Efeitos do silenciamento de duas Triptofanil-tRNA sintetases de Trypanosoma brucei mediante RNA de interferência
Fecha
2001-06-21Autor
Sánchez, Liliana Torcoroma García
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The parasitic Trypanosoma brucei is the causative agent of African
Trypanosomiasis (sleeping sickness) in humans, and nagana in animals and is
responsible for heavy socioeconomic losses in most countries of sub-Saharan
Africa. Therapy against sleeping sickness is unsatisfactory because the toxicity
and arising of the drug resistant parasites. Trypanosomatids contain a single
mitochondrion, the kinetoplast, whose genetic code deviates from the universal
code where a UGA stop codon is used as a Trp codon. A single nuclear-encoded
tRNATrp(CCA), that can decode the canonical Trp codon, is used by both the
nucleus and mitochondria genes. A C to U editing event at position 34 of
tRNATrp(CCA) changes the anticodon from CCA to UCA allowing the decoding of
the UGA stop codon to Trp from the mitochondrial genes. We have identified
two different nuclear-encoded tryptophanyl-tRNA-synthetase (WARSs) genes
(one cytoplasmic (WARS1) and the other to the kinetoplast (WARS2)) in both
Leishmania and Trypanosoma cells. With the purpose to validate the
mitochondrial WARS enzymes of trypanosomatids, as potential drug targets, we
performed gene silencing (RNAi) experiments with both WARSs forms from T.
brucei. In the conditions tested, the expression of dsRNA from a dual promoter
system generated potent RNA silencing, leading to clear phenotypes
characterized by morphologic alterations, severe growth inhibition, reduction in
the levels of oxygen consumed (only TbWARS2) and cellular death. The
efficiency and specificity of silencing were estimated by quantitative PCR and
significant modifications of the specific amount of TbWARSs mRNA were
evidenced.