Investigação da função biológica dos eventos de arm switching de microRNAs e sua potencial associação com a metilação de RNA m6A utilizando zebrafish como organismo modelo
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Data
2021-02-25
Autores
Oliveira, Arthur Casulli de
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Universidade Estadual Paulista (Unesp)
Resumo
Os microRNAs (miRNAs) são atualmente conhecidos como uma ampla classe de moléculas de RNAs reguladores da expressão gênica. Durante a biogênese, uma fita ("braço") do miRNA duplex é preferencialmente selecionada como um miRNA funcionalmente maduro, enquanto a outra fita é usualmente degradada. Eventos de arm switching ocorrem quando há mudança na preferência do braço selecionado. Isso se reflete na alteração do braço identificado como transcrito funcional, e varia de forma bastante dinâmica entre estágios do desenvolvimento, tipos de tecidos e mesmo entre espécies. Devido às particularidades de seu mecanismo de ação, os arm switching concedem aos miRNAs um ampliado potencial regulatório ao modificar o conjunto de alvos e, comumente, os processos biológicos por eles regulados. Entretanto, os mecanismos moleculares que promovem episódios de arm switching ainda necessitam ser melhor caracterizados. A identificação de metilações m6A em pri-miRNAs de linhagens celulares humanas reportada na literatura forneceu indícios de que este tipo de metilação poderia estar envolvida no processo de arm switching, principalmente devido à alteração que m6A promove na estrutura secundária de RNAs. Para testar esta hipótese, utilizamos o zebrafish, um vertebrado modelo, para avaliar se a metilação m6A poderia estar influenciando na ocorrência de arm switching nesta espécie. Para isso, confrontamos o perfil global de expressão dos braços 5p e 3p dos miRNAs pela análise de uma ampla coleção de dados de RNA-seq de períodos iniciais do desenvolvimento embrionário e diversos tecidos adultos de zebrafish obtidos da literatura. Adicionalmente, aplicamos a técnica de sequenciamento de metilações m6A (m6A-seq) no transcriptoma para identificar potencial correlação entre os picos de metilação presentes nos miRNAs e os eventos de arm switching identificados. Este conjunto de análises nos conduziu à inédita identificação de 14 eventos de arm switching em zebrafish. Pelo estudo detalhado destes casos e sua comparação aos eventos descritos em outras espécies verificamos que eventos de arm switching estão majoritariamente associados ao desenvolvimento ontogenético dos vertebrados. Adicionalmente, nossas análises sugerem que a expressão diferencial de isoformas de miRNAs (isomiRs) pode ser um importante modulador na prevalência dos braços. Também demonstramos, via análises de predição de alvos e enriquecimento funcional, que os eventos de arm switching de fato potencializam a regulação exercida pelos miRNAs, ao aumentarem o número de genes alvos, redes regulatórias e funções biológicas aos quais estão associados. No entanto, surpreendentemente, a análise criteriosa dos dados de m6A-seq nos forneceu indícios de que os pri-miRNAs de zebrafish não são efetivamente metilados, ainda que contenham sítios de metilação m6A em suas sequências. A discrepância entre os resultados obtidos neste trabalho e aqueles reportados em humanos e, mais recentemente, em Arabidopsis thaliana, sugerem que pressões seletivas atuantes na biogênese dos miRNAs poderiam estar ocorrendo em diferentes estágios nestas espécies. Ainda, considerando dados recentes da literatura sobre a interação entre a proteína efetora de metilações m6A e moléculas de RNA, conjecturamos que a estrutura secundária dos pri-miRNAs pode ser um fator permissivo desta modulação. Nesse contexto, concluímos que o arm switching é um fenômeno conservado e que está majoritariamente associado ao desenvolvimento ontogenético dos organismos. Também não encontramos evidências de que a metilação m6A interfira na prevalência dos braços de miRNAs em zebrafish, dada sua ausência aparente nos transcritos de pri-miRNAs desta espécie. Os resultados obtidos neste estudo trazem novas contribuições para a compreensão de mecanismos regulatórios inerentes à atividade funcional de miRNAs. Adicionalmente, fornecem subsídios para pesquisas futuras interessadas em explorar os mecanismos moleculares subjacentes às diferentes taxas de metilação e sua interferência na função e dinâmica evolutiva dos miRNAs nos metazoários.
MicroRNAs (miRNAs) are currently known as a large class of regulatory RNAs. During biogenesis, one strand ("arm") of the duplex miRNA is preferably selected as a functionally mature miRNA, while the other strand is usually degraded. Arm switching events occur when there is a change in the preference of the selected arm. This is reflected in the alteration of the arm identified as the most expressed and functional transcript, and has a dynamically variation between stages of development, types of tissues and even between species. Due to the particularities of their mechanism of action, arm switching grants miRNAs an increased regulatory potential by modifying the set of targets and, commonly, the biological processes they regulate. However, the molecular mechanisms that promote arm switching episodes still need to be better characterized. The identification of m6A methylations in pri-miRNAs from human cell lines reported in the literature provided evidence that this type of methylation could be involved in the arm switching process, mainly due to the change that m6A promotes in the secondary structure of RNAs. To test this hypothesis, we used zebrafish, a model vertebrate, to assess whether m6A methylation could be influencing the occurrence of arm switching in this species. This way, we compared the global expression profile of the miRNAs 5p and 3p arms by analyzing a wide collection of RNA-seq data from early periods of embryonic development and various adult zebrafish tissues obtained from the literature. Additionally, we applied the m6A methylation sequencing technique (m6A-seq) in the transcriptome to identify potential correlation between the methylation peaks present in the miRNAs and the identified arm switching events. This set of analyzes led us to the unprecedented identification of 14 arm switching events in zebrafish. Through the detailed study of these cases and their comparison with the events described in other species, we verified that arm switching events are mostly associated with the ontogenetic development of vertebrates. Additionally, our data suggest that the differential expression of miRNA isoforms (isomiRs) may be an important modulator in the prevalence of arms. We have also demonstrated, via target prediction and functional enrichment analyzes, that arm switching events in fact enhance the regulation exercised by miRNAs, by increasing the number of target genes, regulatory networks and biological functions with which they are associated. However, surprisingly, careful analysis of the m6A-seq data provided us with evidence that zebrafish pri-miRNAs are not effectively methylated, even though they contain m6A methylation sites in their sequences. The discrepancy between the results obtained in this work and those reported in humans and, more recently, in Arabidopsis thaliana, sugget that selective pressures acting on the biogenesis of miRNAs may be occurring at different stages in these species. Still, considering recent data from the literature on the interaction between the m6A methylating reader protein and RNA molecules, we conjecture that the secondary structure of the pri-miRNAs may be a permissive factor in this modulation. In this context, we conclude that arm switching is a conserved phenomenon and that it is mostly associated with the ontogenetic development of organisms. We also found no evidence that m6A methylation interferes with the prevalence of miRNA arms in zebrafish, given their aparent absence in pri-miRNA transcripts of this species. The results obtained in this study bring new contributions to the understanding of regulatory mechanisms inherent to the functional activity of miRNAs. Additionally, they provide subsidies for future research interested in exploring the molecular mechanisms underlying the different m6A methylation rates and their interference in the miRNAs function and evolutionary dynamics in metazoans.
MicroRNAs (miRNAs) are currently known as a large class of regulatory RNAs. During biogenesis, one strand ("arm") of the duplex miRNA is preferably selected as a functionally mature miRNA, while the other strand is usually degraded. Arm switching events occur when there is a change in the preference of the selected arm. This is reflected in the alteration of the arm identified as the most expressed and functional transcript, and has a dynamically variation between stages of development, types of tissues and even between species. Due to the particularities of their mechanism of action, arm switching grants miRNAs an increased regulatory potential by modifying the set of targets and, commonly, the biological processes they regulate. However, the molecular mechanisms that promote arm switching episodes still need to be better characterized. The identification of m6A methylations in pri-miRNAs from human cell lines reported in the literature provided evidence that this type of methylation could be involved in the arm switching process, mainly due to the change that m6A promotes in the secondary structure of RNAs. To test this hypothesis, we used zebrafish, a model vertebrate, to assess whether m6A methylation could be influencing the occurrence of arm switching in this species. This way, we compared the global expression profile of the miRNAs 5p and 3p arms by analyzing a wide collection of RNA-seq data from early periods of embryonic development and various adult zebrafish tissues obtained from the literature. Additionally, we applied the m6A methylation sequencing technique (m6A-seq) in the transcriptome to identify potential correlation between the methylation peaks present in the miRNAs and the identified arm switching events. This set of analyzes led us to the unprecedented identification of 14 arm switching events in zebrafish. Through the detailed study of these cases and their comparison with the events described in other species, we verified that arm switching events are mostly associated with the ontogenetic development of vertebrates. Additionally, our data suggest that the differential expression of miRNA isoforms (isomiRs) may be an important modulator in the prevalence of arms. We have also demonstrated, via target prediction and functional enrichment analyzes, that arm switching events in fact enhance the regulation exercised by miRNAs, by increasing the number of target genes, regulatory networks and biological functions with which they are associated. However, surprisingly, careful analysis of the m6A-seq data provided us with evidence that zebrafish pri-miRNAs are not effectively methylated, even though they contain m6A methylation sites in their sequences. The discrepancy between the results obtained in this work and those reported in humans and, more recently, in Arabidopsis thaliana, sugget that selective pressures acting on the biogenesis of miRNAs may be occurring at different stages in these species. Still, considering recent data from the literature on the interaction between the m6A methylating reader protein and RNA molecules, we conjecture that the secondary structure of the pri-miRNAs may be a permissive factor in this modulation. In this context, we conclude that arm switching is a conserved phenomenon and that it is mostly associated with the ontogenetic development of organisms. We also found no evidence that m6A methylation interferes with the prevalence of miRNA arms in zebrafish, given their aparent absence in pri-miRNA transcripts of this species. The results obtained in this study bring new contributions to the understanding of regulatory mechanisms inherent to the functional activity of miRNAs. Additionally, they provide subsidies for future research interested in exploring the molecular mechanisms underlying the different m6A methylation rates and their interference in the miRNAs function and evolutionary dynamics in metazoans.
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Palavras-chave
RNAs não-codificadores, Biogênese de miRNAs, Modificação de RNAs, Bioinformática, RNA-seq, m6A-seq, non-coding RNAs, miRNA biogenesis, RNA modifications, bioinformatics