53) expressa várias isoformas proteicas através da utilização de diferentes promotores, splicing alternativo ou tradução mediada por IRES, que actuam tanto através da modulação da actividade da proteína P53 como de forma independente desta. A sua função primordial na supressão da tumorigénese e o facto de TP53 ser um dos genes mais frequentemente mutados em cancro, faz com que este seja um dos genes mais estudados. Contudo, tem-se vindo a verificar que as funções da família de P53 ainda não são totalmente conhecidas e a descoberta de novos membros tem vindo a adensar a complexidade desta família.
Recentemente foi descoberta uma nova isoforma proteica originada apartir de iniciação da tradução no codão 160, tendo sido designada por Δ160P53. Porém, o mecanismo responsável pela sua expressão assim como a sua função permaneceram um mistério. O trabalho explanado nesta dissertação mostra que a expressão de Δ160P53 é induzida por sobre-confluência celular e em stress do RE através de taxas de tradução aumentadas. Adicionalmente, é aqui identificado um elemento IRES a jusante do codão de iniciação 160, o qual é responsável pela expressão da isoforma proteica Δ160P53. Curiosamente demonstra-se que a 5´UTR de Δ160P53 inibe a actividade deste elemento IRES. Para além disso, mostra-se que o aumento da fosforilação de EIF2α estimula a síntese de Δ160P53 mediada por IRES. Na sequência deste trabalho, um grupo colaborador mostrou que a proteína Δ160P53 inibe a apoptose, promove crescimento celular e induz transformação maligna. O trabalho descrito nesta tese apresenta os IRESs que assistem a síntese de MTOR e Δ160P53 como potenciais novos alvos terapêuticos para o tratamento de várias doenças, tal como cancro, com hiper-activação da via de sinalização do MTOR e expressão aumentada de Δ160P53, respectivamente.

Modifications of ribosomal RNA are present in every livivng organism. The function of rRNA modifications could be studied only when the process of modifications was described. Currently, scientists study not only individual modifications but also the importance of global level of modifications for maturation and function of ribosome. This thesis deals with the influence of 2'-O-methylation of citidine 1639 and adenosine 100 in 18S rRNA and uridine 2729 in 25S rRNA on initiation in yeast Saccharomyces cerevisiae with special attention of translation controlled by internal ribosome entry site (IRES). Strains with deletion in genes snR51, snR70 and duoble deletion in both genes were successfully created during my master study. Pilot experiments showed the importance of products of both genes in translation initiation.

博士
國立成功大學
基礎醫學研究所
104
The transcription factor, Specificity protein-1 (Sp1) is expressed in mammalian cells and involved in many cellular processes. Sp1 was accumulated in several cancer types and the Sp1 levels correlated with tumor stages. Our previous study indicated that Sp1 is accumulated during hypoxia in an internal ribosomal entry site (IRES)-dependent manner. This study provided evidences that Sp1 accumulation is IRES-mediated manner in stressful or pathological conditions. The overall objective of this study is to investigate the regulation of Sp1 levels through IRES-dependent manner in tumorigenesis and metastasis. First, we found that the Sp1 was induced strongly at the protein level, but not in the mRNA level, in lung tumor tissue, indicating that translational regulation might contribute to the Sp1 accumulation during tumorigenesis. A further study showed that the translation of Sp1 was dramatically induced through an IRES-dependent pathway. RNA immuniprecipitation analysis of proteins bound to the 5-untranslated region (5-UTR) of Sp1 identified interacting protein-nucleolin. We found nucleolin positively facilitates Sp1 IRES activation. Further analysis of the interaction between nucleolin and the 5-UTR of Sp1 mRNA revealed that the GAR domain was important for IRES-mediated translation of Sp1. Moreover, gefitinib, LY294002 and MK2206 compounds inhibited IRES-mediated Sp1 translation, implying that activation of the epithelial growth factor receptor (EGFR) pathway via Akt activation triggers the IRES pathway. EGFR activation-mediated nucleolin phosphorylated at Thr641 and Thr707 was recruited to the 5-UTR of Sp1 as an IRES trans-acting factor (ITAF) to modulate Sp1 translation during lung cancer formation. Furthermore, recent studies have indicated that a decrease in Sp1 level is beneficial to the lung cancer malignancy. We also clarified the decrease of Sp1 levels was regulated through IRES-dependent manner in cancer malignancy. Herein, we used in vitro transcript 5-UTR of Sp1 as probe and analyzed interacting proteins by LC/MS/MS. We found nm23 and hnRNPA2/B1 as interacting proteins in Sp1 5-UTR. We demonstrated that nm23 not only increased the phosphorylation of Sp1 at Thr739 to enhance the protein stability of Sp1 but also formed a complex with hnRNPA2/B1 that was recruited to the 5'-UTR of Sp1 mRNA. Knocking down nm23 or hnRNPA2/B1 decreased Sp1 expression in a cap-independent manner, suggesting that nm23 and hnRNPA2/B1 contributed to the IRES-mediated translational activity of Sp1. Knocking down nm23 or hnRNPA2/B1 also increased the migratory activity of lung cancer cell lines. Furthermore, patients with lung cancer with poor prognosis had low levels of Sp1 and nm23, suggesting an association between nm23/Sp1 levels and survival rate. Studies performed to elucidate the mechanism underlying this relationship indicated that a decrease in nm23 levels in the lung cancer cells with more malignant activity inhibited hnRNPA2/B1 protein stability, and thus subsequently decreased the recruitment of hnRNPA2/B1 to the 5'-UTR of Sp1 mRNA, repressing Sp1 expression through inhibition of the cap-independent transcriptional activity. Taken together, these results suggest that understanding the relationship between nucleolin, nm23, hnRNPA2/B1, and Sp1 in regulating lung cancer tumorigenesis and malignancy will be beneficial of lung cancer.

Cellular response to various stress conditions involves regulation of gene expression by different mechanisms. Translation is the final step in the flow of genetic information and regulation at this level allows an early response to changes in physiological conditions. Initiation of translation is the rate-limiting step of protein synthesis and hence is tightly regulated. Translation initiation in mammalian cells is mainly by “cap dependent pathway” wherein the 5’methyl guanosine “cap” structure is recognized by certain canonical initiation factors along with 40S ribosomal subunit and the complex scans the 5’UTR till it recognizes initiator AUG. This leads to the joining of the 60S ribosomal subunit and the initiation of translation. In an alternate mode of translation initiation called as the Internal ribosome entry site mediated translation (IRES), the ribosomes are recruited closer to the initiator AUG in a 5’ cap independent manner. Efficient translation by IRES mode requires some canonical initiation factors like eIF2 and eIF3 and other non-canonical IRES-trans-acting factors (ITAFs), which include human La antigen, polypyrimidine-tract binding protein (PTB),Upstream of N-Ras (Unr), Poly (rC) binding protein (PCBP2) etc. Various types of stress conditions, such as starvation of growth factors, heat shock, hypoxia, viral infection lead to down regulation of protein synthesis. However, translation of a subset of mRNAs continues or is up-regulated. Many of these mRNA may be translated by an IRES mode. It is believed that cellular IRESs become active during such conditions that abrogate the cap-dependent mode of translation so that the pool of vital proteins is maintained in the cell. In this thesis, presence of ‘IRES’ element has been investigated in the 5’UTR of Interferon regulatory factor -2 (IRF2) mRNA and the possible physiological significance has been studied. Further, it has been shown that polypyrimidine tract binding protein or PTB is important for the IRES activity. The probable mechanism of action of PTB has also been investigated which suggests that PTB interaction alters the IRF2 IRES conformation thus facilitating translation initiation. In the first part of the thesis, mRNAs that continue to be translated under heat-shocked condition, which is known to abrogate cap-dependent translation initiation, has been investigated by cDNA micro-array hybridization analysis of the ribosome bound RNA. The global protein synthesis was severely impaired under heat shock; however a number of mRNAs continued translation under this condition. Some of these mRNAs encode proteins that are likely to be involved in the heat shock response. Few of these genes are also reported to contain IRES element. Since the micro-array was performed from the RNA extracted from ribosome bound mRNA fraction in a condition when cap-dependent translation is impaired, it was hypothesized that some of the genes, which are up regulated under such condition, might operate via cap-independent mode of translation initiation. Based on this study, one candidate gene, the ‘interferon regulatory factor 2 (IRF2)’ was selected from the pool of up regulated genes and presence of an IRES element was investigated. Interferon regulatory factors are DNA-binding proteins that control interferon (IFN) gene expression. IRF2 has been shown to function as repressor of IFN and IFN-inducible genes. Real–Time and semi-quantitative RT-PCR assays were performed which validated the micro-array data. In the second part of the thesis, the presence of IRES element in the 5’UTR of IRF2 was investigated. Bicistronic assay showed comparable IRES activity with a known representative IRES, BiP, thus suggesting the presence of an IRES element in the IRF2 5’UTR. Stringent assays were then performed to rule out cryptic promoter activity, re-initiation/scanning or alternative splicing in the 5’UTR of the IRF2. RNA transfections using in vitro synthesized bicistronic RNAs further validated the presence of the IRES element. To understand the physiological significance of an IRES element in IRF2 mRNA, the cells were subjected to various stress conditions and IRES activity was studied. It seems IRF2 IRES function might not be sensitive to eIF4G cleavage, since its activity was only marginally affected in presence of Coxsackievirus 2A protease, which is known to cleave eIF 4G and thus inhibit the cap-dependent translation. Incidentally, Hepatitis A virus IRES was affected under such condition. Additionally, it was observed that compared to HCV or Bip IRES, the effect of Interferon α treatment was not so pronounced on the IRF2 IRES. This was further evidenced by its unchanged protein level post-treatment with interferon α. Furthermore, in cells treated with tunicamycin (a known agent causing ER stress), the IRF2 IRES activity and the protein levels were unaffected, although the cap dependent translation was severely impaired. The observations so far suggested that the IRF2 protein level is practically unchanged under conditions of ER stress and interferon treatment. Metabolic labeling followed by immunoprecipitation of IRF2 in cells treated with either tunicamycin or interferon suggested that de novo synthesis of the protein is continued under the above conditions thus validating our earlier data. In the third part of the thesis, the role of an IRES trans acting factor, PTB, in modulating the IRF2 IRES activity has been investigated. Analysis of the cellular protein binding with the IRF2 IRES suggested that certain cellular factors might influence its function under stress conditions. The IRF2 IRES was found to interact with a known trans-acting factor or PTB. To study the possible role of this trans acting factor, the PTB gene was partially silenced by PTB specific siRNA. This led to a decrease in the IRF2 IRES activity, suggesting that PTB is probably essential for the IRES activity. Interestingly, when Hela cells (with partially silenced PTB) were treated with tunicamycin (inducer of ER stress) the level of IRF2 protein was also found to be less thus pointing to an important role of PTB in IRF2 protein synthesis under such conditions. Western blot analysis and immunofluoroscence assay suggested that there was no significant nuclear-cytoplasmic relocalization of PTB under the condition studied. Primer extension inhibition assay or Toe-printing analysis was performed to detect the contact points of PTB on the IRF2 5’UTR. Many toe-prints were found on the 3’ end of the 5’UTR RNA. A 3’ deletion mutant was generated that showed reduced PTB binding. Incidentally the IRES activity of the mutant was also found to be less than the wt IRF2 RNA. Subsequently, structural analysis of the RNA was performed using enzymatic (CV1, RNase T1) and chemical modification (DMS) agents. Footprinting assay in presence of PTB suggested that there is change in the structure when PTB interacts with the RNA. To investigate this further, CD spectrum analysis of the IRF2 RNA in the presence of PTB was performed which indicated that there was a conformational change under such condition thus validating our earlier observation. The thesis reveals a novel cellular IRES element in the 5’UTR of IRF2 mRNA. The characterization of the IRES and possible role played by PTB protein in modulating its activity suggests that the regulated expression of IRF2 protein by its IRES element under various stress conditions would have major implications on the cellular response. Incidentally, this study constitutes the first report on translational control of interferon regulatory factors by internal initiation. The results might have far reaching implications on the possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general.

碩士
中原大學
生物科技研究所
97
Protein expression systems are important tools in modern biotechnology. Many expression vectors provided for the different expression system to produce protein have been developed, except the fish cell systems. The promoter is one of the elements for the expression vectors that regulate gene expression on transcription level and have specific for different type cells. To finding the promoter that is optimal in the different cells, CMV, Krt4, Hsp70, beta-actin and Cmlc2 promoter was test in Koi Fin cells (KFC), mammalian cells and insect cells. In addition, to produce more than two of proteins in the same vector, dicistronic vector was used for this study. A IRES element was constrstruct into the expression vector to produce the second protein that was translated by a mechanism of cap-independent translation from the same transcript. The IRESs: EV71, EMCV and RhPV IRES were tested in the KFC cells and mammalian cells. The results demonstrate the CMV promoter have strong activity in mammalian cells, especially for the CHO cells and its activity is more than others about fifteen times. Nevertheless, all of the promoters were weak in the KFC by the transient transfection assay. The Hsp70 promoter seemed to be better than other promoters except compare with CMV promoter either in KFC or mammalian cells. In addition, the EV71 IRES activity was stronger than RhPV and EMCV IRES activity in mammalian cells although RhPV IRES as well as EV71 can function well in the KFC.

Hepatitis C virus (HCV) is a worldwide spread pathogen infecting up to 3 % of the human population. Nowadays, research of new drugs against this virus is focused on the individual steps in its life cycle, including the translation initiation. In the case of HCV translation initiation is dependent on the internal ribosome entry site (IRES). Besides of components of the translational machinery also other components of the cell, so called IRES trans-acting factors (ITAF), contribute to its proper progress. This work continues in previous research of our laboratory focused on searching for new ITAF. In order to search for potential ITAF increasing HCV IRES activity new recombinant plasmid vectors and reference strains were prepared and selection conditions of the selection system were optimized. The differences in the growth characteristics of the reference strains were analyzed and quantified under selective and non-selective conditions. A set of pilot high efficiency transformations of the yeast strain pJ69-4A carrying bicistronic construct with HCV IRES were conducted using human expression cDNA library in order to optimize the efficiency of transformation and selection conditions and to attempt to identify new ITAF. Several dozens of randomly selected clones from these transformations obtained under...

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est responsable du syndrome de l’immunodéficience acquise (SIDA). Il faut identifier de nouvelles cibles pour le développement d’agents anti-VIH-1, car ce virus développe une résistance aux agents présentement utilisés. Notre but est d’approfondir la caractérisation de l’étape du changement de cadre de lecture ribosomique en -1 (déphasage -1) nécessaire à la production du précurseur des enzymes du VIH-1. Ce déphasage est programmé et effectué par une minorité de ribosomes lorsqu’ils traduisent la séquence dite glissante à un endroit spécifique de l’ARN messager (ARNm) pleine-longueur du VIH-1. L’efficacité de déphasage est contrôlée par le signal stimulateur de déphasage (SSF), une tige-boucle irrégulière située en aval de la séquence glissante. La structure du SSF est déroulée lors du passage d’un ribosome, mais elle peut se reformer ensuite. Nous avons montré que des variations de l’initiation de la traduction affectent l’efficacité de déphasage. Nous avons utilisé, dans des cellules Jurkat-T et HEK 293T, un rapporteur bicistronique où les gènes codant pour les luciférases de la Renilla (Rluc) et de la luciole (Fluc) sont séparés par la région de déphasage du VIH-1. La Rluc est produite par tous les ribosomes traduisant l’ARNm rapporteur alors que la Fluc est produite uniquement par les ribosomes effectuant un déphasage. L’initiation de ce rapporteur est coiffe-dépendante, comme pour la majorité des ARNm cellulaires. Nous avons examiné l’effet de trois inhibiteurs de l’initiation et montré que leur présence augmente l’efficacité de déphasage. Nous avons ensuite étudié l’effet de la tige-boucle TAR, qui est présente à l’extrémité 5’ de tous les ARNm du VIH-1. TAR empêche la liaison de la petite sous-unité du ribosome (40S) à l’ARNm et module aussi l’activité de la protéine kinase dépendante de l’ARN double-brin (PKR). L’activation de PKR inhibe l’initiation en phosphorylant le facteur d’initiation eucaryote 2 (eIF2) alors que l’inhibition de PKR a l’effet inverse. Nous avons étudié l’effet de TAR sur la traduction et le déphasage via son effet sur PKR en utilisant TAR en trans ou en cis, mais à une certaine distance de l’extrémité 5’ afin d’éviter l’interférence avec la liaison de la 40S. Nous avons observé qu’une faible concentration de TAR, qui active PKR, augmente l’efficacité de déphasage alors qu’une concentration élevée de TAR, qui inhibe PKR, diminue cette efficacité. Nous avons proposé un modèle où des variations de l’initiation affectent l’efficacité de déphasage en modifiant la distance entre les ribosomes parcourant l’ARNm et, donc, la probabilité qu’ils rencontrent un SSF structuré. Par la suite, nous avons déterminé l’effet de la région 5’ non traduite (UTR) de l’ARNm pleine-longueur du VIH-1 sur l’efficacité de déphasage. Cette 5’UTR contient plusieurs régions structurées, dont TAR à l’extrémité 5’, qui peut interférer avec l’initiation. Cet ARNm a une coiffe permettant une initiation coiffe-dépendante ainsi qu’un site d’entrée interne des ribosomes (IRES), permettant une initiation IRES-dépendante. Nous avons introduit cette 5’UTR, complète ou en partie, comme 5’UTR de notre ARNm rapporteur bicistronique. Nos résultats démontrent que cette 5’UTR complète inhibe l’initiation coiffe dépendante et augmente l’efficacité de déphasage et que ces effets sont dus à la présence de TAR suivie de la tige-boucle Poly(A). Nous avons aussi construit un rapporteur tricistronique où les ribosomes exprimant les luciférases utilisent obligatoirement l’IRES. Nous avons observé que cette initiation par l’IRES est faible et que l’efficacité de déphasage correspondante est également faible. Nous avons formulé une hypothèse pour expliquer cette situation. Nous avons également observé que lorsque les deux modes d’initiation sont disponibles, l’initiation coiffe dépendante est prédominante. Finalement, nous avons étudié l’effet de la protéine virale Tat sur l’initiation de la traduction et sur l’efficacité de déphasage. Nous avons montré qu’elle augmente l’initiation de la traduction et que son effet est plus prononcé lorsque TAR est située à l’extrémité 5’ des ARNm. Nous proposons un modèle expliquant les effets de Tat sur l’initiation de la traduction par l’inhibition de PKR ainsi que par des changements de l’expression de protéines cellulaires déroulant TAR. Ces résultats permettent de mieux comprendre les mécanismes régissant le déphasage du VIH-1, ce qui est essentiel pour le développement d’agents anti-déphasage.
The human immunodeficiency virus type 1 (HIV-1) is responsible for the acquired immune deficiency syndrome (AIDS). HIV-1 develops a resistance towards the inhibitors used to treat infected patients. It is thus important to identify new targets for the development of novel antiretroviral agents. The aim of our work was to better characterize the programmed -1 ribosomal frameshift which generates the precursor of HIV-1 enzymes. The frameshift occurs at a specific sequence of HIV-1 full-length messenger RNA (mRNA), the slippery sequence, and is performed by a minority of the ribosomes translating this mRNA. The frameshift efficiency is controlled by the frameshift stimulatory signal (FSS), an irregular stem-loop located downstream of the slippery sequence. FSS structure is unfolded by every ribosome translating this region and can refold afterwards. We showed that HIV-1 frameshift efficiency is affected by changes in the rate of translation initiation. We transfected Jurkat-T and HEK 293T cells with a bicistronic reporter that contains the frameshift region of HIV-1 between the Renilla luciferase (Rluc) and the firefly luciferase (Fluc) genes. Rluc is produced by all ribosomes translating this reporter whereas only ribosomes that make a –1 frameshift produce Fluc. The translation of the reporter is initiated via a cap-dependant mode, like the majority of cellular mRNAs. We first determined the effect of three inhibitors of translation initiation. We showed that their presence increases the frameshift efficiency. We next determined the impact of the TAR stem loop, which is located at the 5’end of every HIV-1 mRNA. TAR is known to impair the binding of the small subunit of the ribosome (40S) to the mRNA. TAR also modulates the activity of the double-stranded RNA-dependent protein kinase (PKR). When PKR is activated, it phosphorylates the eukaryotic initiation factor 2 (eIF2), inhibiting translation initiation. The inhibition of PKR has the opposite effect. We studied the effect of TAR on PKR by positioning TAR at a distance of the 5’ end where it cannot interfere with the binding of the 40S. Our results showed that a small amount of TAR, which activates PKR, increases the frameshift efficiency whereas a large amount of TAR, which inhibits PKR, decreases it. A model is presented where the variations of translation initiation modulate HIV-1 frameshift efficiency by altering the distance between the elongating ribosomes. This influences the probability that these ribosomes encounter or not a folded FSS. We next observed the effect of the 5’ untranslated region (UTR) of HIV-1 full length mRNA on its frameshift efficiency. This 5’UTR contains several structured parts, including TAR at the 5’end, which can inhibit translation initiation. This mRNA has a cap and an internal ribosome entry site (IRES) and could then use a cap dependent and an IRES-dependent mode of translation initiation. We replaced the 5’UTR of our bicistronic reporter mRNA by the complete 5’UTR of HIV-1 full-length mRNA or a part of it. Our results showed that the presence of the complete 5’UTR inhibits cap-dependent initiation of translation and increases the frameshift efficiency. Those effects are mostly due to the presence of TAR followed by a Poly(A) stem-loop. We also constructed a tricistronic reporter where the ribosomes translating the luciferases have to use an IRES-dependent initiation mode. The rate of this initiation was low and the frameshift efficiency obtained was also low. We proposed a hypothesis accounting for this situation. We also observed that when both initiation modes are available, the cap-dependent mode seems to be highly favored. Finally, we studied the impact of the Tat viral protein on translation initiation and frameshift efficiency. We showed that the presence of Tat increases translation initiation and decreases the frameshift efficiency. Those effects are more important when TAR is present at the 5’end of mRNA. We propose a model explaining the effects of Tat on translation initiation by the inhibition of PKR and by changes in the expression of cellular proteins that are able to unfold TAR. Our results allow us to better understand the mechanisms controlling HIV-1 frameshift, which will help in the development of drugs targeting the HIV-1 frameshift.

p53 is a nodal tumor suppressor protein that acts as a major defense against cancers. Approximately 50% of human tumours have mutations in p53 gene. Among its myriad features, the most distinctive is the ability to elicit both apoptotic death and cell cycle arrest. p53 has several isoforms. Most of them are produced by either internal promoter activity of the gene or alternate splicing of the pre-mRNA. Apart from these mechanisms, p53 mRNA has also been shown to be translated into two isoforms, the full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which acts as a dominant-negative inhibitor of FL-p53. Under conditions of cellular stress, the canonical mode of translation initiation is compromised. To maintain the synthesis of proteins important for cell survival and cell-fate decisions, a subset of cellular mRNAs utilizes a non-canonical mode of translation initiation. The 5’ untranslated region of these mRNAs are highly structured and function as Internal Ribosome Entry Site (IRES). Previously, from our laboratory it has been shown that translation of p53 and its N-terminally truncated isoform ΔN-p53 can be initiated by IRES mediated mechanism. IRES mediated translation of ΔNp53 was maximum at G1-S phase but that of FL-p53 was maximum at the G2-M phase. Interestingly in case of a human genetic disorder X-linked dyskeratosis congenita (X-DC), aberrant IRES mediated p53 translation has been reported. It has also been reported that during oncogenic induced senescence (OIS) a switch between cap-dependent to IRES meditated translation occurs in p53 mRNA. From our laboratory, we have also demonstrated that polypyrimidine tract binding protein (PTB) positively regulates the IRES activities of both the p53 isoforms by shuttling from nucleus to the cytoplasm during genotoxic stress conditions. It is very important to understand how these two isoforms are regulated and in turn control the cellular functions. In the first part of the thesis, to investigate the importance of the structural integrity of the cis acting elements within p53 RNA, we have compared the secondary structure of the wild-type RNA with cancer-derived silent mutant p53 RNAs having mutations in the IRES elements such as L22L (CTA to CTG) a natural cancer mutation and Triple Silent Mutation (mutations were present at the wobble position of codon 17, 18, 19). These mutations result in the conformational alterations of p53 IRES RNA that abrogates the IRES function ex vivo significantly. It appears that these mutant RNAs failed to bind some trans-acting factors (p37, p41/44 etc) which might be critical for the IRES function. By super-shift assay using anti hnRNPC1/C2 antibody, we have demonstrated that the TSM mutant showed reduced binding to this protein factor. Partial knockdown of hnRNP C1/C2 showed significant decrease in p53 IRES activity and reduced synthesis of ΔN-p53. Also we have showed that introducing compensatory mutations in TSM mutant RNA rescued the secondary structure as well as function of p53 IRES. Further, the role of another silent point mutation in the coding sequence of p53 was investigated. Silent mutation (CCG to CCA) at codon 36 (P36P) showed decreased IRES activity. The mutation also resulted in differential binding of cellular proteins. Taken together, our observations suggest pivotal role of some specific trans acting factors in regulating the p53-IRES function, which in turn influences the synthesis of different p53 isoforms. In the second part of the thesis, p53 IRES RNA interacting proteins were identified using RNA affinity approach. Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) were identified and their interaction with p53 IRES RNA in vitro and ex vivo was studied. Interestingly, in the presence of Ca2+ ions Annexin A2 showed increased binding with p53 IRES. By competition UV crosslinking we have showed Annexin A2 and PSF interact specifically with p53 IRES. Toe printing assay results showed the putative contact points of Annexin A2 and PSF proteins on p53 IRES RNA. Interestingly, both proteins showed extensive toe-prints in the neighbourhood of the initiator AUG region of p53. Further, competition UV-crosslinking reveals the interplay of these two proteins. Annexin A2 and PSF appear to compete each other for binding with p53 IRES. PSF is known to interact with PTB protein. Since PTB also interacts with p53 IRES and positively regulates the translation, we wanted to study the interplay between PTB and PSF proteins binding with p53 IRES. To address this, we have performed competition UV crosslinking experiment and showed that increasing concentrations of PTB decreases PSF and p53 IRES interaction. However, increasing concentrations of PSF does not decrease or increase in PTB p53 IRES interaction. Results suggest that both Annexin A2 and PSF proteins play important role in regulation of p53 IRES activity. To address the physiological role of Annexin A2 and PSF proteins on p53 IRES activity, these proteins were partially knocked down in cellulo. This in turn showed decrease in p53 IRES activity in dual luciferase assays as well as in the steady state levels of both the p53 isoforms in transient transfection experiments. Heightened or continued expression of p53 protein is very important under stress where IRES-dependent translation supersedes normal cap-dependent translation. Results showed that expression of Annexin A2 under doxorubicin and thapsigargin induced stress are important for maintenance of both p53 IRES activity and steady state levels of p53 isoforms. Earlier from our laboratory we have showed that the IRES responsible for ∆N-p53 translation is active at G1/S phase while the IRES responsible for full length p53 translation is active at G2/M phase. Subcellular localization of the trans-acting factors plays a pivotal role in regulation of IRES activity of cellular mRNA. In this context we wanted to study the nuclear and cytoplasm localization of Annexin A2 under different cell cycle stages. We have seen Annexin A2 protein is dispersed in nucleus and cytoplasm at G1/S boundary, but post-G2 phase it moved from nucleus to cytoplasm. Further we wanted to investigate the effect of Annexin A2 and PSF on expression of p53 transactivated genes. Partial knock down of Annexin A2 and PSF proteins showed decrease in p21 luciferase activity. By real-time PCR analysis, we have also showed decrease in expression of different p53 targets upon silencing of Annexin A2 protein. Taken together, our observations suggest pivotal role of cis acting and trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

碩士
中原大學
化學研究所
95
Baculovirus expression vectors system （BEVS）that based on Autograph californica nucleopolyhedrovirus （AcMNPV） exten¬sively used for protein expression in recent years. Comparing with traditional Prokaryotic （ex. E. coli） and Eukaryotic （ex. Yeast） expression systems, this system has similar post-translation processing with mammalian cells. To extend the applications of BEVS, we have used internal ribosome entry sites （IRES） to construct bi-cistronic baculovirus expression vectors. In this study we explored the activity of PnV539 IRES in insect cells besides IPLB-Sf21. We were also determine the IRES activity of EV71, EMCV and RhPV IRES in mammalian cells （CHO, COS-1, U2-OS ）. The results suggest PnV539 IRES can express EGFP reporter gene in IPLB-Sf21, NTU-LY-3S, IPLB-BmN4 and IPLB-LD652Y. However, the IRES activity of PnV539 IRES in mammalian cells is still unknown. In transfection assay the activity of EV71 IRES in mammalian cells are better than EMCV and RhPV IRES. These results demonstrate the PnV539 IRES might be a good choice for non-lytic bi-cistronic baculovirus expression vectors. In addition the EV71 IRES might be suitable to construct bi-cistronic baculovirus expression vector for gene therapy.

碩士
中原大學
醫學工程研究所
92
IRES (Internal Ribosome Entry Site) provides an alternative, cap-independent translation initiation pathway. This is a typical case of RNA untranslated region (UTR) regulates gene expression. It is interesting to the biologist: How many sequences in the database are structurally similar to those known IRES? In this research, a series of RNA bioinformatics tools have been applied to answer this question. After reviewing the contemporary RNA secondary structure prediction and comparison methods, two RAN bioinformatics tools were implemented to perform two-stage strategy IRES searching flow. In the first stage, the RNALfold program is used to predict locally stable RNA secondary structures. The RNA Align was followed to compare the predefined IRES to the aforementioned RNA structures. The potential IRES regions were screened by the cascaded workflow. To evaluate the workflow performance, four virus complete genomes, including EV71 USA MS strain, Bovine Enterovirus, Human Rhinovirus and Hepatitis C Virus (HCV) were used to searching for IRES domain IV of Enterovirus type 71 (EV71, contain 205 nucleotides). The results shown the workflow can successfully find the IRES of Enterovirus genus. Searching the HCV IRES domain III, which contain 206 nucleotides, in UTRdb virus 5' UTR sequences was also conducted. The prediction sensitivity with the setting of the maximum allowable predicted structure length to 100, 250 and 400 nucleotides was calculated. The results shown that when the length was set to 250, one can find most HCV and Pestivirus IRESes. In addition, it is also found that there are possible target IRES structure in the 5' UTR of Simian Picornavirus 12 (SV45) and Porcine Enterovirus 8 (PEV-8). However, the IRES activity and functional similarity on these regions require further proven by wet-lab experiments.

碩士
長庚大學
醫學生物技術研究所
96
Enterovirus 71 belongs to Picornaviridae family. The 5’untranslated region (5’UTR) of viral genome contains type I internal ribosome entry site (IRES). The main function of the IRES is cap-independent translation. This study attempts to identify the minimal functional sequences of EV71 IRES. The predicted secondary structure of EV71 IRES contains six stem-loops and six spacer regions by M-fold software. The different truncated forms of the IRES were inserted into the pRHF vector that contains a dicistronic expression system. The mutated DNA clone was transfected into HeLa cells, and the results from luciferase assay showed that the deletion of nucleotides 193 to 228 was decreased viral translation activity to about 50%. The deletion of nucleotides 91 to 167(stem-loop II), 242 to 445 (stem-loop IV), 453 to 561 (stem-loop V) and 566 to 636 (stem-loop VI) were loss almost entire IRES activity, suggesting that the stem-loop II, IV, V and VI are important for IRES activity but not the stem-loop III. However, recombinant viruses with deletion mutations generated from infectious clone caused lethality (IF-del II, IF-del IV, IF-del V and IF-del VI.) and deletion III mutation (IF-del III) was rescued. This virus exhibited the same CPE, plaque size and growth rates in comparison with that of wild-type. Taken together, our results suggest that nucleotides 91-636(stem-loop II-VI), but not nucleotides 193 to 228 (stem-loop III) is a major minimal functional region of IRES.

碩士
國立陽明大學
生物化學研究所
88
Hepatitis C virus (HCV), known to be a main causative agent of non-A, non-B hepatitis, is classified in a separate genus of the Flaviviridae family and has a high relationship with liver cirrhosis, chronic hepatitis and hepatocellular carcinoma. HCV is an envelope RNA virus. The genome of HCV consists of a single-stranded, positive-sense RNA of approximately 9500 nucleotides. It is translated in to a single polypetides of ~3010 amino acid in length. The 5’nontranslation region (5’NTR) of the HCV genome is 341 nucleotides in length. Extensive sequence analyses showed that the 5’ noncoding region is highly conserved among different HCV isolates. A complex secondary structure has been proposed for the HCV 5’NTR. These features, indicate that translation of HCV RNA is initiated by binding of the ribosome to an internal ribosome entry site (IRES). According to the experiment evidence derived from expression studies of dicistronic mRNA in vitro, point out that HCV IRES is an independent folding motif (IFM). There are four domains in the IRES, and we suppose that each domain is also an IFM. So we designed four kinds of deletion mutants depends on different domains combination ( 1~371 includes entire IRES and up to 30 nt of coding sequence ; 1~341 is IRES ; 1~300 is domain I~III ; 1~124 is domain I~II ) to test the binding ability of the RNA with HeLa cell extract. These results show that each deletion mutant could specific bind cell extract and the 1~341 RNA segment has strongest binding affinity. It suggests that HCV could control virus replication by modify the IRES secondary structure with 341~471, 30 nt. 英文摘要…………………………………………………………………….ii 壹、緒論………………………………………………………………………1 C型肝炎病毒的發現………………………………………………………….1 C型肝炎病毒基因體之特性………………………………………………….1 C型肝炎病毒對寄主細胞蛋白質合成之影響……………………………….3 獨立摺疊單元…………………………………………………………………4 貳、實驗材料與方法……………………………………………………….12 實驗材料…………………………………………………………………….12 實驗方法…………………………………………………………………….13 質體之小量純化………………………………………………….13 質體之大量純化………………………………………………….14 PCR反應…………………………………………………………….16 RNA合成…………………………………………………………….17 細胞培養………………………………………………………….18 細胞抽出物之製備……………………………………………….18 Band shift assay……………………………………………….19 電泳……………………………………………………………….19 參、結果…………………………………………………………………….23 利用PCR產生所設計的刪除式突變DNA模板……………………………..23 刪除式突變之RNA合成………………………………………………………25 測試刪除式突變之RNA是否與細胞抽出物作用……………………………26 測試刪除式突變之RNA與細胞抽出物作用是否為專一性作用……………28 肆、討論…………………………………………………………………….32 比較各段刪除式突變RNA與細胞抽出物之結合能力………………………32 細胞因子與IRES之作用………………………………………………………35 伍、參考文獻……………………………………………………………….36

Internal ribosome entry sites (IRES) are segments of the mRNA found in untranslated regions, which can recruit the ribosome and initiate translation independently of the more widely used 5’ cap dependent translation initiation mechanism. IRES play an important role in conditions where has been 5’ cap dependent translation initiation blocked or repressed. They have been found to play important roles in viral infection, cellular apoptosis, and response to other external stimuli. It has been suggested that about 10% of mRNAs, both viral and cellular, can utilize IRES. But due to the limitations of IRES bicistronic assay, which is a gold standard for identifying IRES, relatively few IRES have been definitively described and functionally validated compared to the potential overall population. Viral and cellular IRES may be mechanistically different, but this is difficult to analyze because the mechanistic differences are still not very clearly defined. Identifying additional IRES is an important step towards better understanding IRES mechanisms. Development of a new bioinformatics tool that can accurately predict IRES from sequence would be a significant step forward in identifying IRES-based regulation, and in elucidating IRES mechanism. This dissertation systematically studies the features which can distinguish IRES from nonIRES sequences. Sequence features such as kmer words, and structural features such as predicted MFE of folding, Q_MFE, and sequence/structure triplets are evaluated as possible discriminative features. Those potential features incorporated into an IRES classifier based on XGBboost, a machine learning model, to classify novel sequences as belong to IRES or nonIRES groups. The XGBoost model performs better than previous predictors, with higher accuracy and lower computational time. The number of features in the model has been greatly reduced, compared to previous predictors, by adding global kmer and structural features. The trained XGBoost model has been implemented as the first high-throughput bioinformatics tool for IRES prediction, IRESpy. This website provides a public tool for all IRES researchers and can be used in other genomics applications such as gene annotation and analysis of differential gene expression.

碩士
國立陽明大學
醫學生物技術暨檢驗學系暨研究所
99
Enterovirus 71 (EV71), a member of Picornaviridae family, can lead to severe neurological complications, and has caused several large outbreaks in Taiwan since 1998. The translation of enterovirus occurs via internal ribosomal entry site (IRES) at the 5’ untranslated region (5’-UTR) of the virus RNA, and is modulated by certain cellular factors called IRES trans-acting factors (ITAFs). Nowadays, there is no vaccine or antiviral agent known to be effective in treating or preventing EV71 infection. Previously, in our laboratory has showed that knockdown of UNR, one of the known ITAFs, significantly decreased the IRES activity of the EV71, and UNR could bind domain X in the EV71 5’-UTR as analyzed by pulldown assay. In this study, we used the in vitro selection procedure to isolate RNA aptamers capable of binding to the EV71 IRES domain X and UNR. Ten aptames were obtained; among them four isolates contain the same sequences. We also show that the aptamers could block the interaction between UNR and EV71 IRES domain X by pulldown assay. Additionally, we analyzed the inhibitory potency of the aptamers on the the EV71 IRES activity by the in vitro translation/ luciferase reporter assay. We show that aptamers could inhibit the IRES activity in a dose-dependent manner with the IC50 ranging from 0.24~1.28 µM. The results provide the evidence that viral translation initiation may serve as a novel anti-viral stragegy, and that the selected RNA aptamers have great potential as anti-EV71 drugs.

碩士
國立臺灣大學
生物化學暨分子生物學研究所
92
Hepatitis C virus (HCV), the major infectious agent of non-A, non-B hepatitis, often causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV is a positive-sense, single-stranded RNA virus of genomic size 9.6 kb in length. The viral genome consists of a 5’ noncoding region (NCR), a large open reading frame encoding a polyprotein of approximately 3000 amino acids, and a 3’ NCR. The 5’NCR is highly conserved among HCV isolates. Translation initiation of HCV is controlled by the internal ribosome entry site (IRES) encompassing almost the entire 5’NCR and the sequences about 30 nt downstream the AUG codon. The IRES folds into a stable secondary and tertiary structure and functionally replaces several initiation factors by directly recruiting the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). The eIF3 complex is composed of at least ten different proteins with 650 kDa. Two subunits of eIF3 complex, p116 and p170, have been demonstrated to specifically bind to the domain Ⅲ of HCV IRES. According to secondary structure prediction, p116 contains a putative RNA recognition motif (RRM) near the N terminal region (a.a. 185-268). By performing gel retardation assay and site-directed mutagenesis previously, our laboratory has demonstrated the interaction between p116-RRM and the domain Ⅲ of HCV IRES. Nevertheless, the p116 subdomain from amion acid 227 to 320 was recently identified to interact with the domain Ⅱ apical part of HCV IRES by using the technique of SERF (selection of random RNA fragments). In this study, binding ability of eIF3 p116 subunit to HCV IRES domain Ⅱ and domain Ⅲ were further examined to learn the roles of the cis-elements involved in the internal initiation of translation. A similar binding ability of p116-RRM to the HCV IRES domain Ⅱ (nt 65-102) and the domain Ⅲabcd (nt 131-278) was found by filter binding assay. An attempt to set up the SERF technique was also made to determine the minimal binding region of p116-RRM on the HCV IRES. In addition, by adding the RNA representing the apical domain of domain Ⅱor domain Ⅲabcd as competitor in an in vitro translation assay, the synthesis of core protein mediated by the HCV IRES was inhibited. The studies of interaction between the eIF3 subunit and HCV IRES will help us to understand the control mechanisms of the internal initiation of HCV.

The ribosome is a macromolecular machine, present in high copy number in the cell, that synthesizes proteins from information encoded in messenger RNA. It is a universal translator, found in all life forms and in all eras recent enough to bear life. The ribosome is structurally complex and its structure is highly evolutionarily conserved; that conservation reinforces the concept that its function in executing translation is essential. As a subject of study, the ribosome lends itself well to direct imaging, as it is large, asymmetric, dynamic, and it interacts with other heterogeneous agents throughout the translation process; if we are to infer function from structure, then the most certain way to observe the ribosome’s structure is to image it as directly as possible. Cryo-electron microscopy and single particle reconstruction are appropriate tools for this endeavor, as they can produce high-resolution three-dimensional structures of ribosomes or other macromolecular samples, and they can even reveal multiple biologically relevant states of a single sample. Although the ribosome is highly conserved in terms of its presence and core structure and functions, there is considerable variation among taxa, and the function of some of this variation is not yet understood. For example, the ribosome of the unicellular trypanosomatid parasite Leishmania major exhibits unusually large expansion segments of ribosomal RNA, as well as unusual cleavage sites in ribosomal RNA that is otherwise conserved. Here, we present a three-dimensional cryo-electron microscopy reconstruction of the 80S ribosome of Leishmania major and compare it to the available ribosome structures of closely related parasites. There is also structural variation related to the mechanism of translation: certain viruses with RNA genomes employ highly structured segments of RNA called internal ribosome entry sites to initiate translation of viral proteins on host cell ribosomes via noncanonical mechanisms. We explore one instance of this with a reconstruction of the encephalomyocarditis virus internal ribosome entry site bound with necessary protein factors to a eukaryotic 40S ribosomal subunit.

Host-pathogen interactions in Hepatitis C Virus infection: Deciphering the role of host proteins and microRNAs Hepatitis C virus (HCV) is a positive sense single stranded RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV genome consists of a single open reading frame flanked by highly structured 5‟ and 3‟ untranslated regions (UTRs) at both ends. Unlike cellular mRNAs, HCV RNA translation is independent of the cap structure and is mediated by an internal ribosomal entry site (IRES) present in the 5‟UTR. HCV replication begins with the synthesis of a complementary negative-strand RNA using the positive strand RNA genome as a template catalyzed by the NS5B RNA dependent RNA polymerase (RdRp). The de novo priming of HCV RNA synthesis by NS5B occurs at the very end of the 3‟UTR. The 3‟UTR is organized into highly structured regions namely the variable region, poly U/UC region and the 3‟X region. These regions contain cis-acting elements that determine the efficiency of viral replication. In addition, the interaction of trans-acting factors with the 3‟ UTR is also important for regulation of HCV replication. HCV 3‟UTR interacts with several cellular proteins such as the human La protein, polypyrimdine tract binding protein (PTB), poly (rC)-binding protein 2 (PCBP2) and Human antigen R (HuR). However, the molecular basis of regulation of viral replication by these proteins is not well understood. Many proteins that are hijacked by HCV as well as other cytoplasmic RNA viruses, such as La, PCBP2, HuR and PTB are RNA binding proteins (RBPs). They are involved in post transcriptional regulation of cellular gene expression. Thus the subversion of these proteins by the virus can affect their normal physiological functions. In addition to proteins, recent reports also describe the involvement of non-coding RNAs including microRNAs (miRNA) and long non coding RNAs (lncRNA) in HCV infection. miRNAs can either directly bind to the HCV genome and regulate its life cycle or indirectly modulate the expression of host proteins required by the virus. miRNAs that are differentially regulated in virus infected tissues or body fluids of infected patients can also serve as biomarkers for diagnosis of various stages of the disease. Hence, it was planned to study the role of host proteins and miRNAs in the HCV life cycle and pathogenesis to have novel insights into the biology of HCV infection. Riboproteomic studies have identified several host proteins that directly interact with the 5‟ and/or 3‟UTRs of the HCV RNA. One of the RNA binding proteins that predominantly interact with the 3‟UTR of HCV RNA was found to be HuR. In the present study, we have extensively characterized the interaction between HuR and HCV 3‟UTR and studied its functional implications in HCV life cycle along with other host factors. Characterizing the HCV 3’UTR–HuR interaction and its role in HCV replication HuR is a ubiquitously expressed member of the Hu family which shuttles between the nucleus and cytoplasm in response to stress. Whole genome siRNA knockdown and other studies have suggested that HuR is essential for HCV replication. However, the molecular mechanism of its involvement in this process was not clear. We observed that siRNA mediated knockdown of HuR reduces the HCV RNA and protein levels. Immunofluorescence studies indicated that HuR relocalizes from the nucleus to the cytoplasm in HCV infected cells. Through confocal microscopy and GST pulldown assays, we have demonstrated that HuR co localizes with the viral polymerase, NS5B and directly interacts with the NS5B protein. Membrane flotation assays showed that HuR is present in the detergent resistant membrane fractions which are the active sites of HCV replication. In addition to the interaction of HuR with the viral protein NS5B, we also characterized its interaction with the viral RNA. Direct UV cross linking assays and UV cross linking immunoprecipitation assays were performed to demonstrate the interaction of HuR with the HCV 3‟UTR. The RRM3, hinge region and RRM1 of HuR were found to be important for binding. Further, we observed that HuR competes with PTB for binding to the 3‟UTR when cytoplasmic S10 extracts or recombinant proteins were used in UV cross linking assays. In contrast, the addition of HuR facilitated the binding of La protein to the HCV 3‟UTR in the above assays. Competition UV cross linking assays indicated that both HuR and PTB bind to the poly U/UC region of the 3‟UTR while La binds to the variable region. HuR and La showed higher affinities for binding to the 3‟UTR as compared to PTB in filter binding assays. Since HuR and PTB interact with the same region on the 3‟UTR and HuR showed ~4 fold higher affinity for binding, it could displace PTB from the 3‟UTR. Next, we investigated the roles of HuR, PTB and La in HCV translation and replication in cell culture using three different assay systems, HCV sub genomic replicon, HCV bicistronic SGR-JFH1/Luc replicon as well as the infectious HCV full length RNA (JFH1). Results clearly indicated that HuR and La are positive modulators of HCV replication. Interestingly, PTB facilitated HCV IRES mediated translation but appeared to have a negative effect on HCV replication. The positive effectors, HuR and La showed significant co localization with one another in the cytoplasm in immunofluorescence studies. GST pulldown and coimmunoprecipitation experiments indicated protein-protein interactions between HuR and La but not between HuR and PTB. Through quantitative IP-RT assays, we demonstrated that the overexpression of HuR in HCV RNA transfected cells increases the association of La with the HCV RNA while HuR knockdown reduces the association of La with the HCV RNA. Previous studies in our laboratory have shown that La helps in HCV genome circularization. The addition of HuR significantly increased La mediated interactions between the 5‟UTR and the 3‟UTR of HCV RNA as monitored by 5‟-3‟ co precipitation assays, suggesting a possible mechanism by which cooperative binding of HuR and La could positively regulate HCV replication. Taken together, our results suggest a possible interplay between HuR, PTB and La in the regulation of HCV replication. Studying the role of HuR- associated cellular RNAs in HCV infection HuR belongs to the category of mRNA turnover and translation regulatory proteins (TTR-RBPs), which are capable of triggering rapid and robust changes in cellular gene expression. HuR plays a role in several post transcriptional events such as mRNA splicing, export, stability and translation. In the present study, we have investigated the possible consequences of relocalization of HuR on cellular processes in the context of HCV infection. We observed that 72h post transfection of infectious HCV-JFH1 RNA, there is an increase in the mRNA levels of some of the validated targets of HuR including the vascular endothelial growth factor A (VEGFA), dual specificity phosphatise 1 (MKP1) and metastasis - associated lung adenocarcinoma transcript (MALAT1). IP-RT assays demonstrated that the association of HuR with VEGFA and MKP1 was higher in HCV-JFH1 RNA transfected cells as compared to the mock transfected cells indicating that increase in HuR association could probably help in stabilization of these mRNAs. Interestingly, we observed that the association of HuR with the lncRNA MALAT1 decreases in the presence of HCV RNA, while its RNA levels increased. Earlier it has been reported that MALAT1 interacts with HuR and was predicted to interact with La. We confirmed the interaction of both HuR and La proteins with MALAT1 RNA in vitro and in the cell culture system. Results from our time course experiments suggest that relocalization of HuR and La upon HCV infection might decrease their association with the nuclear retained MALAT1 RNA leading to significant reduction in MALAT1 RNA levels at the initial time points. However at later time points, MALAT1 was found to be unregulated through activation of the Wnt/beta-catenin pathway as demonstrated using a chemical inhibitor against β-catenin. Since MALAT1 is a known regulator of epithelial mesenchymal transition (EMT) and metastasis, we further studied the physiological consequence of the observed increase in MALAT1 levels upon HCV infection. Cell migration and cell invasion studies suggested that the knockdown of MALAT1 led to the inhibition of HCV- triggered wound healing and matrigel invasion and also rescued the down regulation of E-Cadherin protein levels, an EMT marker. Our study highlights the importance of the lncRNA, MALAT1 in HCV infection and suggests its possible involvement in HCV induced HCC. Investigating the role of miRNAs in HCV pathogenesis and replication miRNAs can also regulate HCV infection and pathogenesis in multiple ways. It is known that under disease conditions, there is aberrant expression of intracellular as well as circulating miRNAs. We have investigated the expression profile of 940 human miRNAs in HCV infected patient serum samples to identify the differentially regulated miRNAs. miR-320c, miR-483-5p and the previously reported miR-125b were found to be upregulated in the serum of cirrhotic and non-cirrhotic HCV infected patient serum samples. All three miRNAs were also unregulated in the cell culture supernatant of HCV infected cells as well as within the HCV infected cells. miR-483-5p was specifically enriched in the exosomes isolated from patient serum samples. Knockdown of miR-320c and miR-483-5p did not have significant effect on HCV replication while knockdown of miR-125b affected HCV replication through regulation of one of its target genes, HuR. We observed that with time, miR-125b levels in HCV-JFH1 RNA transfected cells increase while the HuR protein levels decrease. Using luciferase reporter constructs, we demonstrated that the decrease in HuR protein levels is indeed mediated by miR-125b. Mutations in the target site of miR-125b in the HuR 3‟UTR prevented the down regulation of luciferase activity. Next we tested the effect of silencing miR-125b on HCV replication. Knockdown of miR-125b prevented the reduction in HuR protein levels but with no significant effect on HCV replication. It appeared that the HuR protein already present in the cytoplasm could be sufficient to support HCV replication. Hence similar experiments were carried out in cells depleted of HuR using either siRNA against HuR or a chemical inhibitor of nucleocytoplasmic transport of HuR, Leptomycin B. We observed that when the intracellular levels of HuR are reduced using either of the two approaches, there is a decrease in HCV replication. This is in accordance with the results obtained in the first part of the thesis. However when miR-125b was silenced in HuR depleted cells, we noticed an upregulation in the HuR protein levels by western blot analysis and a consequent increase in HCV RNA levels as quantified by qRT-PCR. From our findings, we can conclude that miR-125b mediated regulation of HuR plays an important role in HCV replication. We hypothesize that this could be a cellular response to HCV infection to which the virus responds by inducing protein relocalization. Altogether, these studies outline the importance of host factors including cellular proteins and non-coding RNAs in the regulation of HCV life cycle and pathogenesis. Results reveal the mechanistic insights into how HCV infection triggers host defense pathways, which are evaded by the virus by counter strategies.

Background Promoters are key players in gene regulation. They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG http://corg.molgen.mpg.de, a framework for studying upstream regions including untranslated exons (5' UTR). Description The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences. As of now, we have included 5 species in our analysis pipeline (man, mouse, rat, fugu and zebrafish). We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server http://tomcat.molgen.mpg.de:8080/das. The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set. Conclusion The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites).

碩士
國立成功大學
生物資訊與訊息傳遞研究所
100
Ribosome is composed of rRNAs, ribosomal proteins, and many non-ribosomal factors to conduct the translation function. Traditionally, ribosomal proteins were considered as co-factors to execute the protein translation. But, numerous studies have demonstrated that ribosomal proteins not only play as co-factors of translational complex but also regulate the protein synthesis of specific mRNAs. RPL19 is a component of ribosome large subunit which belonged to the L-19E super-family and conserved among eukaryotes. In previous study, RPL19 was reported to have an impact on cyclin D1 protein expression but not on other cell cycle regulators, which indicated RPL19 may be a regulator of specific cell cycle regulators. During cell cycle progression, internal ribosome entry site (IRES) was reported to mediate the translational regulation of many cell cycle regulators. Since cyclin D1 expression was reported to be regulated by RPL19 and the 5’UTR of cyclin D1 mRNA carries a potential IRES element, the hypothesis was reported that RPL19 may regulate the expression of cyclin D1 through IRES. To address it, cells were synchronized and following experiments were conducted. First, western blot analysis showed that RPL19 expression level remained unchanged during cell cycle progression. However, RNA-IP showed that RPL19 interacted with cyclin D1 mRNAs at G1/S boundary. Bicistronic reporter assay showed that the 5’UTR of cyclin D1 had strong IRES activity and was regulated by RPL19. IRES-mediated translation regulation is often facilitated with the help of IRES trans-acting factors (ITAFs). RPL19 cooperated with a known ITAF, hnRNP A1, to regulate the IRES activity of cyclin D1. Furthermore, we observed that down-regulation of RPL19 significantly decreased the proliferation rate of HeLa cells. To sum up, we identified that RPL19, a ribosomal protein, can cooperate with hnRNPA1 to regulate cell cycle progression through regulating the IRES activity of cyclin D1.