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Exploring the Requirement for the CCR4‐NOT Deadenylase Complex Subunit Regena/NOT2 in MicroRNA‐mediated Gene Silencing
Author(s) -
Reinke Catherine
Publication year - 2015
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.29.1_supplement.lb228
Subject(s) - gene silencing , microrna , rna induced silencing complex , biology , genetics , microbiology and biotechnology , rna silencing , rna interference , trans acting sirna , gene , rna , non coding rna
A forward genetic screen was carried out using Drosophila melanogaster to determine the cellular components and molecular 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). Our screen utilized a Green Fluorescent Protein (GFP)‐based reporter to visualize miRNA‐mediated gene silencing. The GFP transgene contains miRNA binding sites, and is regulated by endogenous miRNAs. MiRNA‐mediated silencing is accomplished through translation block, mRNA degradation, or a combination of these mechanisms by miRISC. A requirement for the CCR4‐NOT complex subunit Regena/NOT2 in miRNA‐mediated gene silencing was discovered through genetic analysis of an isolated mutant . We are exploring the specific role of Regena/NOT2 in silencing by determining whether this CCR4‐NOT complex component is required for translational block, transcript degradation, or both. We are further characterizing a putative role for Regena/NOT2 in miRNA biogenesis and maturation, as has been demonstrated in Arabidopsis. Ongoing studies of additional mutants generated in the screen aim to identify the components of miRISC in different cellular contexts and with respect to different miRNA targets, to elucidate the complex molecular mechanisms of miRNA‐mediated silencing.