Investigation of the biological function of microRNA arm switching events and their potential association with the RNA methylation m6A using zebrafish as model organism

Author(s)

Arthur Casulli de Oliveira

• Document type: Doctoral Thesis
• Press: Botucatu. 2021-03-10.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2021-02-25
• Advisor: Danillo Pinhal

Abstract

MicroRNAs (miRNAs) are currently known as a large class of regulatory RNAs. During biogenesis, one strand ("arm") of the duplex miRNA is preferably selected as a functionally mature miRNA, while the other strand is usually degraded. Arm switching events occur when there is a change in the preference of the selected arm. This is reflected in the alteration of the arm identified as the most expressed and functional transcript, and has a dynamically variation between stages of development, types of tissues and even between species. Due to the particularities of their mechanism of action, arm switching grants miRNAs an increased regulatory potential by modifying the set of targets and, commonly, the biological processes they regulate. However, the molecular mechanisms that promote arm switching episodes still need to be better characterized. The identification of m6A methylations in pri-miRNAs from human cell lines reported in the literature provided evidence that this type of methylation could be involved in the arm switching process, mainly due to the change that m6A promotes in the secondary structure of RNAs. To test this hypothesis, we used zebrafish, a model vertebrate, to assess whether m6A methylation could be influencing the occurrence of arm switching in this species. This way, we compared the global expression profile of the miRNAs 5p and 3p arms by analyzing a wide collection of RNA-seq data from early periods of embryonic development and various adult zebrafish tissues obtained from the literature. Additionally, we applied the m6A methylation sequencing technique (m6A-seq) in the transcriptome to identify potential correlation between the methylation peaks present in the miRNAs and the identified arm switching events. This set of analyzes led us to the unprecedented identification of 14 arm switching events in zebrafish. Through the detailed study of these cases and their comparison with the events described in other species, we verified that arm switching events are mostly associated with the ontogenetic development of vertebrates. Additionally, our data suggest that the differential expression of miRNA isoforms (isomiRs) may be an important modulator in the prevalence of arms. We have also demonstrated, via target prediction and functional enrichment analyzes, that arm switching events in fact enhance the regulation exercised by miRNAs, by increasing the number of target genes, regulatory networks and biological functions with which they are associated. However, surprisingly, careful analysis of the m6A-seq data provided us with evidence that zebrafish pri-miRNAs are not effectively methylated, even though they contain m6A methylation sites in their sequences. The discrepancy between the results obtained in this work and those reported in humans and, more recently, in Arabidopsis thaliana, sugget that selective pressures acting on the biogenesis of miRNAs may be occurring at different stages in these species. Still, considering recent data from the literature on the interaction between the m6A methylating reader protein and RNA molecules, we conjecture that the secondary structure of the pri-miRNAs may be a permissive factor in this modulation. In this context, we conclude that arm switching is a conserved phenomenon and that it is mostly associated with the ontogenetic development of organisms. We also found no evidence that m6A methylation interferes with the prevalence of miRNA arms in zebrafish, given their aparent absence in pri-miRNA transcripts of this species. The results obtained in this study bring new contributions to the understanding of regulatory mechanisms inherent to the functional activity of miRNAs. Additionally, they provide subsidies for future research interested in exploring the molecular mechanisms underlying the different m6A methylation rates and their interference in the miRNAs function and evolutionary dynamics in metazoans. (AU)

• FAPESP's process: 17/17510-2 - Investigation of the role of m6A methylation on microRNA biogenesis and arm-switching using zebrafish as biological model.
• Grantee: Arthur Casulli de Oliveira
• Support Opportunities: Scholarships in Brazil - Doctorate