Pervasive transcription is widespread amongst eukaryotic genomes, and produces long noncoding RNAs (lncRNAs) in addition to classically annotated transcripts such as messenger RNAs (mRNAs). LncRNAs are heterogeneous in length and map to intergenic regions or overlap with annotated genes. Analogous to mRNAs, lncRNAs are transcribed by RNA polymerase II, regulated by common transcription factors, and possess 5’ caps and perhaps 3’ poly(A) tails. However, lncRNAs perform distinct functions, acting as scaffolds for ribonucleoprotein complexes or directing proteins to nucleic acid targets. The act of transcribing a lncRNA can also affect the local chromatin environment. Furthermore, whereas mRNAs are predominantly turned over in the cytoplasm, both nuclear and cytoplasmic pathways reportedly participate in lncRNA degradation. In this study, I address the question of when and how lncRNAs and mRNAs are distinguished in the cell. Messenger RNAs interact with a defined series of protein factors governing their production, processing and decay, and I hypothesised that lncRNAs might be similarly regulated. I therefore sought to determine which mRNA-binding proteins, if any, also bind lncRNAs. I reasoned that this would reveal the point at which lncRNAs and mRNAs diverge, and how differences in their biogenesis and turnover equip them for different roles. I selected factors from key stages of mRNA metabolism in Saccharomyces cerevisiae, and identified their transcriptome-wide targets using CRAC (crosslinking and analysis of cDNAs). CRAC can detect interactions with low abundance transcripts under physiological conditions, and reveal where within each transcript a protein is bound. Analyses of binding sites in mature mRNAs and intron-containing pre-mRNAs revealed the order in which the tested factors interact with mRNAs, and which region they bind. The poly(A)-binding protein Nab2 bound throughout mRNAs, consistent with an architectural role, whereas the cytoplasmic decay factors Xrn1 and Ski2 bound to poly(A) tails, which might act as hubs to coordinate turnover. The RNA packaging factors Tho2 and Gbp2, and nuclear surveillance factors Mtr4 and Trf4 bound abundantly to intron-containing premRNAs, indicating that they act during or shortly after transcription. The tested factors bound lncRNAs to various extents. LncRNA binding was most abundant for Mtr4 and Trf4, moderate for Tho2, Gbp2, the cap binding complex component Sto1, and the 3’ end processing factors Nab2, Hrp1 and Pab1, and lowest for Xrn1, Ski2 and the export receptor Mex67. This suggests that early events in lncRNA and mRNA biogenesis are similar, but unlike mRNAs, most lncRNAs are retained and degraded in the nucleus. Analyses of two documented classes of lncRNA, cryptic unstable transcripts (CUTs) and stable unannotated transcripts (SUTs), revealed some differences. SUTs were most similar to mRNAs, with canonical cleavage and polyadenylation signals flanking their 3’ ends, and poly(A) tails bound by the poly(A)-binding protein Pab1. CUTs lacked these characteristics, and in comparison to SUTs bound more abundantly to Mtr4 and Trf4 and less so to Ski2, Xrn1 and Mex67. Furthermore, CUTs accumulated upon Hrp1 depletion, suggesting that Hrp1 functions non-canonically to promote CUT turnover. Mtr4, Trf4 and Nab2 also bound abundantly to promoter-proximal RNA fragments generated from ~1000 protein coding genes. These fragments possessed short oligo(A) tails (hallmarks of nuclear surveillance substrates), were not bound to cytoplasmic factors, and apparently correspond to a population of ~150-200 nt promoter-proximal lncRNAs. Notably, CRAC analyses of Mtr4 and Sto1 targets in yeast subjected to a media shift revealed widespread changes in the abundance and surveillance of mRNAs, promoter-proximal transcripts and CUTs, which at many loci were arranged in a complex transcriptional architecture. Overall, the transcriptome-wide binding analyses presented here reveal that lncRNAs diverge from mRNAs prior to export, and are predominantly retained in the nucleus. Transcript fate is apparently determined during 3’ end processing, with CUTs diverging from mRNAs early in transcription via a distinct termination pathway coupled to rapid turnover, and SUTs diverging during or shortly after cleavage and polyadenylation, making them more stable and perhaps prone to escape to the cytoplasm. Promoter-proximal transcripts might arise from termination associated with an early checkpoint in Pol II transcription. The diverse behaviours of lncRNAs arise from their association with distinct subsets of RNA binding proteins, some of which perform different roles when bound to different types of transcript. In conclusion, my results provide the foundation for a mechanistic understanding of how distinct classes of non-coding Pol II transcripts are produced, and how they can perform diverse functions throughout the nucleus.

Evidências sugerem que somente cerca de 2% do genoma codifica proteínas, mas que a maior parte dos 80% restante possui atividade transcricional. Por não ser codificadora de proteínas, essa fração do genoma foi considerada como \'DNA lixo\'. Entretanto, estudos mais recentes e análises pós-ENCODE vem demonstrando que parte significativa destes RNAs não-codificantes desempenham papéis importantes em processos biológicos essenciais e também em doenças. Os RNAs longos não codificadores (lncRNAs) embora tradicionalmente conhecidos pelo imprintinggenômico, vem demonstrando diversos mecanismos de regulação da expressão gênica, principalmente emnível pós transcricional. Um destes lncRNAs que está envolvido principalmente com a metastase em câncer é o HOTAIR. O melanoma tem sido utilizado como modelo de progressao do câncer por suas etapas bem definidas e por isso já tem apresentado alguns lncRNAs envolvidos na melanomagenese e progressão do melanoma, tal como o HOTAIR. Assim, neste trabalho foi analisado a expressão de lncRNAs de amostras de melanócito e melanoma, sendo que as amostras malignas representam as principais fases de progressão deste tipo de câncer. Foram analisados os níveis de expressão relativa. Além disso, foi realizado a expressão diferencial dos grupos representativos do melanoma. Foram encontrados lncRNAs com valores de expressão e significância (p-ajustado <0,01 e fold change >1) que podem ser indicativos de expressão associada a progressão do melanoma. Os lncRNAs mais diferencialmente expressos foram avaliados quanto a sua capacidade de interação proteína-RNA e literatura científica disponível e então foram selecionados para posteriores ensaios funcionais. / Evidence suggests that only about 2% of the genome encodes protein, but most remaining 80% has transcriptional activity. Since they do not coding for proteins, this fraction of the genome was considered \'junk DNA\', However, recent studies and post-ENCODE analisys has shown that significant part of these non-coding RNAs play important roles in essential biological processes and in disease. Long noncoding RNAs (lncRNAs) although traditionally known for genomic imprinting, has demonstrated several mechanisms of regulation of gene expression, especially at the post transcriptional level. One of these lncRNAs that is involved primarily with metastasis in câncer is HOTAIR. Melanoma has been used as a model of câncer progression by its well-defined steps, and so it has been presented some lncRNAs involved in melanoma progression and melanomagenese, as HOTAIR was demonstrated. In this work it was analyzed the expression of lncRNAs of melanocyte and melanoma samples, and malignant samples represent the main stages of progression of this type of câncer. Relative expression levels were analyzed. Furthermore, it was performed differential expression of representative melanoma groups. lncRNAs found with expression values and significance (p-adjusted <0.01 and fold change> 1) may be indicative of expression associated with melanoma progression. The lncRNAs more differentially expressed were evaluated for their ability to interact protein-RNA and available scientific literature and then were selected for further functional assays.

Evidências sugerem que somente cerca de 2% do genoma codifica proteínas, mas que a maior parte dos 80% restante possui atividade transcricional. Por não ser codificadora de proteínas, essa fração do genoma foi considerada como \'DNA lixo\'. Entretanto, estudos mais recentes e análises pós-ENCODE vem demonstrando que parte significativa destes RNAs não-codificantes desempenham papéis importantes em processos biológicos essenciais e também em doenças. Os RNAs longos não codificadores (lncRNAs) embora tradicionalmente conhecidos pelo imprintinggenômico, vem demonstrando diversos mecanismos de regulação da expressão gênica, principalmente emnível pós transcricional. Um destes lncRNAs que está envolvido principalmente com a metastase em câncer é o HOTAIR. O melanoma tem sido utilizado como modelo de progressao do câncer por suas etapas bem definidas e por isso já tem apresentado alguns lncRNAs envolvidos na melanomagenese e progressão do melanoma, tal como o HOTAIR. Assim, neste trabalho foi analisado a expressão de lncRNAs de amostras de melanócito e melanoma, sendo que as amostras malignas representam as principais fases de progressão deste tipo de câncer. Foram analisados os níveis de expressão relativa. Além disso, foi realizado a expressão diferencial dos grupos representativos do melanoma. Foram encontrados lncRNAs com valores de expressão e significância (p-ajustado <0,01 e fold change >1) que podem ser indicativos de expressão associada a progressão do melanoma. Os lncRNAs mais diferencialmente expressos foram avaliados quanto a sua capacidade de interação proteína-RNA e literatura científica disponível e então foram selecionados para posteriores ensaios funcionais. / Evidence suggests that only about 2% of the genome encodes protein, but most remaining 80% has transcriptional activity. Since they do not coding for proteins, this fraction of the genome was considered \'junk DNA\', However, recent studies and post-ENCODE analisys has shown that significant part of these non-coding RNAs play important roles in essential biological processes and in disease. Long noncoding RNAs (lncRNAs) although traditionally known for genomic imprinting, has demonstrated several mechanisms of regulation of gene expression, especially at the post transcriptional level. One of these lncRNAs that is involved primarily with metastasis in câncer is HOTAIR. Melanoma has been used as a model of câncer progression by its well-defined steps, and so it has been presented some lncRNAs involved in melanoma progression and melanomagenese, as HOTAIR was demonstrated. In this work it was analyzed the expression of lncRNAs of melanocyte and melanoma samples, and malignant samples represent the main stages of progression of this type of câncer. Relative expression levels were analyzed. Furthermore, it was performed differential expression of representative melanoma groups. lncRNAs found with expression values and significance (p-adjusted <0.01 and fold change> 1) may be indicative of expression associated with melanoma progression. The lncRNAs more differentially expressed were evaluated for their ability to interact protein-RNA and available scientific literature and then were selected for further functional assays.

RNA has many diverse functions in living organisms, from serving as genome for many viruses, to regulating DNA replication, transcription, translation and other metabolic processes. Some RNA, like protein, has been shown to have catalytic activity. The great proportion of the mass of RNA in living cells, in the form of ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA), constitutes the machinery of protein synthesis, the remainder (approximately 2%) consists of many heterogeneous RNA species of relatively small size, loosely termed "small RNAs", the functions of many of which are completely unknown. In an attempt to understand some of these functions, three hitherto undescribed small RNAs of the budding yeast Saccharomyces cerevisiae were identified and their genes were cloned. These three small RNAs, which lack polyadenylation at their 3' ends, appear to represent the three most abundant RNA species in this organism after rRNA and tRNA. The most abundant of the three was found to be mainly cytoplasmic and was therefore called "small cytoplasmic RNA 1" (scR1). The other two RNAs, named snR17 and snR30, were found to be enriched in nuclear fractions and to possess trimethyl guanosine cap structures at their 5 ends, identifying them as belonging to the ubiquitous class of "U" small nuclear RNAs (U snRNAs), of which several are required for the endonucleolytic cleavage and splicing reactions in the maturation pathways of nuclear precursor mRNAs (pre-mRNA). Whereas scR1 and snR30 are both encoded by single genes, snR17 is the only yeast small RNA found so far to be encoded by two genes. SnR17 was found to be essential: haploid yeast strains lacking intact copies of one or other of the genes appeared to grow normally, but strains lacking both genes were inviable. The nucleotide sequences of the snR17 genes were determined, and the primary and predicted secondary structures of the RNA, 328 nucleotides in length, were found to show significant similarities to those of U3 snRNA, an abundant U snRNA, the function of which is not known. SnR17 belongs to a family of S. cerevisiae snRNAs which, unlike those involved in pre-mRNA splicing, are located in the nucleolus hydrogen-bonded to pre-rRNA, and are associated with antigenic protein that is recognized by human antibodies specific for a 36 kD polypeptide of the U3 small nuclear ribonucleoprotein (U3 snRNP) in mammals. U3 snRNA is also nucleolar and associated with pre-rRNA. Given their structural similarities, snR17 and U3 snRNA are presumably homologous. Yeast snRNAs associated with the anti-(U3)RNP antigen share with U3 snRNAs a conserved nucleotide sequence element. This sequence element alone, however, when injected into Xenopus oocytes, was not sufficient to direct binding of the antigen. The association of snRNAs with pre-rRNA suggests that they function in ribosomal biogenesis.

Resumo: As técnicas mais usadas atualmente para alterar o fluxo metabólico por uma determinada via são a deleção e/ou inserção de sequências regulatórias ou genes no cromossomo bacteriano. Estes são métodos que alteram permanentemente a via metabólica em questão, perturbando o balanço metabólico durante a fase lag de crescimento, o que muitas vezes leva a um excessivo prolongamento desta fase além de diminuir a viabilidade celular. O metabolismo de purinas em Bacillus subtilis tem grande potencial para manipulação genética e produz metabólitos com valor biotecnológico. Cinco riboswitches (purE, xpt, nupG, pbuE e pbuG) controlam o metabolismo de purina em B. subtilis promovendo a terminação precoce da transcrição em ligação com guanina ou adenina. O GTP gerado nesta via é utilizado na biossíntese da vitamina B2 (riboflavina), apresentando o mesmo mecanismo de regulação com riboswitch de flavina-monofosfato (ribDG). Neste estudo, um novo modelo de controle de metabolismo de purina foi construído visando o aumento reversível do fluxo de carbono pela via em B. subtilis utilizando pequenos RNAs não-codificadores sintéticos (sRNAs). Estes foram desenhados e sintetizados para interferir na regulação da expressão gênica realizada pelos riboswitches, mantendo assim a via biossintética ativa. Os riboswitches foram caracterizados por uma nova metodologia in vitro na presença dos ligantes guanina, adenina ou FMN, e nas células de B. subtilis, sendo possível constatar o papel regulador sobre o op... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The currently employed tools to redirect metabolic flux through a specific pathway are deletion and/or insertion of regulatory sequences or genes in the bacterial chromosome. These methods permanently modify the pathway perturbing the metabolic balance during the lag growth phase, which generally extends this process and also decreases cell viability. The purine metabolism in Bacillus subtilis has great potential for genetic manipulation and produces value-added biotechnological products. Five riboswitches (purE, xpt, nupG, pbuE and pbuG) control the purine metabolism in B. subtilis by promoting the premature transcription termination guided by guanine or adenine levels in the cell. The GTP generated in this pathway is used in the biosynthesis of vitamin B2 (riboflavin), which is regulated by a flavin-monophosphate riboswitch (ribDG). In this study, a new metabolism control model was developed aiming to reversibly increase the carbon flux through selected pathways employing small synthetic non-coding RNAs (sRNAs). These sRNAs were designed and synthesized to interfere with the regulation of gene expression performed by the riboswitches, thus maintaining the biosynthetic pathways active. The riboswitches were characterized by a new in vitro methodology in the presence of the ligands: guanine, adenine or FMN. The riboswitches were characterized in B. subtilis cells using the luxABCDE bioluminescence operon as reporter. Synthetic sRNAs were designed in the Ribomaker software to ... (Complete abstract click electronic access below) / Doutor

The study of platelet messenger and micro-RNAs is of increasing interest owing to the fact that platelets contain the machinery to splice and translate mRNA into proteins in response to inhibitory or activating signals. However, the relatively small size (roughly 4000-5000 transcripts) and short half-life of the platelet transcriptome makes this a technically challenging aspect of platelet biology to investigate. The aims of these thesis investigations were therefore to optimise protocols for the isolation of platelets for downstream RNA analyses and function testing, to investigate the functional capabilities of platelet subpopulations rich in RNA, and to understand the functional and transcriptomic impact of gene mutations predicted to influence platelet function. I found that the optimal method for isolating platelets from whole blood is to use simple single step centrifugation to obtain platelet rich plasma. This method is as effective as more involved methods at reducing white blood cell contamination whilst causing minimal platelet activation. Using this method in combination with flow cytometric cell sorting techniques I was able to isolate the newly formed reticulated platelet sub-population and to confirm the link between reticulation status and increased RNA content. Furthermore, using a range of platelet function assays I demonstrated that reticulated platelets are more reactive than non-reticulated platelets. By obtaining blood samples from a patient with a PLA2G4A mutation I was able to show that loss of cPLA2α enzymatic activity alters both platelet function and the expression of certain mRNA transcripts. My investigations using samples from a range of patients with bleeding tendencies show the benefit of combining deep platelet phenotyping with next generation sequencing to understand the causation of bleeding disorders. Together these investigations highlight the utility of genomic DNA and platelet specific mRNA studies in providing novel insights in to pathways regulating platelet reactivity.

A visao sobre o dogma central da biologia molecular passou por aperfeicoamentos na virada deste seculo. Muito se deve ao interesse por pesquisas feitas para compreensao do que ate entao eram regioes do genoma conhecidas como DNA Lixo. Neste contexto, projetos de transcriptoma, avancos em tecnologias de sequenciamento, bem como analises em bioinformatica, contribuiram para elucidar o que estava sendo transcrito. Tais regioes foram denominadas como RNAs nao-codificadores ou non-coding RNA (ncRNA) que eram transcritas, mas nao traduzidas em proteinas. Apesar da quantidade de metodos para o estudo in silico dos ncRNAs, existem lacunas a serem preenchidas nas pesquisas desta molecula, tais como: metodos de anotacao em geral, caracterizacao de novas classes e mecanismos alternativos de busca por similaridade de sequencia primaria. Alem disso, nao se havia uma ferramenta que reunisse num unico local as informacoes dos bancos de dados publicos de ncRNA disponiveis. Neste trabalho, buscou-se preencher tais lacunas, contribuindo para o desenvolvimento de metodos computacionais nas pesquisas em ncRNAs. Foram utilizados os genomas de Hymenoptera e Diptera como sistema biologico para aplicar e testar os metodos desenvolvidos. / The classical vision of the central dogma of molecular biology was not changes dramatic until the end of the 20th century. At this time the scientific communities were interesting to understand what have in the regions of the genome known as \"Junk DNA\". Transcriptome projects together with sequencing Technologies anda bioinformatics analysis help to elucidate that this transcripts were regions that do not coding proteins and maybe has function. These transcripts are called non-coding RNA (ncRNA). Although there are a lot of computational approaches to the in silico research of ncRNA, there is a gap of research about this molecule such: approaches to the general annotation of ncRNA; identification of new classes of ncRNA; and alternatives search mechanisms of ncRNA. Besides that, there are not any central repository of public non-coding RNA databases that could help search for the information about it. In this report, we fill this gap. We tried to contributing to the development of computational methods in research on ncRNAs. We also used the Hymenoptera and Diptera genomes as a biological system to apply and test our developed approaches.

A visao sobre o dogma central da biologia molecular passou por aperfeicoamentos na virada deste seculo. Muito se deve ao interesse por pesquisas feitas para compreensao do que ate entao eram regioes do genoma conhecidas como DNA Lixo. Neste contexto, projetos de transcriptoma, avancos em tecnologias de sequenciamento, bem como analises em bioinformatica, contribuiram para elucidar o que estava sendo transcrito. Tais regioes foram denominadas como RNAs nao-codificadores ou non-coding RNA (ncRNA) que eram transcritas, mas nao traduzidas em proteinas. Apesar da quantidade de metodos para o estudo in silico dos ncRNAs, existem lacunas a serem preenchidas nas pesquisas desta molecula, tais como: metodos de anotacao em geral, caracterizacao de novas classes e mecanismos alternativos de busca por similaridade de sequencia primaria. Alem disso, nao se havia uma ferramenta que reunisse num unico local as informacoes dos bancos de dados publicos de ncRNA disponiveis. Neste trabalho, buscou-se preencher tais lacunas, contribuindo para o desenvolvimento de metodos computacionais nas pesquisas em ncRNAs. Foram utilizados os genomas de Hymenoptera e Diptera como sistema biologico para aplicar e testar os metodos desenvolvidos. / The classical vision of the central dogma of molecular biology was not changes dramatic until the end of the 20th century. At this time the scientific communities were interesting to understand what have in the regions of the genome known as \"Junk DNA\". Transcriptome projects together with sequencing Technologies anda bioinformatics analysis help to elucidate that this transcripts were regions that do not coding proteins and maybe has function. These transcripts are called non-coding RNA (ncRNA). Although there are a lot of computational approaches to the in silico research of ncRNA, there is a gap of research about this molecule such: approaches to the general annotation of ncRNA; identification of new classes of ncRNA; and alternatives search mechanisms of ncRNA. Besides that, there are not any central repository of public non-coding RNA databases that could help search for the information about it. In this report, we fill this gap. We tried to contributing to the development of computational methods in research on ncRNAs. We also used the Hymenoptera and Diptera genomes as a biological system to apply and test our developed approaches.

Tesis por compendio / Los viroides, los agentes infecciosos más simples de la escala biológica, están constituidos por una molécula circular de RNA monocatenario de aproximadamente 250-400 nucleótios (nt) que no codifica proteína alguna. A pesar de esta simplicidad estructural, los viroides son capaces de replicarse autónomamente, moverse sistémicamente y en muchos casos causar enfermedades en sus plantas huéspedes. Las infecciones producidas por viroides representativos generan la acumulación de pequeños RNAs viroidales (vd-sRNAs) de 21-24 nt con características similares a los pequeños RNA interferentes (siRNAs), la huella dactilar del silenciamiento mediado por RNA. La identificación de los vd-sRNAs implica que los viroides son diana de la primera barrera de silenciamiento mediado por RNA, formada por las RNasas ‘Dicer-like’ (DCLs). Para examinar si los vd-sRNAs se unen a las proteínas AGOs —el componente clave del complejo RISC (‘RNAinduced silencing complex’) que constituye la segunda barrera del silenciamiento mediado por RNA— hojas de Nicotiana benthamiana infectadas por el viroide del tubérculo fusiforme de la patata (PSTVd) se agroinfiltraron con nueve de las diez proteínas AGOs de Arabidopsis thaliana. Inmunoprecipitaciones a partir de los halos agroinfiltrados y análisis ‘Western-’ y ‘Northern-blot’ han mostrado que todas las AGOs se expresaron y, a excepción de AGO6, AGO7 y AGO10, unieron vd-sRNAs: AGO1, AGO2 y AGO3 los de 21 y 22 nt, mientras que AGO4, AGO5 y AGO9 también mostraron afinidad por los de 24 nt. La secuenciación masiva mostró que las AGO1, AGO2, AGO4 y AGO5 agroexpresadas unen los PSTVd-sRNAs en función de su tamaño y nucleótido 5’-terminal, y que los perfiles de los correspondientes vd-sRNAs cargados en las AGOs adoptan una distribución específica a lo largo del genoma viroidal. La agroexpresión de AGO1, AGO2, AGO4 y AGO5 en hojas de N. benthamiana infectadas con PSTVd atenuó la acumulación de los RNAs genómico viroidales, indicando que éstos, o sus precursores, también son diana de RISC. En contraste con los ribovirus, la infección de PSTVd en N. benthamiana no afectó de forma significativa la regulación mediada por miR168 de la AGO1 endógena, que carga vd-sRNAs con especificidad similar a su homóloga de A. thaliana. Mientras se conoce bien la biogénesis de los RNA viroidales, su degradación está restringida a algunos datos que implican al silenciamiento mediado por RNA. En el curso de nuestros estudios sobre el PSTVd, hemos observado consistentemente un patrón de 6-7 RNAs subgénomicos (sgRNAs) de polaridad (+) que aparecen junto con los RNAs monoméricos circulares y lineares en berenjena, un huésped experimental de este viroide. Hibridaciones ‘Northern-blot’ con sondas de tamaño parcial y completo, mostraron que los sgRNAs (+) de PSTVd derivan de diferentes regiones del RNA genómico y que algunos son parcialmente solapantes. Parte de los sgRNAs (+) de PSTVd se observaron también en N. benthamiana y tomate, donde han pasado desapercibidos a causa de su menor acumulación. El análisis por extensión de cebador de sgRNAs (+) de PSTVd representativos excluye que sean productos de terminaciones prematuras de la transcripción, pues carecen del extremo 5’ común que cabría esperar si ésta empezara en una posición específica. Ulteriores análisis mediante 5’- y 3’-RACE indican que los sgRNAs (+) de PSTVd tienen extremos 5’-OH y 3’-P, que probablemente resultan de cortes endonucleolíticos de precursores más largos catalizados por RNasas típicas que generan este tipo de extremos. Análisis de sgRNA (-) de PSTVd, que también se acumulan en berenjena infectada, mostraron que presentan características estructurales muy similares a los sgRNA (+). Nuestros resultados proporcionan una nueva visión de cómo ocurre la degradación in vivo de los RNAs viroidales, posiblemente durante su replicación, y sugieren que síntesis y degradación de las cadenas de PSTVd están conectadas, como se ha observado en los mRNAs. / Minoia, S. (2015). Degradación in vivo de un viroide de replicación nuclear: rutas catalizadas por proteínas Argonauta cargadas con pequeños RNAs viroidales y por otras ribonucleasas que generan RNAs subgenómicos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48553 / Compendio

Tesis por compendio / Viroids, small circular RNAs (246-401 nt) with a high content in secondary structure that until recently have been detected only in higher plants, are the simplest infectious agents in the biological scale and do not encode any protein. Therefore, they depend on their genomic sequence and structural motifs to use the transcription, processing, and trafficking machinery of their hosts in order to be replicated and invade them systemically, leading eventually to economically important diseases. The secondary structure of nuclear viroids (family Pospiviroidae) is generally rod-like, while in some chloroplastic viroids (family Avsunviroidae) it is multi-branched. These conformations are mostly supported by data in silico (resulting from algorithms that predict the secondary structure with minimal free energy) and in vitro (using biophysical or biochemical approaches). The assumption that the conformation of the viroid RNAs in vitro is similar or even identical to that adopted in vivo is questionable due, among other reasons, to the different ionic conditions used in in vitro analyses with respect to those existing in planta, as well as to a number of interactions with the proteins or other factors in the host. Therefore, in the present Doctoral Thesis, the in vivo structures of three viroids have been studied, applying different approaches. In the eggplant latent viroid (ELVd), taking advantage of its high genetic variability, co-variations and compensatory mutations have been screened in natural variants in order to confirm or refine in vivo the structures predicted in silico for both viroid strands and those obtained through in vitro SHAPE (2'-hydroxyl groups analysed by primer extension). The results of the three methodologies are consistent for ELVd (+) RNA and lead to a quasi-rod-like conformation with a bifurcation at each terminal domain. This structure, although similar, is not identical to that of ELVd (-) RNA, because its conformation has a central cruciform motif (confirmed in vivo by the presence of covariations therein) and because, in addition, both RNAs show different electrophoretic mobilities in native polyacrylamide gels. The in vitro results for ELVd (-) RNA were less consistent with those obtained in silico and in vivo. On the other hand, the high accumulation of the monomeric circular (mc) positive RNAs of potato spindle tuber viroid (PSTVd) and avocado sunblotch viroid (ASBVd) in Nicotiana benthamiana, and avocado respectively, allowed the determination of the in vivo structure of both RNAs by SHAPE, enabling their direct comparison with the conformations derived previously in vitro using the same technique, and those predicted in silico. The structures determined in vivo for mc PSTVd (+) and mc ASBVd (+) RNAs are very similar (but not identical) to those observed in silico and by in vitro SHAPE. These results provide the first direct evidence that, in their physiological context, the circular RNAs of two viroids, one nuclear and other chloroplastic, are essentially naked and not strongly associated with host proteins. However, we have observed that the conserved central region of mc PSTVd (+) RNA, particularly the loop-E involved in replication and other functions, shows a lower SHAPE reactivity in vivo, possibly due to interactions with one or more proteins mediating these functions or to structural changes induced by other factors of their natural habitat. The low accumulation of mc ASBVd (-) RNA in its host, only allowed for the examination of its structure in silico and by in vitro SHAPE, leading to a rod-like conformation similar to, but not identical, that of mc ASBVd (+) RNA, since the electrophoretic mobility of both RNAs in native polyacrylamide gels is slightly different. / Los viroides, pequeños RNAs circulares (246-401 nt) con un elevado contenido en estructura secundaria que hasta ahora sólo han sido detectados en plantas superiores, son los agentes infecciosos más simples de la escala biológica y no codifican proteína alguna. Por lo tanto, dependen de motivos de secuencia y estructura de su genoma para utilizar la maquinaria de transcripción, procesamiento y tráfico de sus huéspedes con el fin de ser replicados e invadirlos sistémicamente, llegando a producir enfermedades de importancia económica. La estructura secundaria de los viroides nucleares (familia Pospiviroidae) es en general de tipo varilla, mientras que presenta múltiples ramificaciones en algunos viroides cloroplásticos (familia Avsunviroidae). Estas conformaciones están sostenidas por datos mayoritariamente obtenidos in silico (con algoritmos que predicen la estructura secundaria con menor energía libre) e in vitro (por métodos biofísicos o bioquímicos). La asunción de que la conformación de los RNAs viroidales in vitro es similar o incluso idéntica a la que adoptan in vivo es cuestionable debido a las diferentes condiciones iónicas utilizadas en los análisis in vitro con respecto a las existentes in planta, así como a las interacciones con proteínas u otros factores del huésped. Por ello, en la presente Tesis Doctoral se han estudiado las estructuras in vivo de tres viroides aplicando diferentes metodologías. En el viroide latente de la berenjena (ELVd), aprovechando su gran variabilidad genética, se han rastreado covariaciones y mutaciones compensatorias en variantes naturales que confirmen o afinen in vivo las estructuras de las dos cadenas del viroide predichas in silico y las obtenidas in vitro mediante SHAPE (2'-hidroxilo analizada por extensión del cebador). Los resultados de las tres metodologías son consistentes entre sí para el ELVd (+) RNA y conducen a una conformación en varilla con una bifurcación en cada extremo. Esta estructura es similar pero no idéntica a la del ELVd (-) RNA, ya que su conformación presenta un motivo cruciforme central (confirmado in vivo por la presencia de covariaciones en el mismo) y, además, ambos RNAs muestran movilidades electroforéticas distintas en geles de poliacrilamida nativos. Los resultados in vitro para el ELVd (-) RNA fueron menos consistentes con los obtenidos in silico e in vivo. Por otra parte, la alta acumulación de las formas monoméricas circulares (mc) positivas de los viroides del tubérculo fusiforme de la patata (PSTVd) y del manchado solar del aguacate (ASBVd) en Nicotiana benthamiana y aguacate, respectivamente, ha permitido aplicar una modificación de la metodología SHAPE para determinar la estructura in vivo de ambos RNAs, facilitando su comparación directa con las estructuras previamente derivadas in vitro mediante la misma técnica, y las predichas in silico. Las estructuras in planta de los mc PSTVd (+) y mc ASBVd (+) RNAs son muy similares (pero no idénticas) a las observadas in silico y mediante SHAPE in vitro. Estos resultados aportan las primeras pruebas directas de que los RNAs circulares de dos viroides, uno nuclear y el otro cloroplástico, se encuentran en su contexto fisiológico mayoritariamente desnudos y no fuertemente asociados a proteínas del huésped. Sin embargo, hemos observado que la región central conservada del mc PSTVd (+) RNA, particularmente el bucle E implicado en replicación y otras funciones, muestra una menor reactividad SHAPE in vivo posiblemente debida a la interacción con una o más proteínas que medien dichas funciones o a cambios estructurales motivados por otros factores del hábitat natural. Dada la baja concentración en su huésped del mc ASBVd (-) RNA, su estructura únicamente se ha estudiado in silico y por SHAPE in vitro, conduciendo a una conformación de tipo varilla parecida a, pero no la misma que la del mc ASBVd (+) RNA, ya que la movilidad electroforética de los dos RNAs e / Els viroides, menuts RNAs circulars (246-401 nt) amb un elevat contingut en estructura secundària que fins ara només han estat detectats en plantes superiors, són els agents infecciosos més simples de l'escala biològica, i no codifiquen proteïnes. Per tant, depenen de motius de seqüència i estructura del seu genoma per tal d'utilitzar (i fins i tot modular) la maquinària de transcripció, processament i tràfic dels seus hostes amb la finalitat de ser replicats i envair-los sistèmicament, arribant a produir malalties d'importància econòmica. L'estructura secundària dels viroides nuclears (família Pospiviroidae) és en general de tipus vareta, i presenta múltiples ramificacions en alguns viroides cloroplàstics (família Avsunviroidae). Aquestes conformacions estan majoritàriament sostingudes per dades in silico (mitjançant algoritmes que prediuen l'estructura secundària amb menor energia lliure) i in vitro (amb mètodes biofísics o bioquímics). L'assumpció que la conformació dels RNAs viroidals in vitro és similar, o fins i tot idèntica, a aquella que adopten in vivo és qüestionable a causa de, entre altres raons, les diferents condicions iòniques utilitzades en les anàlisis in vitro pel que fa a les existents in planta, així com a les interaccions amb proteïnes o altres factors de l'hoste. Per això, en la present Tesi Doctoral s'han estudiat les estructures in vivo de tres viroides aplicant diferents metodologies. En el viroide latent de l'albergínia (ELVd), aprofitant la seua gran variabilitat genètica, s'han rastrejat covariacions i mutacions compensatòries en variants naturals que confirmen o afinen in vivo les estructures de les dues cadenes del viroide predites in silico i aquelles obtingudes in vitro mitjançant SHAPE (2'-hidroxil analitzada per extensió del cebador). Els resultats de les tres metodologies són consistents entre si per a l'ELVd (+) RNA, i condueixen a una conformació en vareta amb una bifurcació a cada extrem. Aquesta estructura, si bé similar, no és idèntica a aquella de l'ELVd (-) RNA, ja que la seua conformació presenta un motiu cruciforme central (confirmat in vivo per la presència de covariacions en el mateix) i, a més, tots dos RNAs mostren mobilitats electroforètiques diferents en gels de poliacrilamida natius. Els resultats in vitro per a l'ELVd (-) RNA són menys consistents amb les dades obtingudes in silico i in vivo. D'altra banda, l'alta acumulació de les formes monomèriques circulars (mc) positives dels viroides del tubercle fusiforme de la creïlla (PSTVd) i del tacat solar de l'alvocat (ASBVd) en Nicotiana benthamiana i alvocat respectivament, ha permès aplicar una modificació de la metodologia SHAPE per a determinar l'estructura in vivo de tots dos RNAs, possibilitant la comparació directa amb l'estructura prèviament derivada in vitro amb la mateixa tècnica, i la conformació predita in silico. Les estructures de tipus vareta obtingudes per als mc PSTVd (+) i mc ASBVd (+) RNAs és molt similar (però no idèntica) a les observades in silico i mitjançant SHAPE in vitro. Aquests resultats aporten les primeres proves directes que, en el seu context fisiològic, els RNAs circulars dels viroides nuclears i cloroplàstics es troben majoritàriament nus i no fortament recoberts per proteïnes de l'hoste. No obstant això, hem observat una menor reactivitat SHAPE in vivo de la regió central conservada del PSTVd, particularment del bucle E implicat en replicació i en altres funcions, possiblement a causa de la interacció amb una o més proteïnes que intervenen aquestes funcions o a canvis estructurals motivats per altres factors de l'hàbitat natural. Atesa la baixa concentració del mc ASBVd (-) RNA en el seu hoste, la seua estructura únicament s'ha estudiat in silico i amb SHAPE in vitro, conduint a una conformació de tipus vareta semblant a, tot i que no la mateixa, aquella del mc ASBVd (+) RNA, ja que la mobilitat electroforètica de / López Carrasco, MA. (2017). Disección de la estructura secundaria in silico, in vitro e in vivo de RNAs viroidales nucleares y cloroplásticos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/86145 / Compendio