Caracterização molecular de RNAs teloméricos não codantes em Leishmania major
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Data
2018-03-23
Autores
Morea, Edna Gicela Ortiz
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Universidade Estadual Paulista (Unesp)
Resumo
Os protozoários do gênero Leishmania causam leishmaniose, uma doença tropical negligenciada, que se apresenta de diferentes formas clínicas e que acomete milhões de pessoas no Brasil e no mundo. Até o momento não existe nenhuma vacina ou tratamento eficientes para leishmaniose e por isso, estudos que contribuam para o entendimento da biologia e fisiologia do parasito podem fornecer uma possibilidade de encontrar novos alvos terapêuticos. Este é o caso dos telômeros, cuja função principal é manter a estabilidade do genoma que se perturbada pode afetar diretamente a proliferação ou multiplicação dos parasitos. Os telômeros são estruturas nucleoprotéicas localizadas nas extremidades dos cromossomos, mantidos pela enzima telomerase. Eles são regulados por diversos processos entre os quais se encontram alguns longos RNAs não codificadores (lncRNA). Entre os lncRNAs com função telomérica está o TER (RNA Telomerase) o qual é um dos principais componentes do complexo telomerase, pois contém uma pequena sequência molde a qual é copiada pela telomerase durante a replicação dos telômeros, o TER foi recentemente identificado em Leishmania spp., porém se desconhece a sua função e biogênese. Outro RNA não codificante com função telomérica é o TERRA (Repetições Teloméricas contendo RNA), os quais são essenciais para a manutenção dos telômeros. Até o momento, identificamos a presença do TERRA expressos a partir das extremidades cromossômicas nas diferentes fases de desenvolvimento (amastigota, promastigota e metacíclica) do parasito. Para isso, utilizamos bibliotecas SL-Seq independentes construídas a partir de RNA total das três formas do parasito, em busca de transcritos que apresentassem o sinal da sequência líder (SL) posicionada a montante das repetições subteloméricas, como um marco que determina o processo de maturação do RNA mensageiro. Além do sinal SL, foi identificada a existência de elementos subteloméricos conservados em Leishmania, denominados CSB (Conserved Sequence Boxes), e ilhas CpGs putativas. Usando esta abordagem nós encontramos 7 classes diferentes de extremidades cromossômicas em L. major. Os resultados obtidos in silico foram validados experimentalmente utilizando Northern blot e RT-PCR. Foi confirmada a existência de transcritos TERRA com tamanho e número variável e expressão diferenciada entre as diferentes formas de desenvolvimento. No entanto, esses transcritos foram identificados em alguns cromossomos e não em todos, confirmando os resultados obtidos in silico. Transcritos TERRA maduros poliadenilados originados de alguns cromossomos também foram identificados nas três formas do parasito. Além disso, foi analisado o comprimento dos telômeros em um ciclo de desenvolvimento e nas fases promastigotas e metacíclicas mantidas in vitro até 6 repiques sucessivos (passagem 1-6). Os resultados mostraram que durante um ciclo de desenvolvimento as formas amastigotas apresentam telômero mais curtos que as formas promastigota e metacíclica derivadas da mesma, e que o tamanho dos telômeros diminui progressivamente nas formas promastigotas e metacíclicas delas derivadas, durante repiques sucessivos in vitro. Também observamos que nos parasitos que apresentam encurtamento progressivo dos telômeros se detecta aumento dos níveis de transcritos TERRA. Portanto, esses resultados sugerem que i) há regulação da expressão de TERRA em nível transcricional nos telômero durante o desenvolvimento de L. major, ii) o TERRA pode estar diretamente envolvido com a manutenção dos telômeros nesses parasitas, talvez regulando negativamente a atividade da telomerase e que iii) provavelmente as formas promastigotas e metacíclicas delas derivadas entram em senescência replicativa após vários ciclos de duplicação in vitro. Em relação à identificação das ilhas CpGs putativas em posição subtelomérica, foram realizadas inicialmente análise de metilação de DNA baseado na utilização de enzimas de restrição bloqueadas por metilação. Os resultados sugerem que estas regiões podem estar metiladas, porém ensaios de tratamento de bissulfito seguido de sequenciamento não confirmaram estes achados. Em relação ao componente RNA da telomerase, TER, para ajudar no entendimento da sua função na biologia do parasito, o gene foi inicialmente clonado e sequenciado para posteriormente ser subclonado em vetor de expressão em Leishmania spp. para realizar as análises funcionais. Um vetor utilizando a metodologia CRISPR/Cas9 para edição do TER no genoma de L. major foi desenhado e está em fase de construção. Também foi padronizada a utilização de oligonucleotídeos antisenso do tipo morpholinos, como ferramenta capaz de abolir a maturação do mRNA TER. Esta metodologia é uma ferramenta muito importante para a realização de knockdown em Leishmania spp., pois somente poucas espécies destes parasitos possuem maquinaria de RNA de interferência.
Protozoan from Leishmania genus are the etiologic agents of leishmaniasis, a tropical disease that presents different clinical manifestations and affect million people in Brazil and in the world. There are no eficiente vacines nor treatment protocols against leishmaniasis, therefore, it is crucial to improve the knowledge about Leishmania molecular biology and physiology for the development of new therapies. Actually, telomeres have been considered potential molecular targets as they are responsible to maintain genome stability. Leishmania telomeres similarly to other eukaryotes, are nucleoprotein structures found at the end of the chromosomes maintained by telomerase. They can be regulated by different mechanisms such as the action of long non-coding telomeric RNAs (lncRNAs). Among the telomeric lncRNAs are the telomerase RNAcomponent (TER) which is crucial for telomerase activity because it contains the template sequence that is copied by telomerase during telomere elongation. TER was recently identified in Leishmania although there is no information about its function and biogenesis.TERRA, the Telomere Repeat containing RNA is other telomeric RNA that it is involved with telomere length regulation telomere damage response and alterations in the composition of telomeric chromatin and is essential for telomere maintenance. In this work, we identified TERRA transcripts in the three Leishmania developmental stages (amastigote, promastigote e metacyclic). We interrogate three independent SL-Seq libraries obtained from total RNA obtained from Leishmania developmental stages and search for transcripts that present the sequence leader (SL) signal located upstream the subtelomeric/telomeric repeats as a mark for mRNA maturation. In addition, we had also search the L. major genome for the presence of Leishmania specific subtelomeric elements, the Conserved Sequence Boxes (CSB) and putative subtelomeric CpG islands. Using this approach, we found 7 different types of chromosome ends in L. major. In silico results were validated using Northern blot and RT-PCR/RT-qPCR. We confirmed the existence of TERRA transcripts of variable length and size, which also present different expression profiles depending on the parasite life stage. Our analyses also revealed the existence of polyadenilated TERRA transcripts from some but not all chromosome ends. In addition, telomere length analysis was done using parasites originated from a single developmental cycle and from promastigote and metacyclic forms maintained in vitro for up to 6 successive replicas in culture. The results showed that during a single developmental cycle amastigote forms present shorter telomeres than promastigotes and metacyclic derived there from. In addition telomeres length progressively shorten in promastigote and derived metacyclic during successive in vitro cultivation, or after increasing population doubling. We also observed that parasites showing progressive telomeres shortening have increased levels of TERRA transcripts. Therefore, these results suggest that i) TERRA expression is probably regulated at the transcriptional level during L. major development, ii) TERRA may be directly involved in parasite telomere maintenance, probably acting as a negative regulator of telomerase activity, and iii) probably promastigotes and derived metacyclic forms enter into replicative senescence after several population derived therefrom enter into replicative senescence after several population doubling cycles in vitro. To identified putatives CpG islands at subtelomeric position, we first performed digestion of DNA with methylation sensitive restriction enzymes. Although these results suggested the existence of methylated DNA at the subtelomeric putative CpG islands, bisulfite treatment followed by DNA sequencing did not confirm these findings. We had also initiate functional studies about a L. major TER component. We cloned, sequenced and subcloned TER gene (leishTER) in a Leishmania expression vector. In addition, we also design a CRISPR/Cas9 system to genetic edit the L. major TER in the parasite genome. And finally, we have standardized the use of antisense morpholine oligonucleotides as a tool to inhibit the maturation of the Leishmania TER component. This later approache will be valuable to study the functions of genes encoding protein and lncRNA in these protozoa, since only a few Leishmania species present the RNA interference machinery.
Protozoan from Leishmania genus are the etiologic agents of leishmaniasis, a tropical disease that presents different clinical manifestations and affect million people in Brazil and in the world. There are no eficiente vacines nor treatment protocols against leishmaniasis, therefore, it is crucial to improve the knowledge about Leishmania molecular biology and physiology for the development of new therapies. Actually, telomeres have been considered potential molecular targets as they are responsible to maintain genome stability. Leishmania telomeres similarly to other eukaryotes, are nucleoprotein structures found at the end of the chromosomes maintained by telomerase. They can be regulated by different mechanisms such as the action of long non-coding telomeric RNAs (lncRNAs). Among the telomeric lncRNAs are the telomerase RNAcomponent (TER) which is crucial for telomerase activity because it contains the template sequence that is copied by telomerase during telomere elongation. TER was recently identified in Leishmania although there is no information about its function and biogenesis.TERRA, the Telomere Repeat containing RNA is other telomeric RNA that it is involved with telomere length regulation telomere damage response and alterations in the composition of telomeric chromatin and is essential for telomere maintenance. In this work, we identified TERRA transcripts in the three Leishmania developmental stages (amastigote, promastigote e metacyclic). We interrogate three independent SL-Seq libraries obtained from total RNA obtained from Leishmania developmental stages and search for transcripts that present the sequence leader (SL) signal located upstream the subtelomeric/telomeric repeats as a mark for mRNA maturation. In addition, we had also search the L. major genome for the presence of Leishmania specific subtelomeric elements, the Conserved Sequence Boxes (CSB) and putative subtelomeric CpG islands. Using this approach, we found 7 different types of chromosome ends in L. major. In silico results were validated using Northern blot and RT-PCR/RT-qPCR. We confirmed the existence of TERRA transcripts of variable length and size, which also present different expression profiles depending on the parasite life stage. Our analyses also revealed the existence of polyadenilated TERRA transcripts from some but not all chromosome ends. In addition, telomere length analysis was done using parasites originated from a single developmental cycle and from promastigote and metacyclic forms maintained in vitro for up to 6 successive replicas in culture. The results showed that during a single developmental cycle amastigote forms present shorter telomeres than promastigotes and metacyclic derived there from. In addition telomeres length progressively shorten in promastigote and derived metacyclic during successive in vitro cultivation, or after increasing population doubling. We also observed that parasites showing progressive telomeres shortening have increased levels of TERRA transcripts. Therefore, these results suggest that i) TERRA expression is probably regulated at the transcriptional level during L. major development, ii) TERRA may be directly involved in parasite telomere maintenance, probably acting as a negative regulator of telomerase activity, and iii) probably promastigotes and derived metacyclic forms enter into replicative senescence after several population derived therefrom enter into replicative senescence after several population doubling cycles in vitro. To identified putatives CpG islands at subtelomeric position, we first performed digestion of DNA with methylation sensitive restriction enzymes. Although these results suggested the existence of methylated DNA at the subtelomeric putative CpG islands, bisulfite treatment followed by DNA sequencing did not confirm these findings. We had also initiate functional studies about a L. major TER component. We cloned, sequenced and subcloned TER gene (leishTER) in a Leishmania expression vector. In addition, we also design a CRISPR/Cas9 system to genetic edit the L. major TER in the parasite genome. And finally, we have standardized the use of antisense morpholine oligonucleotides as a tool to inhibit the maturation of the Leishmania TER component. This later approache will be valuable to study the functions of genes encoding protein and lncRNA in these protozoa, since only a few Leishmania species present the RNA interference machinery.
Descrição
Palavras-chave
Leishmania major, Leishmaniose, Telômero, Ácido ribonucleico, Telomerase, Leishmaniosis, Telomere, Ribonucleic acid