Studying the role of m6A methylation in miRNA sorting in extracellular vesicles during obesity and type 2 diabetes

• Grant number: 24/20515-0
• Support Opportunities: Research Grants - Visiting Researcher Grant - International
• Start date: May 21, 2025
• End date: August 18, 2025
• Field of knowledge: Biological Sciences - Physiology - Physiology of Organs and Systems
• Principal Investigator: Marcelo Alves da Silva Mori
• Grantee: Marcelo Alves da Silva Mori
• Visiting researcher: Dr Rajesh Kumari
• Visiting researcher institution: University Of Allahabad/Ua, India
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 21/08354-2 - The interplay between the immune system and metabolism as a key determinant of the aging process, AP.TEM

Abstract

This research proposal aims to discover the role of N6-methyladenosine (m6A) RNA methylation in sorting and packaging of microRNA (miRNA) into the extracellular vesicles during obesity and diabetes. The m6A methylation in eukaryotes is primarily operated by three key protein complexes: "writers" (methyltransferases), "erasers" (demethylases), and "readers" (m6A effector complex). "Writers" are responsible for adding the methyl group, while "erasers" have an opposite function as they remove the methyl group by oxidative demethylation. "Readers" bind to the m6A methylated RNAs and alter their functions. m6A RNA methylation has been shown to regulate most of the important aspects of the RNA biology including nuclear export, splicing, stability, mRNA translation and phase separation into lipid granules for many specific functions. Extracellular vesicles (EVs) are nano carriers regulating intercellular communication by transferring proteins, DNA and RNA including miRNA. The sorting and packaging of different miRNA is not well understood and altered content of EVs is associated with many diseases such as obesity, type 2 diabetes (T2D) and cardiovascular disease. Thus, in this proposal, we aim to discover the role of m6A methylation in conditions of metabolic dysfunction. First, we will isolate EVs from human patient's blood samples (obese with and without T2D) and adipocytes after challenging with metabolic stress. Next, we will be isolating miRNA from these EVs and pool down m6A methylated miRNA using m6A antibodies. The samples will be subjected to methyl RNA immunoprecipitation sequencing and further computational analysis to explore the differentially sorted miRNA profile. Overall, this proposal will not only identify and validate the differential content of EVs from obese patients with established T2D but also unravel the regulatory role of m6A during their content loading under metabolic stress. (AU)