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Characterization of a novel methylation mechanism in Trypanosoma brucei
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O parasita protozoário Trypanosoma brucei (T. brucei) é um agente patogénico que causa a doença do sono em humanos e nagana em animais. O seu ciclo de vida é dividido entre um hospedeiro mamífero e um vector, a mosca tsé-tsé. No mamífero provoca infecções que se prolongam por vários anos graças à variação antigénica, na qual o parasita troca regularmente a sua capa de glicoproteínas variáveis de superfície (VSG, “variant surface glycoprotein”) para escapar ao sistema imune do hospedeiro. Para sobreviver nos dois hospedeiros, o parasita altera o seu metabolismo, morfologia e proliferação entre cada estádio de vida. Na base destas alterações fenotípicas encontra-se a regulação génica. Em T. brucei, os genes estão organizados em unidades policistrónicas, transcritas a partir de um promotor comum num longo ARN mensageiro (ARNm) que é posteriormente processado em transcritos individuais. Como não existe controlo de transcrição, a expressão génica é regulada de forma pós-transcricional, dependendo do processamento, estabilidade, e acção de proteínas reguladoras do ARNm. A cauda poli(A) dos ARNm apresenta também uma função regulatória ao servir de plataforma de interacção com proteínas específicas. As modificações pós-transcricionais consistem em grupos químicos presentes nas moléculas de ARN que afectam a sua função e tempo de vida. Entre estas modificações, distingue-se a N6-metiladenosina (ou m6A), que é a mais abundante no ARNm eucarionte. m6A consiste na metilação da posição N6 em adenosinas e afecta a estabilidade dos transcritos metilados no citoplasma. Para regulação, as modificações de m6A dependem de enzimas que as depositam (metiltransferases), reconhecem (leitoras) e removem (demetilases). Em T. brucei, foi recentemente descoberto que a maioria das modificações de m6A encontram-se na cauda poli(A) de transcritos de VSG. Estes transcritos são os mais abundantes e estáveis na forma do parasita que infecta os mamíferos. Adicionalmente, descobriu-se que a presença de m6A estabiliza estes mesmos transcritos, ao interferir com a sua deadenilação pela enzima CAF1. Noutros eucariontes, m6A encontra-se em regiões internas do ARNm e promove a degradação dos transcritos metilados. Assim, o estudo de m6A em T. brucei permitiu, a descoberta de uma função regulatória nova para esta modificação.
Trypanosoma brucei (T. brucei), a protozoan parasite, is the causative agent of sleeping sickness in humans and nagana in cattle. During its life cycle, T. brucei transitions to different parasite forms to survive in the mammalian and tsetse fly hosts. Differentiation into each life cycle stage relies on stage-specific gene expression to control morphologic, metabolic and proteomic changes. Regulation of gene expression in T. brucei is mainly post-transcriptional and as such, dependent on mRNA processing, stability and protein interactions. These mechanisms allow T. brucei to express highly abundant and stable transcripts such as the ones encoding Variant Surface Glycoproteins (VSG), which are crucial for the parasite to survive in the mammalian host. Recently, our laboratory discovered that in T. brucei, most N6-methyladenosine (m6A) is found in the poly(A) tail of VSG transcripts. m6A is an RNA modification involved in eukaryotic gene expression regulation. The presence of m6A in the poly(A) tail stabilized VSG transcripts by interfering with deadenylation, a process where CAF1 deadenylase degrades poly(A) tails. However, many questions remained open, including how m6A is controlled during the parasite’s life cycle; how many m6A modifications exist per poly(A) tail and where; and how m6A interferes with poly(A) tail deadenylation. In this work, we aimed to address these 3 questions using different biochemical and gene manipulation assays. First, we mapped the methylated transcripts in three stages of the parasite’s life cycle (slender, stumpy and procyclic forms) and found that each of these stages presented a different methylated transcriptome. Non-proliferative stumpy forms exhibit the highest number of methylated transcripts, many of which are related with RNA metabolism. We also found that not all surface proteins, and not all VSG transcripts, are methylated. To map m6A modifications within poly(A) tails, we initiated the development of a novel technique (TAIL-miCLIP). We tested TAIL-miCLIP in a poly(A) oligonucleotide with two known m6A sites and found that only one site was effectively identified, requiring future optimizations. To investigate how m6A interferes with deadenylation, we first showed that m6A levels are regulated by CAF1 in vivo but still cause a delay in CAF1 processivity in vitro. We identified six RNA binding protein candidates that could bind to m6A and further interfere with CAF1 activity, through in silico prediction and an experimental RNA pulldown followed by mass spectrometry. We characterized two of these candidates after inducible depletion: ALBA2 and G1IP2. We discovered that both proteins affect steady-state abundance of methylated and non-methylated transcripts similarly, and ALBA2 depletion does not significantly affect the half-life of tested transcripts. Therefore, G1IP2 and ALBA2 may act as general RNA regulators independently of methylation. Overall, our work contributes to elucidating some of the mechanisms behind poly(A) tail methylation in T. brucei. Future studies can benefit from these results to further understand m6A regulation and function in the cell, and possibly translate these to other eukaryotic organisms.
Trypanosoma brucei (T. brucei), a protozoan parasite, is the causative agent of sleeping sickness in humans and nagana in cattle. During its life cycle, T. brucei transitions to different parasite forms to survive in the mammalian and tsetse fly hosts. Differentiation into each life cycle stage relies on stage-specific gene expression to control morphologic, metabolic and proteomic changes. Regulation of gene expression in T. brucei is mainly post-transcriptional and as such, dependent on mRNA processing, stability and protein interactions. These mechanisms allow T. brucei to express highly abundant and stable transcripts such as the ones encoding Variant Surface Glycoproteins (VSG), which are crucial for the parasite to survive in the mammalian host. Recently, our laboratory discovered that in T. brucei, most N6-methyladenosine (m6A) is found in the poly(A) tail of VSG transcripts. m6A is an RNA modification involved in eukaryotic gene expression regulation. The presence of m6A in the poly(A) tail stabilized VSG transcripts by interfering with deadenylation, a process where CAF1 deadenylase degrades poly(A) tails. However, many questions remained open, including how m6A is controlled during the parasite’s life cycle; how many m6A modifications exist per poly(A) tail and where; and how m6A interferes with poly(A) tail deadenylation. In this work, we aimed to address these 3 questions using different biochemical and gene manipulation assays. First, we mapped the methylated transcripts in three stages of the parasite’s life cycle (slender, stumpy and procyclic forms) and found that each of these stages presented a different methylated transcriptome. Non-proliferative stumpy forms exhibit the highest number of methylated transcripts, many of which are related with RNA metabolism. We also found that not all surface proteins, and not all VSG transcripts, are methylated. To map m6A modifications within poly(A) tails, we initiated the development of a novel technique (TAIL-miCLIP). We tested TAIL-miCLIP in a poly(A) oligonucleotide with two known m6A sites and found that only one site was effectively identified, requiring future optimizations. To investigate how m6A interferes with deadenylation, we first showed that m6A levels are regulated by CAF1 in vivo but still cause a delay in CAF1 processivity in vitro. We identified six RNA binding protein candidates that could bind to m6A and further interfere with CAF1 activity, through in silico prediction and an experimental RNA pulldown followed by mass spectrometry. We characterized two of these candidates after inducible depletion: ALBA2 and G1IP2. We discovered that both proteins affect steady-state abundance of methylated and non-methylated transcripts similarly, and ALBA2 depletion does not significantly affect the half-life of tested transcripts. Therefore, G1IP2 and ALBA2 may act as general RNA regulators independently of methylation. Overall, our work contributes to elucidating some of the mechanisms behind poly(A) tail methylation in T. brucei. Future studies can benefit from these results to further understand m6A regulation and function in the cell, and possibly translate these to other eukaryotic organisms.
Descrição
Tese de doutoramento em Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2025.
Palavras-chave
N6-methyladenosine (m6A) Regulation Differentiation Stability Parasite N6-methyladenosine (m6A) Regulação Diferenciação Estabilidade Parasita