Résumé La Ribonucléase P (RNase P) est une enzyme principalement reconnue pour sa participation à la maturation en 5’des ARN de transfert (ARNt). Cependant, d’autres substrats sont reconnus par l’enzyme. En général, la RNase P est composée d’une sous-unité ARN (le P-ARN, codé par le gène rnpB) qui porte le centre actif de l’enzyme et d’une ou de plusieurs sous-unités protéiques (la P-protéine). Les P-ARN chez toutes les bactéries, la majorité des archéobactéries et dans le génome nucléaire de la plupart des eucaryotes, possèdent généralement une structure secondaire très conservée qui inclut le noyau (P1-P4); l’hélice P4 constitue le site catalytique de l’enzyme et l’hélice P1 apparie les extrémités du P-ARN en stabilisant sa structure globale. Les P-ARN mitochondriaux sont souvent moins conservés et difficiles à découvrir. Dans certains cas, les seules régions de structure primaire qui restent conservées sont celles qui définissent le P4 et le P1. Pour la détection des gènes rnpB, un outil de recherche bioinformatique, basé sur la séquence et le profil de structure secondaire, a été développé dans le laboratoire. Cet outil permet le dépistage de toutes les séquences eucaryotes (nucléaires et mitochondriales) du gène avec une très grande confiance (basée sur une valeur statistique, E-value). Chez les champignons, plusieurs ascomycètes encodent un gène rnpB dans leur génome mitochondrial y compris tous les membres du genre d’Aspergillus. Cependant, chez les espèces voisines, Neurospora crassa, Podospora anserina et Sordaria macrospora, une version mitochondriale de ce gène n’existe pas. Au lieu de cela, elles contiennent deux copies nucléaires du gène, légèrement différentes en taille et en contenu nucléotidique. Mon projet a été établi dans le but d’éclaircir l’évolution de la RNase P mitochondriale (mtRNase P) chez ces trois espèces voisines d’Aspergillus. En ce qui concerne les résultats, des modèles de structures secondaires pour les transcrits de ces gènes ont été construits en se basant sur la structure consensus universelle de la sous-unité ARN de la RNase P. Pour les trois espèces, par la comparaison de ces modèles, nous avons établi que les deux copies nucléaires du gène rnpB sont assez distinctes en séquence et en structure pour pouvoir y penser à une spécialisation de fonction de la RNase P. Chez N. crassa, les deux P-ARN sont modifiés probablement par une coiffe et les extrémités 5’, 3’ sont conformes à nos modèles, ayant un P1 allongé. Encore chez N. crassa, nous avons constaté que les deux copies sont transcrites au même niveau dans le cytoplasme et que la plus petite et la plus stable d’entre elles (Nc1) se retrouve dans l’extrait matriciel mitochondrial. Lors du suivi du P-ARN dans diverses sous-fractions provenant de la matrice mitochondriale soluble, Nc1 est associée avec l’activité de la RNase P. La caractérisation du complexe protéique, isolé à partir de la fraction active sur un gel non dénaturant, révèle qu’il contient au moins 87 protéines, 73 d’entre elles ayant déjà une localisation mitochondriale connue. Comme chez la levure, les protéines de ce complexe sont impliquées dans plusieurs fonctions cellulaires comme le processing de l’ADN/ARN, le métabolisme, dans la traduction et d’autres (par exemple : la protéolyse et le repliement des protéines, ainsi que la maintenance du génome mitochondrial). Pour trois protéines, leur fonction est non déterminée. / Abstract Ribonuclease P (RNase P) is an endonuclease that cleaves 5’- leader sequences from tRNA precursors and a few other small RNAs. In most cases, the enzyme is a ribonucleo-protein complex (ribozyme), containing an RNA subunit (P-RNA; encoded by the rnpB gene) that carries the active centre of the enzyme, plus one or more protein subunits. P-RNAs in Bacteria, Eukarya and Archaea have a highly conserved secondary structure including the core P1 and P4 helices. P4 forms the catalytic site of the ribozyme, and P1 pairs the RNA termini, stabilizing overall structure and protecting from nuclease degradation. For processing of mitochondrial (mt) tRNAs, certain eukaryotic species (e.g., Saccharomyces cerevisiae, Aspergillus nidulans) have separate mtDNA-encoded P-RNAs (of bacterial origin). Mt P-RNAs are often less conserved, and difficult to discover. To identify rnpB genes, we have developed a search tool based on sequence plus secondary structure profiles. It predicts all known eukaryotic (nuclear and organellar) rnpB genes with high confidence (based on E-values). In fungi, many ascomycetes encode a mitochondrial rnpB gene, including all members of Aspergillus. Yet, the closely related Neurospora crassa, Podospora anserina and Sordaria macrospora lack an mtDNA-encoded gene version. Instead, they contain two nuclear gene copies with slightly different sequences. My project aims to elucidate the evolution of mitochondrial RNase P in these three closely related species. We have established secondary structure models based on comparisons with the universal minimum consensus secondary structure for all nuclear gene mtP-RNAs copies in all three species. By comparison of these secondary structure models, we have established that the two nuclear copies of rnpB gene are quite distinct in sequence and structure, suggesting a specialization of function. In N. crassa, both P-RNAs are modified most likely by capping, and 5’- 3’ termini perfectly conform to P-RNA structure models that have an elongated P1 helical pairing. Furthermore, we find that the two nuclear copies of rnpB gene are present at about the same level in the cytoplasm, and that the shorter form of P-RNA (Nc1) translocates into the (soluble) mitochondrial matrix. When tracing P-RNA in different mitochondrial sub-fractions of a native gel, the presence of Nc1 and mitochondrial RNase P activity are associated. A proteomics characterization of a P-RNA complex isolated by native gel electrophoresis reveals that it contains at least 87 proteins, 73 of which are of known mitochondrial localization. Like in yeast, the complex contains proteins potentially involved in other DNA/RNA processing activities, but also in translation, in metabolism, and in protein folding. Only three proteins are of unknown function.

Bien que le Virus Respiratoire Syncytial, responsable de la bronchiolite du nourrisson, soit aujourd’hui un problème de santé publique majeur, il n’existe encore aucun vaccin ou antiviral curatif contre ce pathogène. Le manque de données sur les étapes clés du cycle viral et sur les interactions virus-cellule freine le développement de nouvelles molécules antivirales.Nous avons étudié l’interactome de deux protéines virales : la polymérase L et le facteur de transcription M2-1. Dans ce but, nous avons mis au point un crible s’appuyant à la fois sur des critères d’interactomique et sur des critères fonctionnels.La première étape consistait à identifier des partenaires potentiels de M2-1 et L par des co-immunoprécipitations couplées à une approche de protéomique quantitative. Pour plus de pertinence, ce crible a été réalisé sur cellules infectées, grâce des virus recombinants produits par génétique inverse. Ceci nous a permis d’identifier 45 et 137 partenaires potentiels de L et M2-1 respectivement. Une étude systématique de l’impact de l’inhibition de 15 partenaires potentiels de M2-1 sur la multiplication virale a mis en avant trois candidats : ILF2, PABPN1 et PABPC1.Nous nous sommes par la suite concentrés sur PABPC1. L’inhibition de l’expression de PABPC1 altère la multiplication virale, mais nous n’avons pas pu mettre en évidence un effet spécifique sur la transcription ou la traduction virale. Son interaction avec M2-1 a été confirmée, et le domaine MLLE de PABPC1 a été identifié comme le site de liaison à M2-1. L’interaction entre M2-1 et PABPC1 a été observée à la fois dans le cytoplasme et dans les IBAGs, des sous-structures concentrant les ARNm viraux au sein des corps d’inclusion viraux. Nous avons formulé l’hypothèse que M2-1, liée à PABPC1, accompagne les ARNm viraux après leur sortie des corps d’inclusion. Ceci suggère un rôle de M2-1 dans le devenir des ARNm viraux en aval de leur transcription. / Although the Respiratory Syncytial Virus, responsible of bronchiolitis in infants, represents a major public health problem, there are currently no vaccine or curative antiviral directed against it. The lack of information on key steps of its viral cycle and on virus-cell interactions hinders the development of new antiviral molecules.We chose to study the interactome of two viral proteins: the polymerase L and the transcription factor M2-1. To do so, we developed a screen based on interactomic and functional criteria.The first step consisted in identifying potential binding partners of M2-1 and L by co-immunoprecipitations coupled to quantitative proteomics. For better relevance, this screen was realised on infected cells, thanks to recombinant viruses produced by reverse genetics. 45 and 137 potential binding partners of M2-1 and L respectively were thus identified. A systematic study of the inhibition of 15 potential partners of M2-1 and its impact on viral multiplication enabled the selection of three candidates: ILF2, PABPN1 and PABPC1.We chose to concentrate on PABPC1. The inhibition of PABPC1’s expression reduces viral multiplication, but no specific effect on viral transcription or translation was brought to light. Its interaction with M2-1 was confirmed, and the MLLE domain of PABPC1 was identified as the M2-1 binding site. The interaction between M2-1 and PABPC1 was observed both in the cytoplasm and in IBAGs, substructures of viral inclusion bodies where viral mRNA accumulate. We formulated the hypothesis that M2-1, with PABPC1, stays with viral mRNA after leaving inclusion bodies and during their translation. This suggests a role for M2-1 in the fate of viral mRNA downstream of transcription.

No description available.

No description available.

Le laboratoire a identifié récemment la protéine DDB2 comme ayant une activité dans la régulation de l’expression de gènes cibles tels que SOD2, BCL2 et NFKBIA, conférant ainsi à cette protéine un rôle dans le contrôle de la progression métastatique et dans la réponse thérapeutique des cellules tumorales mammaires. Cependant, l’activité réelle de DDB2 dans la transcription génique reste à définir, car sa structure ne permet pas d’expliquer son influence sur l’expression de ses gènes cibles. Cette activité peut être soit inhibitrice comme pour SOD2 et BCL2, soit activatrice comme NFKBIA qui code la protéine IκBα, suggérant que DDB2 doit s’associer avec des inhibiteurs ou des activateurs de la transcription génique, ou en favoriser le recrutement. Afin d’identifier ces partenaires potentiels, susceptibles de participer à son activité transcriptionnelle, nous avons développé une approche de « DNA pull-down », couplée à une analyse protéomique globale par spectrométrie de masse à partir des cellules tumorales mammaires MCF-7 surexprimant naturellement DDB2. Nous avons révélé la présence du complexe UV-DDB (DDB2, DDB1 et Cul4A) sur le promoteur des gènes SOD2 et NFKBIA. Nous avons également mis en évidence que ce complexe, connu pour participer aux premières étapes de la réparation de l’ADN lésé par des rayonnements UV, favorise le recrutement de l’histone acétyl transférase p300 sur le promoteur du gène NFKBIA, expliquant en partie le rôle activateur de DDB2 sur ce gène cible. Notre analyse protéomique à révéler, avec un score élevé, la présence des protéines hnRNP K et J sur le promoteur du gène NFKBIA et d’une manière indépendante de toute interaction physique avec DDB2. La forme J, très peu décrite, présente une affinité plus grande pour le promoteur du gène NFKBIA que la forme K. De plus, nous l’avons observée strictement nucléaire et liée à la chromatine. De manière intéressante, nous montrons que la forme J est surexprimée dans le noyau des cellules tumorales MDA-MB231 métastatiques, comparativement aux cellules T47D non métastatiques. Par la suite, nous avons évalué l’importance des hnRNP K et J dans la transcription du gène NFKBIA par rapport à DDB2 et un régulateur bien décrit tel que le facteur de transcription Sp1. Nos résultats indiquent que les protéines hnRNP K et J, lorsqu’elles sont surexprimées, jouent un rôle de répresseur du gène NFKBIA et ce même en présence des activateurs DDB2 et Sp1. L’ensemble de ce travail a contribué à montrer la présence de protéines, pouvant participer à l’activité transcriptionnelle de DDB2, telles que le complexe UV-DDB et p300. En dehors de tout partenariat avec DDB2, il a été mis en évidence une relation entre les hnRNP K et J et l’activité constitutive de NF-κB, en particulier avec la forme J, qui, par son expression corrélée à l’agressivité des cellules tumorales mammaires, présente un intérêt clinique potentiel en tant que marqueur prédictif de la progression métastatique, tout comme DDB2 / The laboratory has recently identified the DDB2 (Damaged-DNA Binding 2) protein as a regulator of target gene expression like SOD2, BCL2 and NFKBIA, thus conferring to this protein a role in control of metastatic progression and therapeutic response of breast cancer cells. However, the real activity of DDB2 in gene transcription remains to be defined because its structure cannot entirely explain its influence on target gene expression. This protein can act either as an inhibitor like for the SOD2 and BCL2 genes or as an enhancer like for the NFKBIA gene, encoding IκBα protein. This suggests that DDB2 must associate with, or promote recruitment, of inhibitors or activators of gene transcription. In order to search and identify potential partners that could participate in its transcriptional activity, we developed a “DNA pull-down” approach associated with a global proteomic analysis by mass spectrometry from MCF-7 breast cancer cells overexpressing naturally DDB2. With this approach, we reveal the presence of the UV-DDB complex composed by DDB2, DDB1 and Cullin 4A proteins on the SOD2 and NFKBIA gene promoters. We also highlighted that this complex, known for its role in first steps of UV-induced DNA lesion repair, promotes the recruitment of the p300 histone acetyl transferase on the NFKBIA gene promoter, which may explain, in part, the enhancer activity of DDB2 on this target gene. The proteomic analysis from the “DNA pull-down” also reveals, with originality, the presence of heterogeneous ribonucleoproteins K and J (hnRNP K and J) on the NFKBIA gene promoter with a high recovery score among many other proteins and independently of any physical interaction with DDB2. The J form, very poorly described, shows a higher affinity for NFKBIA gene promoter than the K form. Furthermore, we observed that the J form is strictly nuclear and mostly bound to chromatin, while the K form is also found in cytoplasm. Interestingly, we show that the J form is overexpressed in nucleus of metastatic breast cancer MDA-MB231 cells by comparison with non-metastatic breast cancer T47D cells. Then, we evaluated the importance of hnRNP K and J proteins in NFKBIA gene transcription compared with DDB2 and with a well-known regulator, the Sp1 transcription factor. Our results show that hnRNP K and J proteins, when they are overexpressed, play a repressor role on NFKBIA gene expression by binding on its promoter even in presence of DDB2 and Sp1 activators. Taken together, these data show that some proteins could participate in DDB2 transcriptional activity, like the UV-DDB complex and the p300 protein. Outside of any interaction with DDB2, this work highlights a relationship between the hnRNP K and J proteins, and NF-κB constitutive activity, especially with the J form. This latter has an expression correlated with aggressiveness of breast cancer cells and a potential clinical interest as a predictive marker of metastatic progression, like DDB2

Dans les dernières années, une explosion de la recherche sur les ARN a eu lieue à cause de nombreuses découvertes démontrant l’importance de l’ARN dans plusieurs processus biologiques. Ainsi, de grandes quantités d’ARN sont devenues indispensables au bon déroulement de plusieurs études, notamment pour la biologie structurale et la caractérisation fonctionnelle. Cependant, il existe encore peu de méthodes de purification simples, efficaces, fiables et produisant un ARN sous forme native. Dans les dernières années, le laboratoire Legault a mis au point une méthode de purification par affinité utilisant une étiquette ARiBo pour la purification d’ARN transcrits in vitro par la polymérase à ARN du phage T7. Cette méthode de purification d’ARN a été spécifiquement développée pour maximiser la pureté et le rendement. De plus, elle est très rapide et fonctionne avec plusieurs types d’ARN. Cependant, comme plusieurs autres méthodes de purification, cette méthode produit des ARN avec des extrémités 5′ hétérogènes. Dans ce mémoire, des solutions sont proposées pour remédier au problème d’hétérogénéité en 5ʹ′ des ARN transcrits avec la polymérase à ARN du phage T7 et purifiés par la méthode ARiBo. La première solution consiste à choisir la séquence en 5′ parmi celles des 32 séquences testées qui ne présentent pas d’hétérogénéité en 5ʹ′. La seconde solution est d’utiliser une étiquette clivable en 5ʹ′ de l’ARN d’intérêt, tel que le ribozyme hammerhead, déjà utilisée pour ce genre d’application, ou le système CRISPR/Cse3 que nous proposons dans l’article présenté dans ce mémoire. De plus, nous avons adapté la méthode ARiBo pour rendre possible la purification d’un long ARN de 614 nt, le polycistron miR-106b-25. Nous avons également démontré la possibilité d’utiliser la méthode ARiBo pour l’isolation de protéines qui se lient à un ARN donné, le précurseur de miRNA pre-miR-153-2. En conclusion, ce mémoire démontre la possibilité d’adapter la méthode ARiBo à plusieurs applications. / In recent years, the field of RNA research has exploded due to several discoveries demonstrating the importance of RNA in many biological processes. Along with the increased interest in this field, large amounts of RNA have become essential to the success of several studies, in particular for structural biology and functional characterization. However, there are still very few native purification methods that are simple, efficient and reliable. In the past few years, the Legault laboratory has established an affinity purification method using an ARiBo tag to purify RNAs produced by in vitro transcription with the T7 RNA polymerase. This RNA purification method was specifically developed to maximise purity and yield. In addition, this method is fast and works with several types of RNAs. However, like several other purification methods, this method produces RNAs with 5' heterogeneity. This Master’s thesis propose solutions to overcome the problem of 5' heterogeneity for RNAs transcribed with the T7 RNA polymerase and purified with the ARiBo method. The first solution proposed is to choose a 5' sequence among those of the 32 sequences tested that do not present 5'- heterogeneity. The other possibility is the use of a cleavable tag at the 5'-end of the RNA of interest, such as the hammerhead ribozyme, already used for this purpose or the CRISPR/Cse3 system, which is presented here. Furthermore, we have adapted the ARiBo method to purify an RNA of 614 nt, the miRNAs cluster miR- 106b-25. We also demonstrate the possibility to use the ARiBo method to isolate proteins that bind a given RNA, the miRNA precursor pre-miR-153-2. In conclusion, this Master’s thesis demonstrates the possibility of adapting the ARiBo method for several applications.

Selecionamos consecutivamente 63 pacientes com doença mista do tecido conectivo (DMTC) (Kasukawa, 87) para determinar a relevância do padrão SD. Ter uma capilaroscopia periungueal (CPU) até cinco anos antes do início do estudo foi o principal critério de inclusão. Na entrada, avaliamos o envolvimento de órgãos e os auto-anticorpos. A idade média e o tempo de doença foram 45,3 + 10 e 8,45 + 5,42 anos, respectivamente. O padrão SD foi observado em 41 pacientes na entrada (65%) e em 45 na CPU prévia (71,5%), p = 0,20. Dez pacientes (16%) alteraram a CPU, 7 normalizaram e 3 desenvolveram padrão SD. O tempo de doença, número e freqüência de órgãos envolvidos foram semelhantes em pacientes com e sem padrão SD. Em contraste, a análise de cada parâmetro do padrão SD mostrou uma freqüência significativamente menor de áreas avasculares (AA) moderadas/graves na entrada, comparada com a CPU anterior (26,5 e 53%, p = 0,013). Além disto, 76% dos pacientes com doença intersticial pulmonar (TCAR) tiveram AA na entrada, enquanto apenas 24% dos pacientes com esta alteração não apresentavam este achado à CPU (p = 0,017). Adicionalmente, reduzida densidade capilar foi freqüentemente observada em pacientes submetidos à terapia imunossupressora, quando comparados com o grupo sem este tratamento (66,7 e 33,3%, p = 0,001). A CPU na DMTC é um processo dinâmico e a análise de cada parâmetro do padrão SD parece ser um bom indicador de doença intersticial pulmonar e gravidade de doença. / For determining the clinical relevance of SD-pattern in MCTD, sixty-three MCTD patients (Kasukawa´s criteria) were consecutively selected. The main inclusion criterion was availability of previous nailfold capillaroscopy (NC) 5 years before inclusion. At entry, organ involvement and autoantibody evaluation were performed. The mean age and disease duration were 45.3 + 10 and 8.45 + 5.42 years, respectively. SD-pattern was observed in 41 patients at entry (65%) and in 45 at previous NC (71.5%), p = 0.20. Ten patients (16%) changed NC, 7 normalized, and 3 developed SD-pattern. Disease duration, number and frequency of organ involvement were similar in patients with and without SD-pattern. In contrast, analysis of each SD-pattern parameter revealed a significant lower frequency of moderate/severe avascular areas (AA) at entry compared to previous examination (26.5 vs. 53%, p = 0.013). Moreover, 76% of patients with interstitial lung disease (HRCT) had AA at entry, whereas only 24% of patients with this alteration did not have this NC finding (p = 0.017). Furthermore, reduced capillary density was frequently observed in patients taking immunosuppressive therapy than those without (66.7 vs. 33.3%, p = 0.001). NC in MCTD is a dynamic process and analysis of each SD-pattern parameter seems to be a good indicator of lung involvement and disease severity

Selecionamos consecutivamente 63 pacientes com doença mista do tecido conectivo (DMTC) (Kasukawa, 87) para determinar a relevância do padrão SD. Ter uma capilaroscopia periungueal (CPU) até cinco anos antes do início do estudo foi o principal critério de inclusão. Na entrada, avaliamos o envolvimento de órgãos e os auto-anticorpos. A idade média e o tempo de doença foram 45,3 + 10 e 8,45 + 5,42 anos, respectivamente. O padrão SD foi observado em 41 pacientes na entrada (65%) e em 45 na CPU prévia (71,5%), p = 0,20. Dez pacientes (16%) alteraram a CPU, 7 normalizaram e 3 desenvolveram padrão SD. O tempo de doença, número e freqüência de órgãos envolvidos foram semelhantes em pacientes com e sem padrão SD. Em contraste, a análise de cada parâmetro do padrão SD mostrou uma freqüência significativamente menor de áreas avasculares (AA) moderadas/graves na entrada, comparada com a CPU anterior (26,5 e 53%, p = 0,013). Além disto, 76% dos pacientes com doença intersticial pulmonar (TCAR) tiveram AA na entrada, enquanto apenas 24% dos pacientes com esta alteração não apresentavam este achado à CPU (p = 0,017). Adicionalmente, reduzida densidade capilar foi freqüentemente observada em pacientes submetidos à terapia imunossupressora, quando comparados com o grupo sem este tratamento (66,7 e 33,3%, p = 0,001). A CPU na DMTC é um processo dinâmico e a análise de cada parâmetro do padrão SD parece ser um bom indicador de doença intersticial pulmonar e gravidade de doença. / For determining the clinical relevance of SD-pattern in MCTD, sixty-three MCTD patients (Kasukawa´s criteria) were consecutively selected. The main inclusion criterion was availability of previous nailfold capillaroscopy (NC) 5 years before inclusion. At entry, organ involvement and autoantibody evaluation were performed. The mean age and disease duration were 45.3 + 10 and 8.45 + 5.42 years, respectively. SD-pattern was observed in 41 patients at entry (65%) and in 45 at previous NC (71.5%), p = 0.20. Ten patients (16%) changed NC, 7 normalized, and 3 developed SD-pattern. Disease duration, number and frequency of organ involvement were similar in patients with and without SD-pattern. In contrast, analysis of each SD-pattern parameter revealed a significant lower frequency of moderate/severe avascular areas (AA) at entry compared to previous examination (26.5 vs. 53%, p = 0.013). Moreover, 76% of patients with interstitial lung disease (HRCT) had AA at entry, whereas only 24% of patients with this alteration did not have this NC finding (p = 0.017). Furthermore, reduced capillary density was frequently observed in patients taking immunosuppressive therapy than those without (66.7 vs. 33.3%, p = 0.001). NC in MCTD is a dynamic process and analysis of each SD-pattern parameter seems to be a good indicator of lung involvement and disease severity

<p>Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented.</p><p>Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA.</p><p>Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. <i>Trans</i>-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs.</p><p>In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.</p>

Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented. Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA. Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. Trans-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs. In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.