Dissertations / Theses on the topic 'Nprl3'

Parkinson's disease (PD) is a slowly progressive degenerative disorder of the central nervous system that is classically defined in terms of motor symptoms consequent to degeneration of specific subsets of mesencephalic dopaminergic (DA) cells within substantia nigra (SN) pars compacta. No pharmacological treatment is currently available to slow or arrest the neurodegenerative process. Furthermore, accurate early diagnosis suffers from the lack of reliable biomarkers. By the time motor symptoms appear, PD patients have already lost 60-70% of DA-producing cells (Dauer & Przedborski 2003) proving that sporadic PD is diagnosed many years after the onset. It is therefore reasonable to expect that potential pharmacological treatments could be more effective if patients can benefit from it in the premotor phase. Given the systemic nature of the disease, it is not surprising that many alterations of blood physiology have been described in PD patients (Kim et al., 2004; Shults and Haas, 2005; Bongioanni et al., 1996; Migliore et al., 2002; Petrozzi et al., 2002; Salman et al., 1999; Larumbe et al., 2001; Bessler et al., 1999). In this context, a blood test to predict PD would impact the ability to identify new treatments for this incurable disease. Furthermore, it could be applied to a large number of individuals since blood is commonly used in diagnostics for being easily accessible. Gene expression analysis is a powerful tool to study complex diseases such PD and it has been extensively employed to find peripheral biomarkers (Papapetropoulos et al., 2007). In the laboratory of Prof Gustincich, in collaboration with Dr Carninci at RIKEN, Yokohama, Japan, nanoCAGE technology has been previously used to find alterations in the blood transcriptome of 20 drug naïve de novo PD patients compared to 20 Healthy Controls (HC). NanoCAGE allows the identification of Transcription Start Sites (TSSs) and therefore of the associated promoters providing an unbias quantitative description of the cellular transcriptome targeting virtually any RNA molecule present in the sample. The most up-regulated nanoCAGE tag in PD patients is located in the third intron of the gene Nitrogen Permease Regulator Like Protein 3 (Nprl3). Nprl3 gene lies on the telomeric region of human chromosome 11 and contains in its intron the major regulator elements of α globin (Hughes et al., 2005). Neklesa and Davis in 2009 (in yeast) and Bar-Peled et al. in 2013 (in mammals) proved that Nprl3 is a component of a protein complex that inhibits mTORC1 activity. In eukaryotes TOR is the major sensor of nutrients, energy and stress. Alterations in its pathway have been correlated with diseases and conditions where growth and homeostasis are compromised such as cancer, metabolic diseases and aging. The aim of my PhD thesis was to identify the full-length transcript associated to the nanoCAGE tag, validate it, and to test whether it may represent a peripheral biomarker of PD. Taking advantage of rapid amplification of cDNA ends (RACE) assay, I demonstrated that the tag represents an alternative Transcription Start Site of Nprl3 (TagNprl3). It is associated to a TCT motif (YC+1TYTYY) for initiation of transcription, which has been found to be specific for ribosomal protein coding genes and those involved in protein synthesis. The tag maps to a 29nt minisatellite that is found repeated 16 times in the reference genome. High-tag expression is associated to an allelic genomic variant of 13 repeats. To our knowledge this is the first time that a minisatellite variant is both a TSS and an expression quantitative trait locus (eQTL). Unfortunately, high TagNprl3 expression resulted not to be correlated to PD but to heterozygosity. Furthermore, allelic frequencies were not correlated to PD. I then showed that TagNprl3 is expressed in red blood cells (RBCs) both at mRNA and protein levels giving rise to an isoform truncated at the N-terminal. This is able to interact with its protein partner Nprl2 and its overexpression inhibits cell proliferation. This work provides hints for Nprl3 protein function in blood and may suggest a testable hypothesis linking mTOR activity to genomic polymorphisms in modifier genes.

La plupart des voies de signalisation qui régule la croissance cellulaire et le métabolisme sont sous le contrôle du mécanisme du complexe I de la rapamycine (mTORC1). L'un des régulateurs en amont de mTORC1, impliqués dans la détection des acides aminés et l'autophagie, est complexe SEA, chez la levure, et le complexe GATOR, chez les mammifères. Plusieurs composants de GATOR sont dérégulés dans de nombreux cancers et maladies neurodégénératives. Malgré l'intérêt scientifique vis à vis du complexe SEA / GATOR, de nombreux détails concernant sa fonction et son implication dans différents troubles humains sont encore inconnus et restent à investiguer.L'objectif principal de ma thèse était d’élargir notre connaissance sur le complexe SEA / GATOR, et plus particulièrement en ce qui concerne son rôle dans la modulation des voies de signalisation cellulaire. Étant donné que le SEA / GATOR est très conservé, j'ai effectué les expériences en utilisant deux modèles cellulaires : levure S. cerevisiae et lignées cellulaires humaines. Les résultats obtenus ont permis de démontrer un nouveau rôle pour le NPRL2, composant de GATOR, distinct de sa fonction dans la régulation de la voie mTORC1. Nous avons constaté que l'expression ectopique de la NPRL2 induit un stress oxydant et conduit aux dommages de l'ADN et à l'apoptose. Les études sur la levure ont révélé que le complexe SEA relie la voie mTORC1 et la régulation du contrôle de la qualité des mitochondries. Par conséquent, le complexe SEA / GATOR émerge en tant que régulateur multifonctionnel de plusieurs processus cellulaires
The major signaling pathway that regulates cell growth and metabolism is under the control of the mechanistic target of rapamycin complex 1 (mTORC1). One of the mTORC1 upstream regulators involved in amino acid sensing and autophagy is called the SEA complex in yeast and GATOR in mammalian cells. Several GATOR components are deregulated in many cancers and neurodegenerative diseases. Despite of the growing interest to the SEA/GATOR, many details concerning its function and implication in different human disorders are still unknown.The main objective of my thesis was to extend our knowledge about the SEA/GATOR, especially what concerns its role in the modulating cellular signaling network. Because the SEA/GATOR is highly conserved I performed the experiments using two model systems - budding yeast S. cerevisiae and human cells lines. The results I obtained allowed to demonstrate a new role for the GATOR component NPRL2, distinct from its function in mTORC1 regulation. We found that ectopic expression of NPRL2 induces oxidative stress and leads to the DNA damage and apoptosis. The studies in yeast revealed that the SEA complex connects the TORC1 pathway and the regulation of mitochondria quality control. Therefore, the SEA/GATOR complex is emerging as a multifunctional regulator of several cellular processes

L’integrità genomica è minacciata da danni al DNA che, se non adeguatamente riparati, si convertono in mutazioni, il cui accumulo causa instabilità genomica, una tipica caratteristica tumorale. Le cellule eucariotiche reagiscono ai danni attivando la risposta ai danni al DNA. Le rotture a doppia elica del DNA (DSB) sono tra i danni più pericolosi. In Saccharomyces cerevisiae i DSB sono principalmente riparati tramite ricombinazione omologa (HR), che sfrutta sequenze omologhe come stampo per riparare il danno. La HR necessita il processamento nucleolitico (resection) delle estremità del DSB così da generare code di DNA a singolo filamento (ssDNA). La resection inizia con un taglio endonucleolitico da parte del complesso MRX insieme a Sae2, mentre l’estensione della resection è eseguita dalle nucleasi Exo1 e Dna2. Il checkpoint da danno al DNA è una cascata di trasduzione del segnale che blocca il ciclo cellulare così che le cellule abbiano tempo sufficiente per riparare il danno. In S. cerevisiae il checkpoint è attivato dalle chinasi Tel1 e Mec1, ortologhe di ATM e ATR umane. Una volta attivate, Mec1 e Tel1 fosforilano diversi substrati, tra cui l’adattatore Rad9 e la chinasi effettrice Rad53, che amplificano il segnale. Sia la resection che il checkpoint devono essere finemente regolati per garantire una riparazione efficiente dei DSB, evitando di generare troppo ssDNA, e per coordinare la riparazione con la progressione del ciclo. In questa tesi di dottorato, abbiamo dimostrato un nuovo livello di regolazione della resection, basato sul controllo della quantità di Exo1 da parte della proteina che lega l’RNA (RBP) Npl3. Inoltre, abbiamo studiato il ruolo di Sae2 nella riparazione dei danni e nell’attivazione del checkpoint. Npl3 svolge un ruolo chiave nel metabolismo degli RNA ed è molto conservata nell’uomo. Poiché studi recenti mostrano forti connessioni tra metabolismo degli RNA e mantenimento dell’integrità genomica, abbiamo verificato se Npl3 fosse coinvolta nella risposta ai DSB. Abbiamo dimostrato che l’assenza di Npl3 provoca difetti nel processamento delle estremità del DSB. In particolare, Npl3 promuove la resection estesa, agendo nello stesso pathway di Exo1. Inoltre, sia l’assenza di Npl3 che l’inattivazione dei suoi domini di legame all’RNA causano una riduzione del livello di Exo1. Quindi, Npl3 promuove la resection estesa regolando EXO1 a livello dell’RNA. Infatti, in assenza di Npl3, abbiamo dimostrato la presenza di molecole di RNA di EXO1 non correttamente terminate. Questi dati, oltre al fatto che l’overespressione di EXO1 sopprime parzialmente il difetto di resection di cellule npl3Δ, suggeriscono che Npl3 partecipi alla regolazione della resection promuovendo la corretta biogenesi dell’mRNA di EXO1. Riguardo al secondo progetto, Sae2 promuove l’attività endonucleasica di MRX durante la resection e regola negativamente il checkpoint Tel1-dipendente. Infatti, Sae2 limita l’accumulo di MRX alla lesione, riducendo sia il reclutamento che l’attività di segnalazione di Tel1. Non è ancora chiaro come le funzioni di Sae2 nel promuovere la resistenza ai danni e nell’inibire il checkpoint siano collegate. Tramite screening genetico, abbiamo identificato il mutante sae2-ms che, come accade in assenza di Sae2, iperattiva il checkpoint Tel1-dipendente, aumentando il reclutamento ai DSB sia di MRX che di Tel1. A differenza della delezione di Sae2, Sae2-ms non causa difetti di resection né di tethering, e non provoca sensibilità agli agenti genotossici. Inoltre, Sae2-ms provoca iperattivazione di Tel1, ma non di Rad53. Infatti, l’assenza di Sae2, ma non la presenza di Sae2-ms, aumenta l’interazione tra Rad53 e Rad9. Questi dati dimostrano che Sae2 regola il checkpoint sia controllando la rimozione di MRX dai DSB che limitando l’interazione Rad53-Rad9, e che l’inibizione di Rad53 è la principale responsabile della resistenza ai danni promossa da Sae2.
Genomic integrity is threatened by DNA damage that, if not properly repaired, can be converted into mutations, whose accumulation leads to genomic instability, one of the hallmarks of cancer. Eukaryotic cells deal with DNA damage by activating DNA damage response. DNA double strand breaks (DSBs) are among the most dangerous DNA lesions. In Saccharomyces cerevisiae, DSBs are mainly repaired by Homologous Recombination (HR), which exploits a homologous sequence as a template to repair the damage. HR requires the DSB ends to be nucleolytically degraded in order to generate single-strand DNA (ssDNA) tails, in a process known as DSB end resection. Resection initiates with an endonucleolytic cleavage by the MRX complex together with Sae2, while resection extension is carried out by the nucleases Exo1 and Dna2. DNA damage checkpoint is a signal transduction cascade that halts the cell cycle in order to give cells sufficient time to repair the damage. In S. cerevisiae, DNA damage checkpoint is activated by the kinases Tel1 and Mec1, orthologues of human ATM and ATR. Once activated, Mec1 and Tel1 phosphorylate different substrates including the adaptor Rad9 and the effector kinase Rad53, which allow signal amplification. Both DNA end resection and DNA damage checkpoint must be finely regulated to ensure efficient DSB repair, avoiding excessive ssDNA generation, and to properly coordinate repair with cell cycle progression. In this PhD thesis, we provide evidences of a new level of resection regulation, based on the modulation of Exo1 amount by the RNA-binding protein (RBP) Npl3. We have also studied the role of Sae2 in DNA damage repair and checkpoint activation. Npl3 is a S. cerevisiae RBP, which plays a central role in RNA metabolism and is highly conserved from yeast to humans. Since emerging evidences support strong connections between RNA metabolism and genome integrity, we investigated if Npl3 was involved in DSB response. We demonstrated that the absence of Npl3 impairs the generation of long ssDNA tails at DSB ends. In particular, Npl3 promotes resection extension by acting in the same pathway of Exo1. Moreover, both the lack of Npl3 and the inactivation of its RNA-binding domains cause the reduction of Exo1 protein level. So, Npl3 promotes resection extension by regulating EXO1 at the RNA level. Indeed, we proved that the decrease of Exo1 level is due to the presence of not properly terminated EXO1 RNA species. These findings, together with the observation that EXO1 overexpression partially suppresses the resection defect of npl3Δ cells, suggest that Npl3 participates in DSB end resection regulation by promoting the proper biogenesis of EXO1 mRNA. Concerning the second PhD project, Sae2 promotes MRX endonucleolytic activity during resection and negatively regulates Tel1-dependent checkpoint response. Indeed, Sae2 limits MRX accumulation at the damage site, thus reducing Tel1 recruitment and its signalling activity. How Sae2 functions in supporting DNA damage resistance and in inhibiting the DNA damage checkpoint are connected is still unclear. From a genetic screen, we identified the sae2-ms mutant that, similarly to Sae2 absence, upregulates Tel1 signalling activity by increasing both MRX and Tel1 recruitment to the DSBs. However, unlike SAE2 deletion, Sae2-ms does not cause any resection or tethering defect, nor any sensitivity to genotoxic agents. Moreover, Sae2-ms induces Tel1 but not Rad53 hyperactivation. Indeed Sae2 absence, but not Sae2-ms presence, increases Rad53-Rad9 interaction. These data indicate that Sae2 regulates checkpoint activation both by controlling MRX removal from the DSBs and by limiting Rad53-Rad9 interaction and that Rad53 downregulation is the main responsible for Sae2-promoted DNA damage resistance. Altogether, our results allow to better understand the molecular mechanisms involved in the control of DNA damage response processes.

A cultura da laranja doce é muito importante ao redor do mundo, em especial no Brasil, maior produtor mundial dessas frutas. A produção citrícola sempre esteve ameaçada por muitas doenças de grande importância, tais como, o cancro cítrico, a clorose variegada dos citros (CVC) e pinta preta. Entretanto, em 2004, surgiu o huanglongbing (HLB) ou greening, que tem devastado pomares, e para a qual ainda não foi encontrada uma solução definitiva. A transgenia pode ser uma técnica auxiliar no manejo desta doença com a busca de cultivares mais tolerantes, em especial ao HLB. Neste trabalho, as pesquisas de transgenia não envolveram genes exógenos à planta como, por exemplo, genes de outros organismos ou genes sintéticos, ou seja, foi baseado em tecnologias mais recentes já aplicadas em outras espécies vegetais, nas quais a transgenia é utilizada para super-expressar genes dos sistemas de defesa da própria planta. Estudos indicam que a super-expressão de genes do sistema de Resistência Sistêmica Adquirida (SAR - do inglês, \"Systemic Acquired Resistance\") promove a resistência de plantas a doenças. Um gene importante para esse sistema é o gene npr1 que controla a expressão das proteínas relacionadas à patogênese (PR), em especial a PR1. Junto do gene npr1, os genes npr3 e npr4 também são reguladores desse sistema, atuando sobre o gene npr1 de acordo com os níveis de ácido salicílico presentes na célula, nível este que varia de acordo com o nível de infecção de cada célula. Porém, a avaliação de um evento transgênico de citros pode levar muitos anos. Desta forma, para diminuir esse tempo de avaliação, pensou-se em usar plantas modelos. O sistema escolhido foi o tomateiro Micro-Tom (Solanun lycopersicum L. cv. Micro-Tom). Para a obtenção das construções gênicas, foram identificados os genes Csnpr1, Csnpr3 e Csnpr4 de Citrus sinensis L. Osbeck a partir dos genes Atnpr1, Atnpr3 e Atnpr4 de Arabidopsis thaliana L.. Os genes de citros foram obtidos a partir de uma planta de laranja doce por RT-PCR e clonados no vetor pCambia 2201, que foi então inserido em Agrobacterium tumefaciens para a transformação genética. Foi feita a transformação genética de plantas de laranja doce (Citrus sinensis L. Osbeck) e do tomateiro Micro-Tom. Após o crescimento dos brotos regenerados, foi feita a avaliação das plantas obtidas por meio de PCR. As plantas geneticamente modificadas foram aclimatizadas. As plantas de citros foram enxertadas e mantidas em casa de vegetação. As plantas de tomateiro Micro-Tom foram propagadas por sementes. A progênie foi avaliada aplicando o antibiótico de seleção canamicina, obtendo-se assim uma linhagem transgênica homozigota.
The sweet orange industry is very important worldwide, specially in Brazil, considered the world´s largest producer. The citrus production has always been threatened by several diseases of great importance, such as canker, CVC, and black spot. However, in 2004, the huanglongbing (HLB) or greening has been detected and devastated many citrus groves, and no definitive solution has been found yet. Transgenes may be a helpful tool for the management of this diseases, leading to the production of tolerant cultivars, especially to HLB. In this work, research on transgenic did not include the use of exogenous genes to the plant, such as genes from other organism or synthetic genes, i.e, it was based on new emerging technologies, already used on other crops, in which transgeny is used to super express genes from the plants own defense system. Studies indicate that a super expression of genes from the system called Systemic Acquired Resistance (SAR) promotes disease resistance. One important gene to this system is the npr1 gene, which controls the expression of the pathogen related proteins (PR), in special the PR1. Together with the npr1 gene, the genes npr3 and npr4 are also regulators of this system, regulating the action of the npr1 gene according to the levels of salicylic acid present in the cell, this level varies with the level of infection in each cell. Nevertheless, evaluating a citrus transgenic event may take several years. In order to shorten this time, model plants were used. The model chosen was the Micro-Tom tomato (Solanun lycopersicum L. cv. Micro-Tom). In order to obtain the genetic constructions, the genes Csnpr1, Csnpr3 e Csnpr4 were identified in Citrus sinensis L. Osbeck from the genes, Atnpr1, Atnpr3 and Atnpr4 present in the Arabidopsis thaliana L. genome. The citrus genes were obtained from the citrus genome using RT-PCR procedure and cloned separately into the pCambia 2201 vector, which was inserted into Agrobacterium tumefaciens in order to perform the genetic transformation. Sweet orange (Citrus sinensis L. Osbeck) and Micro-Tom plants were genetically modified. After the growth of the regenerated shoots, the evaluation of the obtained plants was done through PCR analysis. The genetically modified plants were acclimatized, the citrus plants were grafted and kept in the greenhouse, the Micro-Tom plants were propagated trough seeds and its progeny was evaluated by applying the selection antibiotic kanamycin, thus obtaining a homozygous transgenic line.

Le diabète de type 2 (DT2) représente un enjeu de santé publique au regard de ses complications, qui sont des maladies complexes, où interagissent des déterminants génétiques et environnementaux.L'objectif de ce travail était d'étudier ces déterminants dans trois populations indépendantes de patients DT2 en couplant études transversales (DIAB2NEPHROGENE) et longitudinales (SURDIAGENE et DIABHYCAR) totalisant 7767 sujets. Via une approche gène-candidat, nous avons focalisé nos recherches sur le système des peptides natriurétiques, le gène NPR3 (codant le récepteur de clairance aux peptides natriurétiques) et les apports sodés puis la voie métabolique des hormones sexuelles, le gène CYP19A1 (codant l’aromatase) et les concentrations de stéroïdes sexuels. Nous avons montré que l'allèle G du rs2270915 du NPR3 est un allèle de risque de pression artérielle (PA) plus élevée et de moindre sensibilité pressive à la réduction sodée qui ne confère pas d'augmentation significative de risque d'évènements cardiovasculaires (ECV) contrairement au rs6889608. Enfin, la survie sans ECV est significativement modulée par les apports en sel avec un risque de morbi-mortalité réduit chez les sujets diabétiques consommant le plus de sel malgré un niveau de PA plus élevé.Nous avons confirmé que le sexe masculin est un facteur de risque pour la néphropathie diabétique (ND) mais également pour la survenue d'ECV. Nous avons montré, chez les hommes, que des concentrations plus élevés d'oestradiol s'associent à une prévalence plus importante de ND mais ne se traduisent pas par une augmentation des événements rénaux oucardiovasculaires. CYP19A1 n'est associé ni avec les niveaux d'oestradiol, ni avec la prévalence ou la sévérité de la ND. Deux SNP s'associent toutefois significativement avec la survenue d'insuffisance rénale chronique terminale.Au total, nous avons identifié dans 2 voies métaboliques distinctes des déterminants génétiques de complications du DT2 ainsi qu'une interaction gène-environnement
Type 2 diabetes (T2D) is a public health issue because of vascular and renal complications, which are complex diseases with interaction between genetic and environmental determinants.The objective of this work was to study these determinants in three independent populations of T2D patients by coupling cross-sectional (DIAB2NEPHROGENE) and longitudinal studies (SURDIAGENE and DIABHYCAR). Through a candidate-gene approach, we first focused on the natriuretic peptides system, NPR3 gene and sodium intake and then on the metabolic pathway of sex hormones, CYP19A1 gene (coding for aromatase) and sex steroid levels.Our first results showed that NPR3 rs2270915 G Allele was associated with high blood pressure (BP) and a reduced salt-sensitivity of BP. However, this SNP was not associated with any significant risk of cardio-vascular events (CVE) or death, at variance with rs6889608. Ultimately, CVE-free survival was impacted by salt intake with a reduced risk of morbi-mortality in those patients having the greatest intake, though a higher BP.In our second study, we confirmed that male gender was a risk factor for diabetic nephropathy (DN), but also for the occurrence of CVE. In men, we showed higher levels of estradiol (E2) associated with a higher prevalence of ND but without any significant increase in renal or CVE during follow-up. CYP19A1 variants were not associated with either E2 levels or the prevalence of ND. However, 2 SNPs tested, were significantly associated with the occurrence of end stage renal failure. Altogether, we have identified 2 different metabolic ways contributing to the genetic determinants of complications associated with T2D including a gene-environment interaction

碩士
國立陽明大學
腦科學研究所
107
The neurodevelopmental disorder focal cortical dysplasia (FCD) is the most common cause of medically refractory epilepsy in both children. Several genes have been identified to be involved in the pathogenesis of this disease, including NPRL2, NPRL3 and DEPDC5, the components of GATOR1 complex. GATOR1 complex acts as a negative regulator of mTORC1 in mTOR signaling pathway, which regulates cell growth, metabolism, autophagy, and proliferation. Although mutations in these genes have been reported to cause FCD and focal epilepsy, the functions of NPRL2/3 in neural development is still not fully understood. To investigate the roles of NPRL2/3 in cortical development, we delivered shRNA by in utero electroporation (IUE) to knock down NPRL2/3 in neural progenitors of mouse embryos. We found that NPRL2/3 knockdown during development caused neuronal migration delay. Furthermore, we observed dendritic morphological changes in the NPRL2/3-knockdown neurons in postnatal mice. Meanwhile, we identified potential novel mutations on NPRL2 and NPRL3 in patients with focal epilepsy. To study whether these mutations will cause neuronal defects or not, we electroporated wild type or mutant NPRL2/3 into mouse embryonic neural progenitors. However, expression of both mutant and wildtype NPRL2 did not cause apparent neural migration defects. Our study may help us understand the roles of NPRL2/3 in neuronal development and provide information for developing effective treatment to NRRL2/3-related neural developmental disorders.

To gain a broader view of the mechanistic consequences of inefficient spliceosome assembly, I have previously carried out an open genetic screen for alleles that improve splicing of 5'SS-G5a introns, which are defective in binding to both U1 and U6 snRNAs. The screen identified alleles of npl3 and mtr10, encoding two functionally linked proteins, Npl3 and Mtr10. Npl3 is primarily nuclear, shuttling SR-like mRNA binding protein implicated in many steps of mRNA biogenesis, including splicing and export, and Mtr10 is its karyopherin (importin). In this work I analyzed both the previously identified and newly generated mutants, and showed that both mtr10 and npl3 suppressors of the defective splicing disrupt nucleocytoplasmic shuttling of Npl3. This results in reduced nuclear Npl3 levels and improved splicing of mutant, suboptimal introns. Interestingly, alterations of nuclear export also improve splicing of suboptimal introns. I propose that the observed splicing effects result from two mechanisms. First, destabilization of Npl3-Mtr10 interactions leads to inefficient re-import of Npl3 to the nucleus. Second, defective interactions of Npl3 with the export machinery inhibit RNA transport to the cytoplasm. Both these mechanisms give more time for the spliceosome to assemble on suboptimal substrates, thus allowing for the completion of splicing before RNA export to the cytoplasm. Notably, RNA sequencing analyses of the selected suppressor strains suggest that increased usage of cryptic splice sites caused by e.g., npl3 mutants is counterbalanced by a general inhibition of splicing due to limiting levels of certain spliceosomal components (e.g., of Prp5 splicing factor). Additionally, non-canonical/non-physiological splicing signals, used more frequently in the mutant strains, may sequester spliceosomal proteins from canonical introns, leading to the observed general decrease of splicing efficiency. Together, my results indicate that reduction of nuclear Npl3 levels leads to a decreased stringency of splice site selection, uncovering a previously unknown role of Npl3 in the modulation of splicing specificity. Thus, Npl3 acts by analogy to eukaryotic SR proteins that modulate alternative splicing patterns.
W celu lepszego zrozumienia mechanistycznych konsekwencji niewydajnego tworzenia spliceosomu, podczas wykonywania swojej pracy magisterskiej przeprowadziłam selekcję supresorów mutacji G5a w intronie, która zaburza parowanie substratu zarówno z U1 snRNA, jak i z U6 snRNA. W wyniku selekcji zidentyfikowałam mutanty w białkach Npl3 oraz Mtr10. Oba białka są ze sobą powiązane funkcjonalnie. Npl3 to białko podobne do eukariotycznych białek SR. Wędruje ono pomiędzy cytoplazmą a jądrem i wpływa na praktycznie wszystkie etapy biogenezy mRNA, m.in. splicing oraz eksport z jądra do cytoplazmy. Mtr10 to karioferyna odpowiedzialna za re-import Npl3 z cytoplazmy do jądra. W tej rozprawie scharakteryzowałam zarówno poprzednio uzyskane, jak i nowo-zidentyfikowane mutanty, oraz pokazałam, że zarówno mutanty npl3 jak i mtr10 zaburzają nukleocytoplazmatyczną wędrówkę białka Npl3. Powoduje to obniżenie poziomu jądrowego Npl3 i poprawę splicingu niekanonicznych (suboptymalnych) substratów pre-mRNA. Co ciekawe, zmiany w eksporcie RNA również poprawiają splicing intronów o suboptymalnych sekwencjach miejsc splicingowych. Zaproponowany przeze mnie model zakłada, że obserwowana poprawa splicingu jest uzyskiwana na dwa sposoby. Po pierwsze, destabilizacja oddziaływania Npl3-Mtr10 prowadzi do niewydajnego re-importu Npl3 do jądra. Po drugie, defektywne interakcje pomiędzy Npl3 a maszynerią eksportową spowalniają transport mRNA do cytoplazmy. Oba te mechanizmy dają więcej czasu na utworzenie spliceosomów na suboptymalnych intronach, zostawiając więcej czasu na dokończenie splicingu suboptymalnych substratów przed ich eksportem do cytoplazmy. Co ciekawe, analiza RNA-seq wybranych szczepów supresorowych sugeruje, że zwiększona częstość wyboru potencjalnie nieprawidłowych miejsc splicingowych, powodowana np. przez mutanty npl3, jest modulowana przez mechanizm zwrotny hamujący splicing poprzez ograniczenie dostępności ważnych białkowych składników spliceosomu, np. białka Prp5. Ponadto, nieprawidłowe miejsca splicingowe wybierane w szczepach supresorowych mogą angażować kompleksy spliceosomowe, skutkując zmniejszeniem dostępności spliceosomów dla pozostałych, kanonicznych intronów, powodując generalne obniżenie wydajności splicingu. Podsumowując, moje wyniki wskazują, że obniżony poziom białka Npl3 w jądrze komórkowym prowadzi do mniej rygorystycznego wyboru miejsc splicingowych przez spliceosom, ujawniając nieznaną poprzednio funkcję tego białka w modulacji specyficzności działania spliceosomu. Białko to zachowuje się więc jak białka SR wyższych eukariontów, które modulują wzory alternatywnego splicingu.

博士
國立臺灣大學
藥理學研究所
103
Renal cell carcinoma (RCC) represents approximately 90% of the adult kidney cancer. In America, it is the seventh most common cancer in men and the ninth most common in women and the incidence and mortality rates in men are almost twice that in women. Although surgical innovation is the most effective treatment in early stage, 30% of patients have metastases when they are diagnosed and do not apply to surgery. Because RCC is resistant to chemotherapy and radiotherapy, scientists are dedicated and focus on the investigation of target therapy of RCC. In this thesis, we explored the anticancer mechanisms of natural or synthetic compounds, and provided the advantages of developing the new treatment strategy of RCC. Physalin F is extracted and purified from Physalis angulata L which has been widely used to treat malaria, asthma, hepatitis, dermatitis, and for rheumatism. The goal of this study, was to investigate the mechanisms of physalin F associated with cell apoptosis in the renal carcinoma cells, A498. The results of this study showed that physalin F induced ROS generation and caused cell apoptosis. Because of the loss of the mitochondria membrane potential, cytochrome c was released into the cytosol and induced caspase activation resulting in apoptosis. Moreover, the phosphorylation of IĸBα was inhibited and prevented NF-ĸB nuclear translocation in physalin F-treated A498 cells. These phenomena were reversed by NAC and GSH. As measured by EMSA, physalin F blocked NF-ĸB activation in A498 cells. These findings suggest that ROS/NF-ĸB is involved in the physalin F-induced A498 cell apoptosis pathway. NPRL-Z-1 is a podophyllotoxin derivative and designed to enhance TOP2 inhibition, overcome drug resistance, and modulate water solubility of etoposide analogues by extending the bulky substituent at C7. In this study, NPRL-Z-1 induced DNA DSBs, TOP2 cleavage complexes formation, and ROS production in A498 cells. When ATM was activated by DSBs, p53 and p21 expression increased and cell cycle was arrested. Ultimately, NPRL-Z-1 induced cell apoptosis. In addition, NPRL-Z-1 inhibited the Akt signaling pathway and induced reactive oxygen species (ROS) generation. These results demonstrated that NPRL-Z-1 appeared to be a novel TOP2 poison and ROS generator, and had better cytotoxicity in RCC cell lines. Heteronemin is a bioactive marine sesterterpene isolated from the sponge Hyrtios sp. Previous reports have shown that heteronemin possesses anticancer activity. Here heteronemin displayed potent cytotoxic effects in A498 human renal carcinoma cells. Heteronemin initiates apoptotic cell death by down-regulating Bcl-2 and Bcl-xL, and up-regulating Bax, leading to the disruption of the mitochondrial membrane potential and the release of cytochrome c from the mitochondria. These effects were associated with the activation of caspase-3/-8/-9, followed by PARP cleavage. Furthermore, heteronemin inhibited the phosphorylation of ERK and Akt signaling pathways and activated p38 and JNK. The specific inhibition of the p38 pathway by SB203580 or p38 siRNA treatment reversed the heteronemin-induced cytotoxicity and apoptotic signaling. Heteronemin also induced autophagy in A498 cells, and treatment with chloroquine (autophagy inhibitor) or SP600125 (JNK inhibitor) inhibited autophagy and increased heteronemin-induced cytotoxicity and apoptotic signaling. Taken together, this study proposes a novel treatment paradigm in which the combination of heteronemin and autophagy inhibitors leads to enhanced RCC cell apoptosis.