Characterization of the mitochondrial pathway of thiolation of tRNAs in Trypanosoma cruzi

Abstract

The parasite that causes Chagas' disease, Trypanosoma cruzi, is a single-celled pathogen that affects about 7 million people worldwide. This parasite has unique characteristics related to its biology, including a single mitochondrion, which houses the structure called kinetoplast containing mitochondrial genome (kDNA). For the translation of genes encoded by kDNA to occur, the importation of all the transport RNAs (tRNAs) from the cytosol is necessary. tRNAs are the most abundantly post-transcriptionally modified RNA species, and these modifications can range from simple methylations to base editing or the addition of complex molecules, such as sugars. These modifications perform different functions such as maintaining the stability of the tRNAs, increasing the codon-anticodon pairing capacity, preventing the change of reading frame during translation and regulating gene expression. Among the post-transcriptional modifications identified so far in tRNAs, thiolation can be found at positions 33 and 34 of tRNAs, whereas uridine thiolation at position 33 (sU33) has been identified only in trypanosomatids. It has been shown that sU33 negatively modulates tRNATrp editing in the mitochondrion of Trypanosoma brucei, which directly affects mitochondrial gene expression. The thiolation of uridine in the mitochondrion requires the protein complex formed by a cysteine desulfurase (Nfs; EC:2.8.1.7), a tRNA-specific 2-thiouridylase (Mtu1; EC:2.8.1.14) and an accessory protein (Isd11), besides using the amino acid cysteine as a thiol group donor. The T. cruzi genome encodes putative orthologs of genes involved in the mitochondrial pathway of thiolation of tRNAs, but the biological role of this pathway has not yet been investigated in this parasite. Thus, the present project aims to investigate the impact of these enzymes on the biology of T. cruzi through the generation of knockout strains for the coding sequences of the genes putatively involved in the mitochondrial thiolation pathway using the CRISPR-Cas9 bar-seq method with the subsequent phenotypic evaluation of the mutant strains throughout the parasite's life cycle.