Identification and characterization of miRNA-target interactions in the metamorphosis of Apis mellifera

Author(s)

Natalia Helena Hernandes

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2016-03-31

Examining board members

Zila Luz Paulino Simoes; Karina Rosa Guidugli Lazzarini; Tiago Campos Pereira

• Advisor: Zila Luz Paulino Simoes

Abstract

Insect metamorphosis is one of the most complex and beautiful of known biological events; it consists of successive morphological and physiological alterations. This intricate process is coordinated by various molecular components, including ecdysteroids (20E), juvenile hormone (JH), transcription factors and microRNAs (miRNAs). The miRNAs regulate gene expression, which in turn orchestrates physiological and anatomical changes necessary for successful insect ontogeny. Despite enormous efforts, the endocrine and genetic circuits that regulate metamorphosis in social insects, such as honey bees (Apis mellifera), are far from being completely elucidated. The miRNAs are a substantial component of this molecular machinery and seem to be ubiquitously involved in the control of biological processes. Disclosing new miRNA-target interactions involved in metamorphosis and in the regulation of 20E and JH cascades can shed light on these poorly understood events. In this study, we provide new pieces to this puzzle. We investigated the roles of miR-34, miR-281, miR-252a and miR-252b, known to be important regulators of insect metamorphosis, in the A. mellifera model. All of these miRNAs revealed a high degree of phylogenetic conservation and responded to treatment with 20E, which altered transcript abundance. Using available information and our databases, we identified interactions involving these miRNAs and the component genes of JH and 20E pathways: ultraspiracle (Usp), fushi tarazu-transcription factor 1 (ftz-f1), ecdysone receptor (EcR), calponin (chd64), insulin receptor 2 (inr2), and Krüppel homolog 1 (Kr-h1). Prediction of miRNA-target interactions revealed that the ecdysteroid receptors EcR and Usp and the transcription factor ftz-f1 are highly targeted by miRNAs involved in metamorphosis; they presented binding sites for all four miRNAs. We also observed that all six-protein coding genes are putatively targeted by miR-34. Using the luciferase assay, we were able to validate the interactions of miR-34 with the targets Krh1, chd64 and inr2; miR-252a with the targets ftz-f1 and EcR; miR-252b with the targets chd64 and ftz-f1; and miR-281 with the targets ftz-f1, EcR and Usp. Investigation of miRNA expression profiles during larval (L3-PP3) and pupal (Pw) development, as a function of the profiles of their respective targets, demonstrated many cases of positive miRNA-mRNA relationships. These results complemented the validation results, showing how the miRNAs regulate their targets. In conclusion, we identified various previously unknown miRNA-mRNA interactions involved in the metamorphosis of A. mellifera. The regulatory pathways proposed and validated by us, as well as their characterizations and relationships with metamorphosis regulator hormones, are unique and add to the understanding of the regulation of metamorphosis in A. mellifera. In this context, our research contributes to a better understanding of the molecular events involved in honey bee metamorphosis. (AU)

• FAPESP's process: 14/18091-5 - Identification and characterization of miRNA-target interactions in the metamorphosis of Apis mellifera
• Grantee: Natália Helena Hernandes
• Support Opportunities: Scholarships in Brazil - Master