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RNA interference may result in unexpected phenotypes in Caenorhabditis elegans.

ABSTRACT: RNA interference (RNAi) is a valuable technique to determine gene function. In Caenorhabditis elegans, RNAi can be achieved by feeding worms bacteria carrying a plasmid expressing double-stranded RNA (dsRNA) targeting a gene of interest. The most commonly used plasmid vector for this purpose is L4440. However, it has been noticed that sequences within L4440 may elicit unspecific effects. Here, we provide a comprehensive characterization of these effects and their mechanisms and describe new unexpected phenotypes uncovered by the administration of unspecific exogenous dsRNA. An example involves dsRNA produced by the multiple cloning site (MCS) of L4440, which shares complementary sequences with some widely used reporter vectors and induces partial transgene silencing via the canonical and antiviral RNAi pathway. Going beyond transgene silencing, we found that the reduced embryonic viability of mir-35-41(gk262) mutants is partially reversed by exogenous dsRNA via a mechanism that involves canonical RNAi. These results indicate cross-regulation between different small RNA pathways in C. elegans to regulate embryonic viability. Recognition of the possible unspecific effects elicited by RNAi vectors is important for rigorous interpretation of results from RNAi-based experiments.

SUBMITTER: De-Souza EA

PROVIDER: S-EPMC6486631 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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RNA interference may result in unexpected phenotypes in Caenorhabditis elegans.

De-Souza Evandro A EA Camara Henrique H Salgueiro Willian G WG Moro Raíssa P RP Knittel Thiago L TL Tonon Guilherme G Pinto Silas S Pinca Ana Paula F APF Antebi Adam A Pasquinelli Amy E AE Massirer Katlin B KB Mori Marcelo A MA

Nucleic acids research 20190501 8

RNA interference (RNAi) is a valuable technique to determine gene function. In Caenorhabditis elegans, RNAi can be achieved by feeding worms bacteria carrying a plasmid expressing double-stranded RNA (dsRNA) targeting a gene of interest. The most commonly used plasmid vector for this purpose is L4440. However, it has been noticed that sequences within L4440 may elicit unspecific effects. Here, we provide a comprehensive characterization of these effects and their mechanisms and describe new unex ...[more]

PMID: 30838421

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Project description:UNLABELLED:Antiviral RNA-mediated silencing (RNA interference [RNAi]) acts as a powerful innate immunity defense in plants, invertebrates, and mammals. In Caenorhabditis elegans, RNAi is systemic; i.e., RNAi silencing signals can move between cells and tissues. Furthermore, RNAi effects can be inherited transgenerationally and may last for many generations. Neither the biological relevance of systemic RNAi nor transgenerational RNAi is currently understood. Here we examined the role of both pathways in the protection of C. elegans from viral infection. We studied the Orsay virus, a positive-strand RNA virus related to Nodaviridae and the first and only virus known to infect C. elegans. Immunity to Orsay virus infection requires the RNAi pathway. Surprisingly, we found that genes required for systemic or transgenerational RNAi did not have a role in antiviral defense. Furthermore, we found that Orsay virus infection did not elicit a systemic RNAi response even when a target for RNAi was provided by using transgenes. Finally, we show that viral siRNAs, the effectors of RNAi, are not inherited to a level that provides any significant resistance to viral infection in the next generation. We conclude that systemic or transgenerational RNAi does not play a role in the defense against natural Orsay virus infection. Furthermore, our data suggest that there is a qualitative difference between experimental RNAi and antiviral RNAi. Our data are consistent with a model of systemic and transgenerational RNAi that requires a nuclear or germ line component that is lacking in almost all RNA virus infections. IMPORTANCE:Since its discovery in Caenorhabditis elegans, RNAi has proven a valuable scientific tool in many organisms. In C. elegans, exogenous RNAi spreads throughout the organism and can be passed between generations; however, there has been controversy as to the endogenous role(s) that the RNAi pathway plays. One endogenous role for which spreading both within the infected organism and between generations would be advantageous is a role in viral defense. In plants, antiviral RNAi is systemic and the spread of RNAi between cells provides protection against subsequent viral infection. Here we investigated this by using the only naturally occurring virus known to infect C. elegans, Orsay virus, and surprisingly found that, in contrast to the exogenous RNAi pathway, the antiviral RNAi response targeted against this virus does not spread systemically throughout the organism and cannot be passed between generations. These results suggest that there are differences between the two pathways that remain to be discovered.

Dataset Information

RNA interference may result in unexpected phenotypes in Caenorhabditis elegans.

Publications

RNA interference may result in unexpected phenotypes in Caenorhabditis elegans.

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