Submission Title

An In Vivo Approach to the Discovery and Characterization of Cellular Mechanisms Regulating microRNA-Mediated Gene Silencing

Location

Jereld R. Nicholson Library

Subject Area

Biology

Description

Our goal is to determine the cellular mechanisms that regulate microRNA (miRNA)-mediated gene silencing. MiRNAs are non-coding RNA molecules that interact with target-gene messenger RNA transcripts via complementary base pairing and silence target gene expression via interaction with the miRNA-induced silencing complex (miRISC). Silencing is accomplished through translation block, mRNA degradation, or a combination of these mechanisms. A forward genetic screen was carried out to identify requirements for miRNA-mediated silencing in Drosophila melanogaster. miRNA function was assayed using Green Fluorescent Protein (GFP)-based reporters of gene silencing. The GFP transgenes contain miRNA binding sites and are regulated by endogenous miRNAs. We have genetic evidence demonstrating roles for both Regena (NOT2), a subunit of the CCR4-NOT deadenylase complex, and the protein kinase dAkt1, in miRNA-mediated gene silencing. Further studies have implicated insulin-signaling in general and specific components of the insulin-signaling pathway in particular in the regulation of miRNA activity. We are investigating the necessity of these genes in miRNA-mediated gene silencing at various stages of Drosophila development. Investigation of the composition, abundance, and localization of known components of the silencing machinery is revealing distinct mechanisms for the regulation of silencing.

Comments

1st place award

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An In Vivo Approach to the Discovery and Characterization of Cellular Mechanisms Regulating microRNA-Mediated Gene Silencing

Jereld R. Nicholson Library

Our goal is to determine the cellular mechanisms that regulate microRNA (miRNA)-mediated gene silencing. MiRNAs are non-coding RNA molecules that interact with target-gene messenger RNA transcripts via complementary base pairing and silence target gene expression via interaction with the miRNA-induced silencing complex (miRISC). Silencing is accomplished through translation block, mRNA degradation, or a combination of these mechanisms. A forward genetic screen was carried out to identify requirements for miRNA-mediated silencing in Drosophila melanogaster. miRNA function was assayed using Green Fluorescent Protein (GFP)-based reporters of gene silencing. The GFP transgenes contain miRNA binding sites and are regulated by endogenous miRNAs. We have genetic evidence demonstrating roles for both Regena (NOT2), a subunit of the CCR4-NOT deadenylase complex, and the protein kinase dAkt1, in miRNA-mediated gene silencing. Further studies have implicated insulin-signaling in general and specific components of the insulin-signaling pathway in particular in the regulation of miRNA activity. We are investigating the necessity of these genes in miRNA-mediated gene silencing at various stages of Drosophila development. Investigation of the composition, abundance, and localization of known components of the silencing machinery is revealing distinct mechanisms for the regulation of silencing.