CRISPR mediated-tagging of endogenous RMEL3 lncRNA for study of its molecular interactions and subcellular localization

• Grant number: 22/09764-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2022
• End date: December 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Enilza Maria Espreafico
• Grantee: Vitor Merino Loes
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 13/08135-2 - CTC - Center for Cell-Based Therapy, AP.CEPID
• Associated scholarship(s): 24/09537-1 - RMEL3 long noncoding RNA as a modulator of the immune response in melanoma, BE.EP.IC

Abstract

Long non-coding RNAs (lncRNA) constitute a class of RNAs with function in the regulation of gene expression and cell signaling. Several lncRNAs play roles in cancer and one of them, RMEL3 (Restricted to Melanoma 3), was identified by our laboratory in melanoma, a cancer originated from melanocytes, pigment cells of the skin. RMEL3 is overexpressed in melanomas carrying the BRAFV600E mutation and its expression is dependent on the activity of this kinase. BRAF, the first kinase of the MAPK signaling cascade, is dysregulated in most cancers. Previous data show that RMEL3 silencing reduces the viability of BRAFV600E cells, while overexpression increases the proliferation rate and viability under mitogen deprivation conditions, as well as tumor growth in experimental animals. This evidence makes RMEL3 a potential therapeutic target. In order to characterize the mechanism of action of this lncRNA, our laboratory focused on determining the interactions of endogenous RMEL3 with proteins. Some of the identified candidates are proteins directly involved in the MAPK pathway (ARAF, FAM83D) and in the Hippo tumor suppressor pathway (STK3/Mst2), among others involved in DNA repair, apoptosis, cytoskeleton and mRNA translation. Therefore, the aims of this project are: i) use algorithms for in silico structural modeling of RNA-protein interaction to obtain information on the RMEL3 sites involved in interaction with proteins; ii) tag the RMEL3 lncRNA with the MS2 stem-loop, to validate the previously identified molecular interactions and characterize the intracellular dynamics of this lncRNA in association with target proteins in melanoma cells. With this project, we hope to contribute to the creation of new molecular tools to understand the role of this lncRNA in the regulation of signaling pathways involved in melanoma progression.