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RNA sequencing of exogenous RNAi mutants under CPL-1* expression

ABSTRACT: The goal of this experiment was to identify RNAs that are regulated by the exogenous RNAi pathway under ER stress conditions.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE202291 | GEO | 2022/09/23

REPOSITORIES: GEO

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RNA sequencing of exogenous RNAi mutants under CPL-1* expression

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Project description:Double stranded RNAs are used to induce gene silencing in functional studies. In Apis mellifera green fluorescent protein (GFP) dsRNA (dsGFP) has been used as an exogenous control as its sequence has no homology in honeybee genome. However, some undesirable effects are observed after dsGFP treatment. A microarray approach comparing gene expression differences between untreated and dsGFP treated groups, containing honeybees workers in two different developmental stages, pre-pupae and light-brown eyed pupae, were used to test the reliability of dsGFP as a control for RNAi experiments. According to these microarrays results dsGFP can be used as a control in RNAi assays as long as the affected genes are taken into account in the analysis.

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The Caenorhabditis elegans RDE-10/RDE-11 complex regulates RNAi by promoting secondary siRNA amplification

Project description:From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10 and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, as well as other candidate RNAi factors. The newly identified RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage, but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10, rde-11, and the dosage-sensitive RNAi defective genes rsd-2, rsd-6 and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways.

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Project description:A green fluorescence protein (GFP)-derived dsRNA (dsRNA-GFP) has been used as an exogenous control for Apis mellifera RNAi assays by multiple research groups. Its sequence does not share any significant homology with any known honey bee genes. Although dsRNA-GFP is not expected to trigger an RNAi response in treated bees, undesirable effects on gene expression, pupal pigmentation or developmental timing have been routinely observed. To better understand the multiple molecular and phenotypic effects of dsRNA-GFP in honey bees and to evaluate its use as a control for RNAi studies, we examined the impact of dsRNA-GFP on global gene expression patterns in developing workers. We found that dsRNA-GFP causes large-scale changes in gene expression associated with multiple biological processes. Furthermore, dsRNA-GFP exposure tended to preferentially decrease, rather than increase, expression of genes compared to controls.

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