Microrna regulation of bursicon in drosophila melanogaster
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Bursicon is a heterodimeric neuropeptide hormone composed of two cystine knot subunits, bursicon α (burs α) and its partner, bursicon β (burs β). In Drosophila melanogaster, the heterodimer regulates wing expansion and the cuticle tanning (sclerotization and melanization) process in newly emerged adult flies via a leucine-rich repeat-containing G protein coupled receptor 2 (DLGR2) while the homodimers mediate expression of genes encoding antimicrobial peptides (AMPs) and stress proteins by activating relish, a transcriptional factor in the immune deficiency (Imd) pathway. However, the regulatory mechanism of bursicon expression remains unknown. MicroRNAs (miRNAs) are small noncoding RNAs, acting as post-transcriptional repressors by base-pairing to the 3´untranslated region (3’UTR) of their target mRNAs. Recent studies show that miRNAs play important roles in regulating insect molting and innate immune response. We hypothesize that miRNAs are involved in the regulation of bursicon gene expression. To verify this hypothesis, we first obtained 3’UTR sequences of burs α and burs β. Then using computational approaches, we predicted a set of miRNAs targeting to the 3’UTR of burs α and burs β genes and created miRNA expression plasmids and luciferase 3’UTR reporters for burs α and burs β. Using these plasmid constructs, we tested their roles in regulating the target gene expression using in vitro dual luciferase assays. Dual luciferase assay results revealed that mir-3642 is the most likely miRNA which regulates burs α gene expression while mir-966, mir-1002, mir-4974 and mir-289 may inhibit burs β gene expression. The developmental expression and tissue distribution profiles of mir-966, mir-1002 and mir-289 revealed that they are expressed in all tested developmental stages and tissues, not just in the CNS where burs α and burs β are expressed. This result indicates that they might have other functions in addition to bursicon regulation during D. melanogaster development and may not be good candidates for investigating the regulation of bursicon expression in the CNS. The miRNA over-expression mutant flies of mir-966, mir-1002 and mir-289 did not show any phenotypes. We did not identify any miRNA which mediates bursicon expression in this study; however, we developed an experimental method to identify miRNAs using miRNA prediction software based on 3’UTR of target gene and verify the functions of the identified miRNAs in vitro via a dual luciferase assay and in vivo in the miRNA over-expression mutants.
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