Mim-tRNAseq: Um manãtodo que mede com precisão a abunda¢ncia e o status de modificação de diferentes tRNAs
Os naveis de tRNAs individuais são regulados dinamicamente em diferentes tecidos e durante o desenvolvimento, e os defeitos do tRNA estãoligados a doenças neurolégicas e ca¢ncer.
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)
Representação artastica de tRNAs © Heather Jenkins
Os RNAs de transferaªncia (tRNAs) entregam aminoa¡cidos específicos aos ribossomos durante a tradução do RNA mensageiro em proteanas. A abunda¢ncia de tRNAs pode, portanto, ter um impacto profundo na fisiologia celular, mas medir a quantidade de cada tRNA nas células tem sido limitada por desafios técnicos. Pesquisadores do Instituto Max Planck de Bioquímica agora superaram essas limitações com mim-tRNAseq, um manãtodo que pode ser usado para quantificar tRNAs em qualquer organismo e ajudara¡ a melhorar nossa compreensão da regulação de tRNA na saúde e na doena§a.
Uma canãlula contanãm várias centenas de milhares de moléculas de tRNA, cada uma das quais consiste em apenas 70 a 90 nucleotadeos dobrados em um padrãosemelhante a uma folha de trevo. Em uma extremidade, os tRNAs carregam um dos vinte aminoa¡cidos que servem como blocos de construção de proteanas, enquanto a extremidade oposta emparelha com o ca³don especificando este aminoa¡cido no RNA mensageiro durante a tradução. Embora existam apenas 61 ca³dons para os 20 aminoa¡cidos, as células de diferentes organismos podem conter centenas de moléculas de tRNA exclusivas, algumas das quais diferem umas das outras por apenas um aºnico nucleotadeo. Muitos nucleotadeos em tRNAs também são decorados com modificações químicas, que ajudam os tRNAs a dobrar ou ligar o ca³don correto.
Os naveis de tRNAs individuais são regulados dinamicamente em diferentes tecidos e durante o desenvolvimento, e os defeitos do tRNA estãoligados a doenças neurolégicas e ca¢ncer. As origens moleculares dessas ligações permanecem obscuras, porque quantificar a abunda¢ncia e as modificações de tRNAs nas células sempre foi um desafio. A equipe de Danny Nedialkova no MPI of Biochemistry desenvolveu agora mim-tRNAseq, um manãtodo que mede com precisão a abunda¢ncia e o estado de modificação de diferentes tRNAs nas células.
Roadblocks e resoluções de modificação
Para medir os naveis de vários RNAs simultaneamente, os cientistas usam uma enzima chamada transcriptase reversa para primeiro reescrever o RNA em DNA. Milhaµes dessas ca³pias de DNA podem ser quantificadas em paralelo por sequenciamento de alto rendimento. Reescrever tRNAs em DNA tem sido extremamente difacil, pois muitas modificações de tRNA bloqueiam a transcriptase reversa, fazendo com que ela pare de sintetizar DNA.
"Muitas pesquisas propuseram soluções elegantes para este problema, mas todas elas aliviam apenas uma fração dos obsta¡culos de modificação em tRNAs", explica Danny Nedialkova, lider do grupo de pesquisa Max Planck no Instituto Max Planck de Bioquímica. "Percebemos que uma transcriptase reversa especafica parecia ser muito melhor na leitura de satios de tRNA modificados. Otimizando as condições de reação, poderaamos melhorar significativamente a eficiência da enzima, permitindo-lhe ler quase todos os bloqueios de modificação de tRNA", acrescenta Nedialkova. Isso tornou possível construir bibliotecas de DNA a partir de ca³pias de tRNA de comprimento total e usa¡-las para sequenciamento de alto rendimento.
O kit de ferramentas computacionais mim-tRNAseq
A análise dos dados de sequenciamento resultantes também apresentou desafios significativos. "Identificamos dois problemas principais: o primeiro éa extensa similaridade de sequaªncia entre diferentes transcrições de tRNA", explica Andrew Behrens, Ph.D. estudante do grupo de Nedialkova e primeiro autor do artigo. "O segundo vem do fato de que um nucleotadeo incorreto (uma incorporação incorreta) éintroduzido em muitos locais modificados durante a transcrição reversa. Ambos tornam extremamente desafiador atribuir cada DNA lido a molanãcula de tRNA da qual se originou", acrescenta Behrens.
A equipe abordou essas questões com novas abordagens computacionais, incluindo o uso de anotações de modificação para orientar o alinhamento de leitura preciso. O kit de ferramentas abrangente resultante éempacotado em um pipeline disponavel gratuitamente para alinhamento, análise e visualização de dados de sequenciamento derivados de tRNA . Os pesquisadores podem usar mim-tRNAseq não apenas para medir a abunda¢ncia do tRNA, mas também para mapear e quantificar as modificações do tRNA que induzem a incorporação incorreta de nucleotadeos pela transcriptase reversa. "mim-tRNAseq abre inaºmeras possibilidades no futuro", diz Nedialkova. "Esperamos que isso ajude a nose a outros a resolver muitas questões pendentes sobre a biologia do tRNA na saúde e na doena§a."