Submission Title

Regena/NOT2 Is Essential for Gene Silencing by MicroRNAs

Subject Area

Biology

Description

A forward genetic screen in Drosophila melanogaster has yielded genetic evidence demonstrating a requirement for the CCR4/NOT complex subunit Regena/NOT2 in gene silencing. A single mutation in Regena/NOT2 generated by ethyl methanesulfonate mutagenesis leads to increased expression of a GFP reporter that is fused to the 3′UTR of the Drosophila Bearded gene and sensitive to microRNA-mediated gene silencing. MicroRNAs 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). MicroRNA-mediated silencing is accomplished by miRISC through translation block, target mRNA degradation, or a combination of these mechanisms. To determine whether the observed requirement of Regena/NOT2 for gene silencing is dependent upon the activity of microRNAs, the effect of a mutation in Regena/NOT2 on a GFP-based reporter with disrupted microRNA-binding sites and insensitive to microRNA-mediated gene silencing was assayed. No change in reporter expression was observed in the presence of a Regena/NOT2 mutation when the reporter lacked microRNA-binding sites, supporting a microRNA-dependent role for Regena/NOT2 in gene silencing. We are further exploring the specific role of Regena/NOT2 in gene silencing by determining whether this CCR4-NOT complex component is required for translational block, transcript degradation, or both, and characterizing a putative role for Regena/NOT2 in microRNA biogenesis and maturation, as has been demonstrated in Arabidopsis. Ongoing studies of additional mutants generated in the forward genetic screen aim to identify additional essential miRISC components, to elucidate the complex molecular mechanisms of microRNA-mediated silencing.

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Regena/NOT2 Is Essential for Gene Silencing by MicroRNAs

A forward genetic screen in Drosophila melanogaster has yielded genetic evidence demonstrating a requirement for the CCR4/NOT complex subunit Regena/NOT2 in gene silencing. A single mutation in Regena/NOT2 generated by ethyl methanesulfonate mutagenesis leads to increased expression of a GFP reporter that is fused to the 3′UTR of the Drosophila Bearded gene and sensitive to microRNA-mediated gene silencing. MicroRNAs 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). MicroRNA-mediated silencing is accomplished by miRISC through translation block, target mRNA degradation, or a combination of these mechanisms. To determine whether the observed requirement of Regena/NOT2 for gene silencing is dependent upon the activity of microRNAs, the effect of a mutation in Regena/NOT2 on a GFP-based reporter with disrupted microRNA-binding sites and insensitive to microRNA-mediated gene silencing was assayed. No change in reporter expression was observed in the presence of a Regena/NOT2 mutation when the reporter lacked microRNA-binding sites, supporting a microRNA-dependent role for Regena/NOT2 in gene silencing. We are further exploring the specific role of Regena/NOT2 in gene silencing by determining whether this CCR4-NOT complex component is required for translational block, transcript degradation, or both, and characterizing a putative role for Regena/NOT2 in microRNA biogenesis and maturation, as has been demonstrated in Arabidopsis. Ongoing studies of additional mutants generated in the forward genetic screen aim to identify additional essential miRISC components, to elucidate the complex molecular mechanisms of microRNA-mediated silencing.