Gene expression is the conduit by which genetic information is connected into cellular phenotypes. Recently, it was shown that gene expression in mammalian cells is governed, at least in part, by the expression of short double stranded RNA (dsRNA). This mode of gene regulation is influenced by a large group of dsRNA binding proteins that could either stabilize or trigger the degradation of dsRNA. Indeed, double stranded RNA (dsRNA) specific ribonucleases (RNases) play an important role in regulating gene expression. In most eukaryotes, members of the dsRNA specific RNase III family trigger RNA degradation and initiate cellular immune response. Disruption of human . RNase III (Dicer) deregulates fetal gene expression and promotes the development of cancer. However, very little is known about the housekeeping function of eukaryotic RNase III and the mechanism by which they distinguish between exogenous and endogenous cellular RNA species. This thesis elucidates how dsRNAs are selected for cleavage and demonstrates their contribution to RNA metabolism in yeast as model eukaryote. Initially, the reactivity determinants of yeast RNase III (Rnt1p) were identified in vitro and used to study the global impact of Rnt1p on the processing of non-coding RNA. The results indicate that Rnt1p is required for the processing of all small nucleolar RNAs (snoRNAs) involved in rRNA methylation and identify a new role of Rnt1p in the processing of intronic snoRNAs. It was shown that Rnt1p cleavage helps to coordinate the expression of some ribosomal protein genes hosting intronic snoRNAs. Direct snoRNA processing from the pre-mRNA blocks the expression of the host gene, while delayed snoRNA processing from the excised intron allows the expression of both genes. In this way, the cell can carefully calibrate the amount of snoRNA and ribosomal proteins required for ribosome biogenesis. In addition, a global analysis of snoRNA processing identified new forms of Rnt1p cleavage signals that do not exhibit a conserved sequence motif but instead use a new RNA fold to recruit the enzyme to the cleavage site. This finding led to the conclusion that Rnt1p may use a wide combination of structural motifs to identify its substrates and thus increases the theoretical number of potential degradation targets in vivo . To evaluate this possibility, a new search for snoRNA independent Rnt1p cleavage targets was performed. Interestingly, many Rnt1p cleavage signals were identified in intergenic regions devoid of known RNA transcripts. In vivo , it was shown that Rnt1p induce the termination of non-polyadenylated transcripts and functions as a surveillance mechanism for transcription read-through. This finding directly links Rnt1p to the transcription machinery and provides a new mechanism for polyadenylation independent transcription termination. Together the work described in this thesis presents an example of how eukaryotic RNase III may identify its substrates and present a case study where transcription, RNA processing and stability are linked.

Submitted by Claudia Rocha (claudia.rocha@udesc.br) on 2018-03-01T12:12:19Z No. of bitstreams: 1 PGPV17MA218.pdf: 1720230 bytes, checksum: 6b5c28d9052e3150143609b5794ee96a (MD5) / Made available in DSpace on 2018-03-01T12:12:19Z (GMT). No. of bitstreams: 1 PGPV17MA218.pdf: 1720230 bytes, checksum: 6b5c28d9052e3150143609b5794ee96a (MD5) Previous issue date: 2017-02-02 / PROMOP / CAPES / Due to the gametophytic self-incompatibility (GSI), the occurrence of cross-pollination between genetically compatible plants is necessary for the naturally fructification of apple trees (Malus domestica Borkh.). The GSI is governed by the multiallelic locus called S, which encodes a family of RNases thats act on the pistil and prevents the formation of the pollen tube when the S alleles presented in the pollen grain is the same as that is presented in the diploid tissue of the pistil. From the identification of the S alleles of plants of interest it is possible to guide the combinations between parents to obtain segregant populations and to predict the efficiency of apple tree genotypes when they are used as pollinators in commercial orchards. The objectives of this dissertation were to validate the use of DNA markers in the identification of the genetic constitution of the S locus of apple tree genotypes, as well as the definition of pollinators genotypes to be adopted in commercial orchards, serving as an auxiliary tool for the apple breeding. The study of the segregation of the S alleles carried out in segregating populations of apple trees by DNA markers (Chapter 2); the locus S of 28 genotypes of apple trees were analyzed by DNA markers and the genetic dissimilarity analysis was done based on the characterization of the same genotypes for based on the minimum descriptors required for the protection of new cultivars (Chapter 3); and the determination of the pollinators genotypes for three apple was done based on the genotyping of the S alleles associated with cross testing (Chapter 4). In the study of segregation of S alleles in segregating populations, the progeny of the cross between Fred Hough (S5S19) vs. Monalisa (S2S10) followed the expected segregation ratio for genotype compatibility: 1:1:1:1. In the other population evaluated, the same pair of S alleles, S3S5, were identified in both parents (M-11/01 and M-13/91), and the progeny showed the same genotype. In 26 of the 28 genotypes evaluated, both alleles S were identified, and in the remaining two genotypes only one allele was identified in each genotype. The average dissimilarity of the 28 genotypes obtained by the morphoagronomic characterization was 35 %. Considering the total genetic compatibility between the genotypes and the five major dissimilarities obtained, 15 crosses were suggested to increase the genetic base of the Epagri's Genetic Apple Breeding Program. Regarding the selection of pollinators, the field pollination tests did not show a significant difference between the cultivars and their respective pollinators tested for characters the fruit set and seed number, but when the S alleles present in each of the genotypes, were identified the presence of semi-compatibility cases between them. This fact can be explain by the high concentration of pollen grains applied on the pistil of the flowers on artificial pollination, which may mask the existing semi-compatibility. Considering the results obtained in this study, DNA markers can be used to identify the locus S genotype in apple trees as an auxiliary tool to the Epagri's Genetic Apple Breeding Program, both for the definition of combinations between parents for the formation of segregant populations as to chose pollinators to fruit-producing cultivars to be adopted in commercial orchards / Devido a autoincompatibilidade gametofítica (AIG), para a formação de frutos em plantas de macieira (Malus domestica Borkh.) é necessária a ocorrência de polinização cruzada entre plantas geneticamente compatíveis. A AIG é governada pelo loco multialélico S, que codifica para uma família de RNases atuantes no pistilo da planta e impede a formação do tubo polínico quando os alelos S presentes no grão de pólen forem iguais àqueles presentes no tecido diploide do pistilo. A partir da identificação dos alelos S de plantas de interesse é possível orientar as combinações entre genitores para a obtenção de populações segregantes via hibridações dirigidas e prever a eficiência de genótipos de macieira quando utilizados como polinizadores em pomares comerciais. Os objetivos dessa dissertação foram validar o uso de marcadores de DNA na identificação da constituição genética do loco S de genótipos de macieira e na definição de genótipos polinizadores a serem adotados em pomares comerciais, servindo como ferramenta auxiliar ao melhoramento genético de macieira. Realizou-se: o estudo da segregação dos alelos S em populações segregantes de macieira mediante genotipagem via marcadores de DNA (Capítulo 2); a genotipagem dos alelos S de 28 genótipos elite de macieira via marcadores de DNA e análise de dissimilaridade genética com base na caracterização quanto aos descritores mínimos requisitados para a proteção de novas cultivares (Capítulo 3); e a determinação dos genótipos polinizadores para três cultivares de macieira baseando-se na genotipagem dos alelos S associada à realização de cruzamentos teste a campo (Capítulo 4). No estudo da segregação dos alelos S em populações segregantes, a progênie do cruzamento entre Fred Hough (S5S19) vs. Monalisa (S2S10) apresentou a proporção de segregação esperada para compatibilidade entre genótipos: 1:1:1:1. Na segunda população avaliada, em ambos os genitores (M-11/01 e M-13/91) foi identificado o mesmo par de alelos S: S3S5, sendo que a progênie apresentou esse mesmo genótipo. Mediante a análise molecular, em 26 dos 28 genótipos avaliados foram identificados ambos os alelos do loco S, sendo que nos dois restantes apenas um alelo foi identificado em cada genótipo. A dissimilaridade média dos 28 genótipos identificada pela caracterização morfoagronômica foi de 35 %. Considerando a compatibilidade genética total entre os genótipos e as cinco maiores dissimilaridades obtidas, foram sugeridos 15 cruzamentos para ampliação da base genética do Programa de Melhoramento Genético da Epagri. Quanto a seleção de polinizadoras, os testes de polinização a campo não demonstraram diferença significativa entre as cultivares e suas respectivas polinizadoras para as características avaliadas, porém quando os alelos S foram identificados constatou-se a presença de casos de semicompatibilidade entre alguns dos genótipos avaliados. Sugere-se que a não identificação dos genótipos semicompatíveis ocorre devido a alta concentração de grãos de pólen aplicada sobre o pistilo das flores via polinização artificial das cultivares, que pode mascarar a semicompatibilidade existente. Considerando os resultados obtidos nesse estudo, a utilização de marcadores de DNA para identificação do genótipo do loco S em macieira pode ser empregada como ferramenta auxiliar ao programa de melhoramento genético de macieira da Epagri, tanto para a definição de combinações entre genitores para a formação de populações segregantes que serão alvo de seleção quanto para a escolha de polinizadoras geneticamente compatíveis com as cultivares produtoras de frutos a serem adotados em pomares comerciais, minimizando as perdas de produção em pomares comerciais devido a semicompatibilidade genética ou incompatibilidade entre os genótipos de macieira (cultivar copa x polinizadora)

No description available.

Bacterial pathogens rely on precise regulation of gene expression to coordinate host infection processes and resist invasion by mobile genetic elements. An interconnected network of protein and RNA regulators dynamically controls the expression of virulence factors using a variety of mechanisms. In this thesis, the role of selected regulators, belonging to the class of small RNAs (sRNAs), is investigated. Streptococcus pyogenes is a pathogen responsible for a wide range of human diseases. Genome-wide screenings have indicated that S. pyogenes encodes numerous sRNAs, yet only a limited number have been characterized. A major goal of this study was to identify and characterize novel sRNAs and antisense RNAs (asRNAs) using RNA sequencing analysis. We validated 30 novel sRNAs and asRNAs, and identified 9 sRNAs directly cleaved by the ribonucleases RNase III and/or RNase Y. Previous work from the laboratory has highlighted the role of sRNAs from the type II Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) systems in S. pyogenes. CRISPR-Cas systems provide adaptive immunity to prokaryotes against infection by mobile genetic elements. Two sRNAs, forming a complementary duplex (dual-RNA), are effectors of this system: the mature CRISPR RNAs (crRNAs) and the trans-activating crRNA (tracrRNA). The dual-RNA guides the Cas9 endonuclease to cleave both strands of the invading DNA in a sequence-specific manner. This RNA-programmable CRISPR-Cas9 system is now utilized for genome editing and engineering in a wide range of cells and organisms. To expand the potentialities of this tool, we both, searched for Cas9 orthologs and predicted numerous tracrRNA orthologs. We defined tracrRNA as a new family of sRNAs sharing the ability to base-pair to cognate crRNAs, without conservation of structure, sequence or location. We show that Cas9 and the dual tracrRNA:crRNAs are only interchangeable between closely related type II CRISPR-Cas systems. In summary, this thesis presents new insights into RNA-mediated regulatory mechanisms in S. pyogenes. We identified and described the expression of novel sRNAs, highlighting potential antisense RNAs. Focusing on the dual-RNA programmable type II CRISPR-Cas system, we provided evidence for co-evolution of the Cas9 enzyme with tracrRNA:crRNA, a basis for Cas9 multiplexing in genome editing.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.