Submission Title

Is the Observed Silencing Activity of Regena/NOT2 Dependent upon the Activity of MicroRNAs?

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). A forward genetic screen was carried out to identify requirements for miRNA-mediated silencing in Drosophila melanogaster. Genetic analysis revealed that the CCR4-NOT deadenylase-complex subunit Regena (NOT2) is required for miRNA-mediated silencing. The reporter system used to identify mutants with altered silencing utilized Green Fluorescent Protein (GFP) fused to the Brd gene 3’UTR, which is regulated by endogenous miRNAs. We are investigating whether Regena/NOT2’s activity in silencing is indeed dependent upon miRNA activity. Two reporter systems, one with functional miRNA binding sites in Brd 3’UTR and one with non-functional miRNA binding sites in the Brd 3’ UTR, are being used to analyze Regena’s activity in silencing the reporter transcript. If Regena/NOT2 acts via miRNAs, then the absence of Regena/NOT2 will lead to an increase in GFP expression and fluorescence when using the reporter with miRNA biding sites. However if Regena’s role in silencing is not dependent on miRNA activity, then the same increase in GFP expression and fluorescence will be observed in the absence of Regena/NOT2, even with the non-functional reporter system, which lacks miRNA binding sites. Observing no change in the reporter lacking miRNA binding sites in the absence of Regena/NOT2 will support the hypothesis that Regena/NOT2 activity is depending upon miRNA activity and Regena/NOT2 is not acting as a repressor of transcription. Further studies will aim to determine where Regena/NOT2 acts in the miRNA mediated gene silencing pathway.

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Is the Observed Silencing Activity of Regena/NOT2 Dependent upon the Activity of MicroRNAs?

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). A forward genetic screen was carried out to identify requirements for miRNA-mediated silencing in Drosophila melanogaster. Genetic analysis revealed that the CCR4-NOT deadenylase-complex subunit Regena (NOT2) is required for miRNA-mediated silencing. The reporter system used to identify mutants with altered silencing utilized Green Fluorescent Protein (GFP) fused to the Brd gene 3’UTR, which is regulated by endogenous miRNAs. We are investigating whether Regena/NOT2’s activity in silencing is indeed dependent upon miRNA activity. Two reporter systems, one with functional miRNA binding sites in Brd 3’UTR and one with non-functional miRNA binding sites in the Brd 3’ UTR, are being used to analyze Regena’s activity in silencing the reporter transcript. If Regena/NOT2 acts via miRNAs, then the absence of Regena/NOT2 will lead to an increase in GFP expression and fluorescence when using the reporter with miRNA biding sites. However if Regena’s role in silencing is not dependent on miRNA activity, then the same increase in GFP expression and fluorescence will be observed in the absence of Regena/NOT2, even with the non-functional reporter system, which lacks miRNA binding sites. Observing no change in the reporter lacking miRNA binding sites in the absence of Regena/NOT2 will support the hypothesis that Regena/NOT2 activity is depending upon miRNA activity and Regena/NOT2 is not acting as a repressor of transcription. Further studies will aim to determine where Regena/NOT2 acts in the miRNA mediated gene silencing pathway.