Functional characterization of lncRNA52 and its interaction with LbrISWI in Leishmania braziliensis

• Grant number: 25/09918-8
• Support Opportunities: Scholarships in Brazil - Master
• Start date: October 01, 2025
• End date: February 28, 2027
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Angela Kaysel Cruz
• Grantee: Brenda Nobile de Freitas
• 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

Leishmania (Viannia) braziliensis is the principal etiological agent of tegumentary leishmaniasis in Brazil. During its digenetic life cycle, the parasite alternates between the sand fly vector and the mammalian host, undergoing drastic environmental and physiological transitions. In contrast to higher eukaryotes, gene expression in Leishmania is primarily regulated at the post-transcriptional level. Within this regulatory framework, non-coding RNAs (ncRNAs), either functioning independently or as components of ribonucleoprotein complexes, play crucial roles in modulating gene expression. Comparative transcriptomic analyses across the three major developmental stages of L. braziliensis have identified long non-coding RNAs (lncRNAs) that are differentially expressed (DE) in a stage-specific manner. Among them, lncRNA52, transcribed from an intergenic region on chromosome 1, was identified in silico as being predominantly expressed in amastigotes, the intracellular form that resides within mammalian host macrophages. Functional characterization through in vitro RNA pulldown assays and in vivo RNA immunoprecipitation (RIP) revealed that lncRNA52 interacts with several proteins, most notably LbISWI, a chromatin remodeling ATPase belonging to the ISWI (Imitation Switch) family. This project aims to elucidate the functional role of lncRNA52 and its molecular interaction with the LbISWI protein across the parasite's life cycle. Specific objectives include: (i) validating the stage-specific expression patterns of lncRNA52 and LbISWI across the three main life stages of the parasite; (ii) determining the subcellular localization of lncRNA52 and LbISWI across the life stages; (iii) identifying the interaction partners of LbISWI recovered through RIP assay; (iv) identifying proteins that interact with lncRNA52 in amastigotes; (v) assessing the effects of lncRNA52 knockout (KO) and overexpression (OE) on LbISWI expression and subcellular distribution; (vi) investigating the reciprocal impact of LbISWI KO and LbISWI OE on lncRNA52 abundance and localization. By dissecting the functional interplay between a long non-coding RNA and a chromatin remodeling factor, this study seeks to unveil novel regulatory mechanisms underpinning Leishmania gene expression, thereby advancing our understanding of parasite biology and revealing potential targets for therapeutic intervention. (AU)