Validation of RNAi knockdown of tRNA synthetases by Real-Time PCR in T.brucei
García, L.T 1; Thiemann, O.H 1.
1Laboratory of Protein Crystallography and Structural Biology, IFSC, Universidade de São Paulo, Av. Trabalhador Sãocarlense 400, PO Box: 369, 13566-590, São Carlos - SP – Brazil.
Diseases caused by trypanosomatid parasites significantly burden the quality of life in developing countries throughout the world. These diseases include African sleeping sickness (T. brucei), Chagas disease (T. cruzi), and Leishmaniasis (Leishmania sp.). Unfortunately, few drugs are available and the few available are toxic or poorly effective. In T. brucei, gene-specific silencing by RNA interference (RNAi) is a valuable tool for determining whether a gene is essential for growth and viability, and additionally, it contributes to reveal information on gene function. Those are basic issue for the validation of drug targets and the development of new drugs. Kinetoplastida are early diverged flagellates that differ from other eukaryotes by a number of features. The mitochondrial (kinetoplast) genetic code of kinetoplastids deviates from the universal code in that the stop codon UGA is used as a codon for tryptophan. All mitochondrial tRNAs of this protists are encoded in the nucleus and imported into the mitochondria by a yet not fully understood mechanism. A single nuclear-encoded tRNATrp(CCA) is used for both nuclear and mitochondrial genes. The imported mitochondrial tRNATrp is edited at the first anticodon position from CCA to UCA, allowing the decoding of specific UGA stop codon to Trp.
In both Leishmania and Trypanosoma, we have identified two different nuclear-encoded tryptophanyl-tRNA-synthetase (WARSs) genes. One is localized to the cytoplasm (WARS1) and the other to the kinetoplast (WARS2). With the purpose to validate the mitochondrial WARS enzymes of trypanosomatids as potential drug target and their physiological role in cellular mechanism, we used the RNAi technique to knockdown the expression of these two enzymes. The full length ORFs encoding WARS1 and WARS2 were cloned into the tetracycline-regulated RNAi vector, pZJM. Procyclic forms (PCF) of T. brucei 2913 were electroporated with these plasmids, allowing the expression of dsRNA from opposing promoters flanking the insertion site.
In this work, the efficiency and specificity of RNAi knockdown were evaluated by Real Time PCR. The knockdown phenotypes observed reveal a significant modification in the induced cell lines showing, unambiguously the central role that WARSs play in cell viability. This work validates the WARS proteins as targets for inhibitor development.
Supported by: CAPES, CEPID-FAPESP.
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