RNA Interference in<i>Caenorhabditis elegans</i>

Conte, Darryl; MacNeil, Lesley T.; Walhout, Albertha J.M.; Mello, Craig C.

doi:10.1002/0471142727.mb2603s109

Cited by 125 publications

(106 citation statements)

References 68 publications

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“…Alleles used in this study listed by chromosome: LGI: prg-1(tm872) , glp-4(bn2) ; LGII: parn-2(tm1339) , neSi[ cb-unc-119( + ); wago1p::parn-1::gfp ], neSi[ cb-unc-119( + ); flag::prg-1 ]; LGIII: henn-1(tm4477) ; LGV: parn-1(tm869) ; unmapped: rfp::pgl-1 . Depletion of candidate nucleases was conducted by feeding worms bacteria expressing dsRNA as described (Conte et al, 2015). …”

Section: Methodsmentioning

confidence: 99%

The RNase PARN-1 Trims piRNA 3′ Ends to Promote Transcriptome Surveillance in C. elegans

Tang

Lee

et al. 2016

Cell

133

132

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SUMMARY piRNAs engage Piwi proteins to suppress transposons and are essential for fertility in diverse organisms. An interesting feature of piRNAs is that, while piRNA lengths are stereotypical within a species, they can differ widely between species. For example piRNAs are mainly 29 and 30 nucleotides in humans, 24 to 30 nucleotides in D. melanogaster, and uniformly 21 nucleotides in C. elegans. However, how piRNA length is determined and whether length impacts function remains unknown. Here we show that C. elegans deficient for PARN-1, a conserved ribonuclease, accumulate untrimmed piRNAs with 3′ extensions. Surprisingly, these longer piRNAs are stable and associate with the Piwi protein, PRG-1, but fail to robustly recruit downstream silencing factors. Our findings identify PARN-1 as a key regulator of piRNA length in C. elegans and suggest that length is regulated to promote efficient transcriptome surveillance.

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Section: Methodsmentioning

confidence: 99%

The RNase PARN-1 Trims piRNA 3′ Ends to Promote Transcriptome Surveillance in C. elegans

Tang

Lee

et al. 2016

Cell

133

132

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“…To validate the fluorescent signal of the GFP::DPFF‐1 transgene, we silenced the dpff‐1 gene in xmSi09 animals using RNAi by feeding. As a negative control for the RNAi studies, we used an empty pPD129.36 plasmid (EP) as previously recommended by Conte and Mello (). xmSi09 animals were grown under control (EP) or dpff‐1(RNAi) conditions at 24°C.…”

Section: Resultsmentioning

confidence: 99%

DPFF‐1 transcription factor deficiency causes the aberrant activation of MPK‐1 and meiotic defects in the Caenorhabditis elegans germline

Villanueva-Chimal

Salinas

Fernández-Cárdenas

et al. 2017

Genesis

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The d4 family of transcription factors consists of three members in mammals. DPF1/neuro-d4 is expressed mainly in neurons and the peripheral nervous system, and is important for brain development. DPF2/requiem/ubi-d4 is expressed ubiquitously and presumably functions as an apoptotic factor, especially during the deprivation of trophic factors. DPF3/cer-d4 is expressed in neurons and in the heart, and is important for heart development and function in zebrafish. In Drosophila, there is only one member, dd4, whose function is still unknown, but it is expressed in many tissues and is particularly abundant in the brain of developing embryos and in adults. Here, we present DPFF-1, the only member of this family of proteins in the nematode C. elegans. DPFF-1 is similar to its mammalian homolog DPF2/requiem/ubi-d4 because it is ubiquitously expressed during embryogenesis and in adult tissues, and because it is important for the induction of germ cell apoptosis during stress. Here, we show that dpff-1 null mutant animals produce less progeny than wild-type nematodes, presumably due to meiotic defects. Gonads of dpff-1 deficient animals showed more germ cells in pachytene and overexpressed the P-MPK-1 signal. Additionally, these animals presented higher levels of p53-induced germ cell apoptosis than wild-type animals. Furthermore, we observed that dpff-1 deficient animals are more sensitive to heat shock. This is the first report showing that the d4 family of transcription factors could be involved in meiosis and stress protection.

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“…C. elegans is particularly amenable to RNAi because, in this system, silencing is heritable 122 . dsRNA that is introduced into hermaphrodites depletes both maternal and zygotic RNAs in all F1 progeny, generating large populations of animals that can be examined for phenotypes 120,123 .…”

Section: Lof Approaches Across Organismsmentioning

confidence: 99%

“…dsRNA that is introduced into hermaphrodites depletes both maternal and zygotic RNAs in all F1 progeny, generating large populations of animals that can be examined for phenotypes 120,123 . The delivery of RNAi reagents to worms is also straightforward: dsRNA can be delivered by injection, soaking or feeding of Escherichia coli strains that are engineered to express dsRNA against specific genes 122,124 . The effects spread from tissue to tissue (known as ‘systemic RNAi’) 120,125 .…”

Section: Lof Approaches Across Organismsmentioning

confidence: 99%

“…The effects spread from tissue to tissue (known as ‘systemic RNAi’) 120,125 . However, it should be noted that some tissues are refractory to RNAi 126 , and the degree of inactivation is sensitive to the experimental conditions 122 . Despite these caveats, the experimental ease of RNAi in C. elegans quickly made it the method of choice for large-scale screens 127 .…”

Section: Lof Approaches Across Organismsmentioning

confidence: 99%

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Loss-of-function genetic tools for animal models: cross-species and cross-platform differences

et al. 2016

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Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.

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RNA Interference inCaenorhabditis elegans

Abstract: RNAi has become an essential tool in C. elegans research. This unit describes procedures for RNAi in C. elegans by microinjecting with dsRNA, feeding with bacteria expressing dsRNA and soaking in dsRNA solution, as well as high-throughput methods for RNAi-based screens.

Cited by 125 publications

References 68 publications

The RNase PARN-1 Trims piRNA 3′ Ends to Promote Transcriptome Surveillance in C. elegans

The RNase PARN-1 Trims piRNA 3′ Ends to Promote Transcriptome Surveillance in C. elegans

DPFF‐1 transcription factor deficiency causes the aberrant activation of MPK‐1 and meiotic defects in the Caenorhabditis elegans germline

Loss-of-function genetic tools for animal models: cross-species and cross-platform differences

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