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MiRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms

ABSTRACT: microRNAs (miRNAs) regulate a wide variety of biological processes by silencing their target genes. Argonaute (AGO) proteins load miRNAs to form RNA-induced silencing complex (RISC), which mediates translational repression and/or mRNA decay of the targets. A scaffold protein called GW182 directly binds AGO and the CCR4-NOT deadenylase complex, initiating the mRNA decay reaction. Although previous studies have demonstrated the critical role of GW182 in vitro and in cultured cells, its biological significance in living organisms remains largely unexplored. Here, we generated gw182-null flies using the CRISPR/Cas9 system and found that, unexpectedly, they can survive until an early second instar larval stage. Moreover, in vivo miRNA reporters can be effectively repressed even in the absence of GW182. Nevertheless, gw182-null flies have defects in expression of chitin-related genes and formation of the larval trachea system, preventing them to complete the larval development. Our results highlight the importance of both GW182-depedent and -independent silencing mechanisms in vivo.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE192811 | GEO | 2024/09/29

REPOSITORIES: GEO

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miRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms

Project description:microRNAs (miRNAs) regulate a wide variety of biological processes by silencing their target genes. Argonaute (AGO) proteins load miRNAs to form RNA-induced silencing complex (RISC), which mediates translational repression and/or mRNA decay of the targets. A scaffold protein called GW182 directly binds AGO and the CCR4-NOT deadenylase complex, initiating the mRNA decay reaction. Although previous studies have demonstrated the critical role of GW182 in vitro and in cultured cells, its biological significance in living organisms remains largely unexplored. Here, we generated gw182-null flies using the CRISPR/Cas9 system and found that, unexpectedly, they can survive until an early second instar larval stage. Moreover, in vivo miRNA reporters can be effectively repressed even in the absence of GW182. Nevertheless, gw182-null flies have defects in expression of chitin-related genes and formation of the larval trachea system, preventing them to complete the larval development. Our results highlight the importance of both GW182-depedent and -independent silencing mechanisms in vivo.

2024-09-29 | GSE267756 | GEO

Interactome of Nematostella GW182 in HEK293 cells

Project description:Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria. Despite this remarkable mechanistic conservation Argonaute and GW182 differentially co-evolved in bilaterians and cnidarians seemingly leading to distinct interaction surfaces.

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Expression data from Tnrc6a (GW182) mutant E18.5 lungs

Project description:GW182 (Tnrc6a) is a key component of RISC (miRNA-Induced Silencing Complex) that plays a critical role in miRNA-mediated gene silencing. Here, we show that GW182 is expressed in airway multiciliated cells (MCCs), and that gene-trap disruption of GW182 leads to defective multicilia formation and downregulation of broad miRNA targets To investigate roles of GW182 in airway multiciliated cells (MCCc), we assessed changes in mRNA expression in E18.5 Tnrc6a mutant lungs using microarrays (Affymetrix).

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Expression data from Tnrc6a (GW182) mutant yolk sac

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Expression data from Tnrc6a (GW182) mutant yolk sac

Project description:GW182 (Tnrc6a) is a key component of RISC (miRNA-Induced Silencing Complex) that plays a critical role in miRNA-mediated gene silencing. Here, we show that GW182 is expressed in the yolk sac endoderm, and that gene-trap disruption of GW182 leads to growth arrest of yolk sac endoderm, impaired hematopoiesis and embryonic lethality. To investigate roles of GW182 in the yolk sac endoderm, we assessed changes in mRNA expression in the yolk sac of E9.5 GW182gt/gt embryos using microarrays (Affymetrix). Yolk sac of wild type littermates and GW182gt/gt embryos at E9.5 was collected for total RNA isolation using Trizol (Invitrogen). RNAs were purified according to the manufacturer’s protocol before subjected to Mouse Gene 1.0 ST Whole Genome Array (Affymetrix) for mRNA expression profiling. Experiments were performed in triplicate. Differentially expressed mRNAs were identified using a two-sample t-test (P<0.05 considered significant).

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RNAi knock down of GW182 in Drososphila cells compared to cells with knock down of AGO1, a key effector of miRNA-mediated gene silencing

Project description:RNA silencing pathways are conserved gene regulation mechanisms that lead to degradation, translational repression or transcriptional silencing of target-transcripts selected based on complementarity with small RNA molecules. Using firefly luciferase reporters, we previously were able show that the RNA binding protein GW182 plays a role in microRNA-mediated gene silencing (Rehwinkel, J. et al., RNA J. 2005). In this study, we provide further evidence for the role GW182 in the miRNA pathway by determining targets regulated by this protein at the genomic level. We examined expression profiles in Drosophila cells depleted of GW182 and compared these profiles to data obtained from cells depleted of AGO1, a key effector of miRNA-mediated gene silencing.

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miRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms

Project description:miRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms

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miRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms

Project description:miRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms

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Defining an evolutionarily conserved role of GW182 in circular RNA degradation.

Project description:By using dsRNA screening, we found that GW182 is critical for the degradation of circRNA in the Drosophila cells. To further confirm our finding genome widely, S2 cells were treated with either β-gal dsRNA (as a control) or GW182 dsRNA for 3 days and total RNA was extracted and sequenced after ribosome depletion. By comparing the expression level of both circRNAs and linear mRNAs between controls and GW182 depleted samples, we confirmed that GW182 depletion led to accumulation circRNAs but not their parental linear mRNAs, indicating that GW182 may play an import role in the degradation of circRNAs.

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