Analysis of the impact of knocking out Nfs and Mtu1 on thiolation of tRNAs in Trypanosoma cruzi

• Grant number: 24/22253-2
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: August 25, 2025
• End date: February 24, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Ariel Mariano Silber
• Grantee: Marilene Souza Braga
• Supervisor: Zdenek Paris
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: Czech Academy of Sciences (CAS), Czech Republic
• Associated to the scholarship: 23/16035-0 - Functional Characterization of NFS, ISD11, and MTU1 Proteins: involvement in Mitochondrial Biology in Trypanosoma cruzi., BP.DD

Abstract

Trypanosoma cruzi, the causative agent of Chagas disease, affects around 7 million people worldwide. It has unique features, including a single-branched mitochondrion, where the kinetoplast structure houses the mitochondrial genome (kDNA). For the translation of kDNA-encoded genes, a complete set of transfer RNAs (tRNAs) must be imported from the cytosol. These tRNAs undergo post-transcriptional modifications, which serve various functions such as stability and gene expression regulation. Among the modifications, thiolation has been found at positions 33 and 34 of the tRNAs, with thiolation of uridine at position 33 (sU33) being identified exclusively in trypanosomatids. The sU33 thiolation negatively modulates the editing of tRNATrp in the mitochondrion of Trypanosoma brucei, impacting mitochondrial gene expression. The protein complex responsible for this modification comprises a cysteine desulfurase (NFS; EC.:2.8.1.7), its accessory protein (ISD11), and a tRNA-specific 2-thiouridylase (MTU1; E.C.:2.8.1.14), using the amino acid cysteine as the thiol donor. The genome of T. cruzi encodes putative orthologs of these proteins, but their role in the mitochondrial biology of this parasite has not yet been investigated. Then, aiming to understand the function of the proteins of interest in T. cruzi biology, knockout strains of the coding sequences of NFS, ISD11 and MTU1 were generated using CRISPR-Cas9. In this project, the main goal is to evaluate the alterations of the levels of thiolation in the cytoplasmic and mitochondrial tRNAs in these knockout cell lines produced: Nfs-/+ and Mtu1-/- and their respective addbacks, in the different life forms of the T. cruzi. This study could shed light on the role of NFS and MTU1 in T. cruzi biology, mainly filling the gap in the tRNA thiolation pathway in this parasite.Keywords: Trypanosoma cruzi, tRNAs, thiolation, cysteine, CRISPR-Cas9. (AU)