RuvB-like 1: essentiality and function in Leishmania braziliensis

• Grant number: 23/14086-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: January 01, 2024
• End date: January 31, 2026
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Angela Kaysel Cruz
• Grantee: Fernanda Hergert Voigt
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 18/14398-0 - UK:Brazil Joint Centre Partnership in Leishmaniasis (JCPiL), AP.TEM

Abstract

The Leishmaniases are caused by trypanosomatid parasites of the genus Leishmania and represent a global public health issue. These parasites have particularities regarding their genetic organization and regulation of gene expression: their genes are arranged in polycistronic units without the presence of specific promoters, and the transcripts undergo the process of trans-splicing to originate mature mRNAs, therefore concentrating their gene expression control at the post-transcriptional level. This control can be done by mRNA-binding proteins, also known as RBP, which can be affected by post-translational modifications (PTM), such as the methylation of arginine residues catalyzed by PRMTs (protein arginine methyltransferases) enzymes, which alter the affinity of RBPs for their substrates and interaction partners. In a previous project developed in the laboratory, the RuvB-like 1 protein of L. braziliensis was co-immunoprecipitated alongside LbrPRMT5, during the search for the proteins affected by PRMTs. RuvB is a putative helicase possibly involved in reversing damage in the replication fork and during the formation of the Holliday structure. In our laboratory, the LbrRuvB-like 1 enzyme has been tagged with myc on the N-terminal portion, and its subcellular location has been determined. Unexpectedly, the enzyme was found mainly in the cytoplasm, apparently with a fraction present in the nucleus of promastigote forms; this distribution needs to be reevaluated throughout the cell cycle. Gene knockout attempts for LbrRuvB-like 1 by CRISPR/Cas9 were unsuccessful, so to evaluate the essentiality of LbrRuvB-like 1 and its functionality, we will use an inducible knockout strategy by DiCre (Dimerised Cre recombinase), with subsequent cellular analysis of the effect of turning off this gene by defining the growth rate and the phosphorylation rate of histone c-H2A, and its involvement in resistance to genotoxic stress using the MTT method. Furthermore, methodologies such as immunofluorescence and western blot will be used to define the subcellular localization of LbrRuvB-like 1 during the parasite's life cycle phases.