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Understanding the role of parasites' epitranscriptome
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Trypanosoma brucei (T. brucei) é um parasita unicelular que causa a doença do sono. Durante o seu ciclo de vida, T. brucei alterna entre dois hospedeiros: um mamífero e a mosca tsetse. Para sobreviver às diferentes condições ambientais encontradas no interior de cada um dos dois hospedeiros, o parasita passa por processos de diferenciação; os estágios do ciclo de vida do parasita diferem entre si morfologicamente e fisiologicamente, consequência de uma expressão génica diferencial ao longo das diferentes fases do ciclo. Dado que o genoma do parasita está maioritariamente organizado em unidades policistrónicas, a regulação da expressão génica é fundamentalmente pós-transcricional. Recentemente, as modificações de RNA têm sido associadas a funções regulatórias, inclusive ao nível da expressão génica e por isso foram denominadas epitranscriptoma. Com este estudo, conseguimos identificar vinte modificações de RNA em RNA total de T. brucei, sendo que nove delas ainda não tinham sido detetadas nos transcritos deste parasita. Uma dessas modificações é a N6-methiladenosina (m6A) que noutros organismos foi associada a processos envolvidos na regulação da expressão génica. Com isto em mente, procurámos por genes, no genoma do parasita, que possivelmente codificassem metiltransferases (MT) de m6A. Após essa pesquisa, realizámos um mutante knock out (KO) para uma das sequências candidatas obtidas. Esta linha celular mutante (MT KO) não exibiu qualquer defeito quando se encontrava na forma presente na corrente sanguínea (slender) ou durante a transição para a forma transmissível para a mosca (stumpy). Contudo, durante a diferenciação para o estágio encontrado no intestino da mosca (procyclic) MT KO exibiu um fenótipo ao nível do crescimento, sendo possível destacar um aumento da percentagem de células em G2-M ou poliploides. Desta forma, este estudo permitiu identificar vinte possíveis reguladores da expressão génica de T. brucei e a descoberta duma possível MT que está envolvida no processo de diferenciação, que possivelmente poderá associar o epitranscriptoma a funções regulatórias no parasita.
Trypanosoma brucei (T. brucei) is the unicelular parasite that causes sleeping sickness. Throughout its life cycle, T. brucei alternates between two hosts: a mammal and the tsetse fly. In order to survive the different environmental conditions found within each of the hosts, this parasite undergoes complex differentiation processes, so that the distinct life cycle stages show morphological and physiological differences between them. These result from a differential gene expression regulation among the different life cycle phases. Given that the parasite’s genome is mostly organized in polycistronic units, gene expression regulation is mainly due to post-transcriptional mechanisms. Recent studies have been associating RNA modifications to possible regulatory roles, including at the gene expression level. Therefore, they are collectively denominated the epitranscriptome. In this study, we were able to detect twenty RNA modifications in total RNA from T. brucei, nine of which were never described before in these parasite’s transcripts. Among the modified nucleosides found, there was N6-methyladenosine (m6A) that in other organisms seems to be involved in several biological processes known to affect gene expression. All things considered, we looked for putative m6A methyltransferases encoded in the parasite’s genome. Subsequently, we generated a knock out (KO) cell line for one of the retrieved sequences. This mutant cell line (MT KO) did not show any defects while in the bloodstream slender form (found in the blood vessels) nor during transition to the transmissible to the fly bloodstream stumpy form. However, during differentiation to the insect procyclic form, MT KO parasites growth is impaired and it is possible to notice an increase in the percentage of cell cycle-arrested cells in G2-M and of polyploid cells. Overall, this study allowed us to detect twenty potential regulators of gene expression in T. brucei and the discovery of a putative MT that is involved in the differentiation process, thus possibly associating the epitranscriptome to regulatory roles in the parasite.
Trypanosoma brucei (T. brucei) is the unicelular parasite that causes sleeping sickness. Throughout its life cycle, T. brucei alternates between two hosts: a mammal and the tsetse fly. In order to survive the different environmental conditions found within each of the hosts, this parasite undergoes complex differentiation processes, so that the distinct life cycle stages show morphological and physiological differences between them. These result from a differential gene expression regulation among the different life cycle phases. Given that the parasite’s genome is mostly organized in polycistronic units, gene expression regulation is mainly due to post-transcriptional mechanisms. Recent studies have been associating RNA modifications to possible regulatory roles, including at the gene expression level. Therefore, they are collectively denominated the epitranscriptome. In this study, we were able to detect twenty RNA modifications in total RNA from T. brucei, nine of which were never described before in these parasite’s transcripts. Among the modified nucleosides found, there was N6-methyladenosine (m6A) that in other organisms seems to be involved in several biological processes known to affect gene expression. All things considered, we looked for putative m6A methyltransferases encoded in the parasite’s genome. Subsequently, we generated a knock out (KO) cell line for one of the retrieved sequences. This mutant cell line (MT KO) did not show any defects while in the bloodstream slender form (found in the blood vessels) nor during transition to the transmissible to the fly bloodstream stumpy form. However, during differentiation to the insect procyclic form, MT KO parasites growth is impaired and it is possible to notice an increase in the percentage of cell cycle-arrested cells in G2-M and of polyploid cells. Overall, this study allowed us to detect twenty potential regulators of gene expression in T. brucei and the discovery of a putative MT that is involved in the differentiation process, thus possibly associating the epitranscriptome to regulatory roles in the parasite.
Descrição
Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016
Palavras-chave
Trypanosoma brucei Epitranscriptoma Forma procyclic Diferenciação Ciclo celular Teses de mestrado - 2016