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Exploration of the Role of the CCR4‐NOT Deadenylase Complex Subunit Regena/NOT2 in MicroRNA‐mediated Gene Silencing
Author(s) -
Knox James,
Rees Katie,
Zbornik Tika,
Morin Michael,
Bowden Sean
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1060.3
Subject(s) - gene silencing , biology , rna induced silencing complex , microrna , rna interference , genetics , argonaute , reporter gene , rna silencing , gene , gene expression , microbiology and biotechnology , regulation of gene expression , non coding rna , rna
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. We are investigating the necessity of this gene in miRNA‐mediated gene silencing at various stages of Drosophila development by determining differences in gene expression in mutant larva lacking functional NOT2. NOT2‐mediated reporter silencing has been shown to be dependent upon the activity of miRNAs as demonstrated by the use of an additional GFP‐based reporter of gene silencing. Reporter gene expression in larval fat body tissues is being assessed to determine whether NOT2 is required for silencing at different developmental time points and in different tissues of Drosophila .. Investigation of the composition, abundance, and localization of known components of the silencing machinery is revealing distinct mechanisms for the regulation of silencing.