RESUMO
The functional characterisation of essential genes in apicomplexan parasites, such as Toxoplasma gondii or Plasmodium falciparum, relies on conditional mutagenesis systems. Here we present a novel strategy based on U1 snRNP-mediated gene silencing. U1 snRNP is critical in pre-mRNA splicing by defining the exon-intron boundaries. When a U1 recognition site is placed into the 3'-terminal exon or adjacent to the termination codon, pre-mRNA is cleaved at the 3'-end and degraded, leading to an efficient knockdown of the gene of interest (GOI). Here we describe a simple method that combines endogenous tagging with DiCre-mediated positioning of U1 recognition sites adjacent to the termination codon of the GOI which leads to a conditional knockdown of the GOI upon rapamycin-induction. Specific knockdown mutants of the reporter gene GFP and several endogenous genes of T. gondii including the clathrin heavy chain gene 1 (chc1), the vacuolar protein sorting gene 26 (vps26), and the dynamin-related protein C gene (drpC) were silenced using this approach and demonstrate the potential of this technology. We also discuss advantages and disadvantages of this method in comparison to other technologies in more detail.
Assuntos
Inativação Gênica , Ribonucleoproteína Nuclear Pequena U1/genética , Toxoplasma/genética , Sequência de Bases , Sítios de Ligação , Cadeias Pesadas de Clatrina/genética , Éxons , Expressão Gênica , Marcação de Genes , Genes Reporter , Loci Gênicos , Vetores Genéticos/genética , Recombinação Homóloga , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Plasmodium falciparum/genética , Ligação Proteica , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Alinhamento de SequênciaRESUMO
Apicomplexan parasites are single eukaryotic cells with a highly polarised secretory system that contains unique secretory organelles (micronemes and rhoptries) that are required for host cell invasion. In contrast, the role of the endosomal system is poorly understood in these parasites. With many typical endocytic factors missing, we speculated that endocytosis depends exclusively on a clathrin-mediated mechanism. Intriguingly, in Toxoplasma gondii we were only able to observe the endogenous clathrin heavy chain 1 (CHC1) at the Golgi, but not at the parasite surface. For the functional characterisation of Toxoplasma gondii CHC1 we generated parasite mutants conditionally expressing the dominant negative clathrin Hub fragment and demonstrate that CHC1 is essential for vesicle formation at the trans-Golgi network. Consequently, the functional ablation of CHC1 results in Golgi aberrations, a block in the biogenesis of the unique secretory microneme and rhoptry organelles, and of the pellicle. However, we found no morphological evidence for clathrin mediating endocytosis in these parasites and speculate that they remodelled their vesicular trafficking system to adapt to an intracellular lifestyle.
