Characterization and functional analysis of Lupus La protein from Leishmania braziliensis

• Grant number: 24/20016-3
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2025
• End date: January 31, 2026
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Angela Kaysel Cruz
• Grantee: Isabela D'Auria Antuniassi
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 23/03015-0 - Of epigenetics, genomic variability and gene expression regulation in Leishmania, AP.TEM

Abstract

Protozoa of the genus Leishmania cause a group of neglected diseases known as leishmaniasis, and the study of these parasites is essential for identifying potential therapeutic targets. During its life cycle, this organism goes through distinct environments such as the gut of the phlebotomine vector and the cells of the immune system of the mammalian host. Thus, necessary aspects for its survival include the genetic organization and regulation of gene expression, which is closely related to post-transcriptional modifications. In this context, RNA-binding proteins (RBPs) play an important role in determining the fate of messenger RNAs (mRNAs) by recognizing specific sequences. Recent studies of our lab have identified the Lupus La protein as an RBP binding to long non-coding RNA (lncRNA), differentially expressed in the life cycle of the protozoan. This protein proved to be interesting because it was also found to be associated with the Grumpy lncRNA in T. brucei, which is involved in ribosome biogenesis as well as in the morphological differentiation of the trypanosomatid. Therefore, the main objective of this project is to investigate and characterize Lupus La in Leishmania braziliensis. To achieve this, the CRISPR-Cas9 technique will be used to tag the protein and knock out the Lupus La gene. This will allow for the verification of its subcellular localization through immunofluorescence microscopy, in addition to evaluating phenotypic changes and protein abundance in different life cycle stages of the parasite. Finally, preliminary results of a pull-down assay will be analyzed to visualize the interactions of Lupus La with other proteins and with non-coding RNAs (ncRNAs).