Evidence of putative non-coding RNAs from Leishmania untranslated regions

Freitas-Castro, Felipe; Ruy, Patrícia de Cássia; Zeviani, Karina Nogueira; Santos, Ramon F.; Toledo, Juliano Simões de; Cruz, Ângela K.

doi:10.1016/j.molbiopara.2017.04.002

Cited by 15 publications

(20 citation statements)

References 23 publications

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“…The regulation occurs mainly at the post-transcriptional level through cis-and trans-acting elements (C. Clayton & Shapira, 2007). These elements have a role for example in the splicing and stability/instability of mRNAs and/or in the efficiency of translation (Freitas Castro et al, 2017;Pasion, Hines, Ou, Mahmood, & Ray, 1996). Taking into account the importance of endogenous UTRs for certain genes, we designed a new strategy in which instead of closely flanking the gene with LoxP sequences, we would target the least transcribed intergenic regions (Figure 2), therefore preserving endogenous UTRs.…”

Section: Utr Preservation Strategymentioning

confidence: 99%

Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

Yagoubat

Crobu

Berry

et al. 2020

Cellular Microbiology

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Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR‐Cas9 has been applied successfully to these parasites providing a robust tool to study non‐essential gene functions. Here, we have developed a versatile inducible system combining Di‐Cre recombinase and CRISPR‐Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre‐recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di‐Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed “universal” template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR‐based (molecular cloning‐free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.

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Section: Utr Preservation Strategymentioning

confidence: 99%

Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

Yagoubat

Crobu

Berry

et al. 2020

Cellular Microbiology

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“…foram sintetizadas na presença de [α-32 P] dCTP como descrito previamente (Feinberg e Vogelstein, 1983), alternativamente para os RNAs menores que 500 nt, sondas (3' endlabeled) foram sintetizadas na presença de [γ -32 P] ATP (Freitas-Castro et al, 2017).…”

Section: Northern Blotunclassified

“…Nos últimos anos, os estudos com RNAs não codificadores provocaram grandes mudanças em todos os campos da biologia e medicina, identificando uma nova camada nos mecanismos conhecidos de regulação da expressão gênica (Hüttenhofer et al, 2005; Cech e Steitz, 2014; Calin, 2019). Em Leishmania, o estudo com ncRNAs ainda é pouco explorado, apesar como a atuação de elementos cis e trans nos processos de regulação da expressão gênica desses parasitos (Rochette et al, 2008;Vasconcelos et al, 2012;Terrão et al, 2017Mercer et al, 2011Freitas-Castro et al, 2017) . As camadas dentro de cada fatia (cromossomo) representam de fora para dentro: CDS; ncRNAs putativos diferencialmente expressos (DE) em PROvsMETA (P/M); METAvsAMA (M/A); e AMAvsPRO (A/P); ncRNAs putativos selecionados para análises funcionais (**); ncRNAs DE nas três comparações entre os estágios de vida (3xDE) e na camada mais interna, Tamanho/localização (L) dos ncRNAs.…”

Section: Identificação E Triagem De Ncrnas Putativos Em L Braziliensisunclassified

“…Caracteristicamente, um grande reportório de snoRNAs está presente em tripanossomatídeos, desempenhando funções específicas no processamento de rRNAs (Michaeli et al, 2012). Mais recentemente em nosso grupo, transcritos derivados de 3'UTR tiveram seus potenciais como ncRNAs regulatórios sugeridos em L. major e L. donovani (Freitas-Castro et al, 2017). Fruto deste trabalho em autoria partilhada com Patrícia Ruy, foram identificados uma pletora de ncRNAs L. braziliensisrepresentando 15% do genoma -; sendo alguns diferencialmente expressos durante o ciclo de vida do parasito (Ruy et al, 2019).…”

Section: Introductionunclassified

“…Fruto deste trabalho em autoria partilhada com Patrícia Ruy, foram identificados uma pletora de ncRNAs L. braziliensisrepresentando 15% do genoma -; sendo alguns diferencialmente expressos durante o ciclo de vida do parasito (Ruy et al, 2019). No entanto, investigações mais detalhadas, e a caracterização funcional de diferentes classes de ncRNAs nesses tripanossomatídeos requerem investigação para entender biogênese, papéis e mecanismos e possíveis relações entre a regulação da expressão gênica e diferenças na patogenicidade desses parasitos (Cupolillo et al, 1995;Freitas-Castro, et al, 2017).…”

Section: Introductionunclassified

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Investigação de RNAs não codificadores em Leishmania (Viannia) braziliensis

Teles¹

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Leishmania is a genus of trypanosomatid protozoan parasites, causative agents of leishmaniases. Viannia sub-genera species as Leishmania (Viannia) braziliensis are the causative agents of cutaneous and muco-cutaneous leishmaniasis in Central and South America. The gene expression in this parasites is regulated via post-transcriptional mecanisms comprising the action of cis and trans regulatory elements and RNA binding proteins. In this context, non coding RNAs poorly explored as putative factors involved in regulation of gene expression in Leishmania must be investigated. In this study the transcriptome of coding and ncRNAs of L. braziliensis were analysed. Differential expression analysis, including gene ontology enrichment analysis, during the parasite development revealed the expected paterns for gene expression in Leishamnia spp. A comparative analysis, considering up-regulated genes suggested a contribution of paralogues genes to diversity between Leishmania. spp. To uncover putative ncRNAs, a computational pipeline was previous designed identifying and characterizing 11,372 putative ncRNAs in L. braziliensis, allowing a classification into different ncRNAs classes. The differential expression analysis revealed similar patterns to those observed for protein coding genes. Thirty-five putative ncRNAs were categorized, selected and subjected to Northern blotting being 6 of them selected to functional analysis. These selected group was investigated conducting and stablishing knockout lines, that revealed phenotypic differences concerning diminishing in promastigote growth and in vitro macrophage infection, suggesting possible functional roles of ncRNAs in L. braziliensis. Finnaly, the protein interactions of these ncRNAs were revealed and must be explored in the future to uncover possible regulatory roles of this ncRNAs until now, putative in L. braziliensis. This work represents an outline of L. braziliensis transcriptome contributing to improve the understanding of coding and noncoding RNA content in the parasite.

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Genome and transcriptome analyses of Leishmania spp.: opening Pandora’s box

Cruz

Freitas-Castro

2019

Current Opinion in Microbiology

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Evidence of putative non-coding RNAs from Leishmania untranslated regions

Cited by 15 publications

References 23 publications

Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

Investigação de RNAs não codificadores em Leishmania (Viannia) braziliensis

Genome and transcriptome analyses of Leishmania spp.: opening Pandora’s box

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