DigitalCommons@Linfield

Deciphering the Role of Insulin Signaling in microRNA-Mediated Gene Silencing in Drosophila Melanogaster

Jereld R. Nicholson Library

microRNAs (miRNAs) are small, non-coding RNA molecules that silence gene expression. miRNAs bind to target-gene messenger RNA (mRNA) transcripts via complementary base pairing and silence target gene expression by interacting with the RNA-induced silencing complex (RISC). miRNA-mediated gene silencing is accomplished through translation block, mRNA degradation, or a combination of these mechanisms. Though many aspects of miRNA-mediated gene silencing are understood, the regulation of miRNA function and the downstream effectors of gene silencing are poorly characterized. Previously, a forward genetic screen for mutants with altered miRNA function was conducted in Drosophila melanogaster, demonstrating a requirement for the protein kinase AKT1 in miRNA-mediated gene silencing. miRNA function was assayed using an eye-specific Green Fluorescent Protein (GFP)-based reporter of gene silencing. GFP was fused to the Brd gene 3’ untranslated region (UTR), which contains miRNA binding sites and is regulated by miRNA-mediated gene silencing in the Drosophila eye. AKT1 is a key component of the insulin-signaling pathway, thus, current efforts aim to elucidate the role of insulin signaling in miRNA-mediated gene silencing. Previous work suggested that disruption of additional genes in the insulin-signaling pathway influence the silencing of the eye-specific GFP reporter. Transgenic Drosophila lines are being constructed to assay the requirement of insulin signaling for silencing additional reporters of miRNA function, and for gene silencing at different stages of the Drosophila life cycle. These transgenic Drosophila lines will be used to determine the role of relevant genes of interest in the assembly and function of miRNA-specific RISC complexes (miRISCs).