Dual role of R-loops-associated ncRNAs in response to replicative stress and gene expression regulation in Leishmania major

• Grant number: 25/05210-0
• Support Opportunities: Scholarships in Brazil - Master
• Start date: August 01, 2025
• End date: January 31, 2027
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Angela Kaysel Cruz
• Grantee: Amanda Cristina Severino
• 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

Leishmaniasis is a neglected disease caused by protozoa of the Leishmania genus, with visceral and tegumentary forms, affecting more than 90 countries. In addition to its medical relevance, the parasite serves as a genetic model due to its evolutionary position. Gene expression regulation is crucial for its life cycle, involving non-coding RNAs (ncRNAs) in post-transcriptional regulation. Recently, ncRNAs associated with DNA forming R-loops have been identified, potentially playing roles in genome stability, replication, and genetic plasticity in these organisms. This proposal will study ncRNAs identified in association with R-loops in Leishmania major. Among them, two ncRNAs, ncRNA_ODD3 and ncRNA_c12, have been detected in both the nucleus and cytoplasm, acting as gene expression regulators and localizing to genome regions enriched for R-loops. Based on the hypothesis that these transcripts, and similar ones, may have distinct roles as free ncRNAs or in association with R-loops, we aim to investigate their potential dual function, including a possible involvement in replication stress and their role in gene expression regulation in L. major. Two additional ncRNAs, ncRNA19 and ncRNA25, which have not yet been studied, will be included to identify potential common features among ncRNAs associated with R-loops. The dual function of two of these ncRNAs, whether in the R-loop context or as free molecules, will be examined through in vitro pulldown assays. The participation of ncRNAs in R-loops will be confirmed by smRNA FISH, using a conditional RNAse H1 knockout strain. Additionally, the potential trans-regulatory role of ncRNA19 and/or ncRNA25 will be investigated using knockout and overexpressing parasites generated by CRISPR/Cas9. (AU)