RESUMO
The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3'-5' exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection.
Assuntos
Exorribonucleases/genética , MicroRNAs/genética , Nucleotidiltransferases/genética , Estabilidade de RNA , RNA Mensageiro/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sequência de Bases , Biotinilação , Linhagem Celular Tumoral , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Exorribonucleases/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Dados de Sequência Molecular , Nucleotidiltransferases/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Polinucleotídeo Adenililtransferase , RNA Mensageiro/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Transgenesis is an essential tool to investigate gene function and to introduce desired characters in laboratory organisms. Setting-up transgenesis in non-model organisms is challenging due to the diversity of biological life traits and due to knowledge gaps in genomic information. Some procedures will be broadly applicable to many organisms, and others have to be specifically developed for the target species. Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects. Here, we report a compilation of the transgenesis tools that we have designed for the malaria vector Anopheles gambiae, including new docking strains, convenient transgenesis plasmids, a puromycin resistance selection marker, mosquitoes expressing cre recombinase, and various reporter lines defining the activity of cloned promoters. This toolbox contributed to rendering transgenesis routine in this species and is now enabling the development of increasingly refined genetic manipulations such as targeted mutagenesis. Some of the reagents and procedures reported here are easily transferable to other nonmodel species, including other disease vector or agricultural pest insects.
Assuntos
Anopheles/genética , Técnicas de Transferência de Genes , Animais , Animais Geneticamente Modificados , Clonagem Molecular , Códon/genética , Elementos de DNA Transponíveis/genética , Embrião não Mamífero/metabolismo , Citometria de Fluxo , Genes de Insetos , Genes Reporter , Vetores Genéticos/metabolismo , Integrases/metabolismo , Larva/genética , Microinjeções , Mosaicismo , Óvulo/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Transgenes , Transposases/metabolismoRESUMO
Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control.