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. 2017 Jul;206(3):1403-1416.
doi: 10.1534/genetics.116.198812. Epub 2017 May 22.

The RNAi Inheritance Machinery of Caenorhabditis elegans

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Free PMC article

The RNAi Inheritance Machinery of Caenorhabditis elegans

George Spracklin et al. Genetics. .
Free PMC article

Abstract

Gene silencing mediated by dsRNA (RNAi) can persist for multiple generations in Caenorhabditis elegans (termed RNAi inheritance). Here we describe the results of a forward genetic screen in C. elegans that has identified six factors required for RNAi inheritance: GLH-1/VASA, PUP-1/CDE-1, MORC-1, SET-32, and two novel nematode-specific factors that we term here (heritable RNAi defective) HRDE-2 and HRDE-4 The new RNAi inheritance factors exhibit mortal germline (Mrt) phenotypes, which we show is likely caused by epigenetic deregulation in germ cells. We also show that HRDE-2 contributes to RNAi inheritance by facilitating the binding of small RNAs to the inheritance Argonaute (Ago) HRDE-1 Together, our results identify additional components of the RNAi inheritance machinery whose conservation provides insights into the molecular mechanism of RNAi inheritance, further our understanding of how the RNAi inheritance machinery promotes germline immortality, and show that HRDE-2 couples the inheritance Ago HRDE-1 with the small RNAs it needs to direct RNAi inheritance and germline immortality.

Keywords: Caenorhabditis elegans; epigenetic inheritance; small RNAs.

Figures

Figure 1
Figure 1
Genetic screen identifies hrde genes. Animals of the indicated genotype expressing a pie-1p::gfp::h2b transgene were exposed to bacteria producing gfp dsRNA. F1 embryos were isolated by hypochlorite treatment and grown on bacteria not expressing gfp dsRNA. GFP expression in oocytes of animals exposed to gfp dsRNA (RNAi generation) and the progeny of these animals (inheriting generation) were visualized by fluorescence microscopy. Percentage of animals expressing GFP is indicated (n > 50). Note: morc-1 is marked with dpy-17(e164).
Figure 2
Figure 2
hrde genes are required for germline immortality. Brood sizes for animals of indicated genotype were counted as detailed in Materials and Methods. Data are mean ± SD (n = 3).
Figure 3
Figure 3
Genetic damage is not likely to be the cause of Mrt in RNAi inheritance defective animals. (A) Brood sizes for animals of indicated genotype were counted as detailed in Materials and Methods. Shift in temperature is indicated by changing color scheme (n = 6, ± SD). (B) hrde-1;hrde-2 animals from the fifth generation at 25° were singled and grown at 20° and their brood sizes were scored (B, top). Animals whose parents had a low brood size (indicated by red bars) were singled, grown at 20°, and their brood sizes were scored (B, bottom).
Figure 4
Figure 4
SET-32 is required for nuclear RNAi-directed H3K9me3. (A) H3K9me3 ChIP was conducted on animals of indicated genotype as described in Materials and Methods. qPCR of H3K9me3 coprecipitating DNA using primers detecting five genes thought to be targeted by nuclear RNAi and three genes not thought to be targeted by nuclear RNAi. Data are relative to coprecipitating eft-3 DNA and expressed as a ratio of signal from indicated mutant to signal from wild-type (n = 6, ± SD). (B) Animals exposed to oma-1 dsRNA (+ oma-1 dsRNA) or control bacteria with L4440 vector (− oma-1 dsRNA) were subjected to H3K9me3 ChIP. Coprecipitating oma-1 DNA was quantified by qPCR. Data were normalized to coprecipitating eft-3 DNA and expressed as a ratio of signals in animals subjected to oma-1 RNAi over signals from no RNAi (−) animals (“1” denotes no change) (n = 3, ± SD). On the x-axis, 0 denotes the predicted start codon of oma-1.
Figure 5
Figure 5
hrde-2 encodes a germline factor required for RNAi inheritance. (A) Predicted hrde-2 gene structure. Arrows indicate mutant alleles identified in genetic screen and black bar indicates deletion allele created by CRISPR/CAS9. (B) HRDE-2 localizes to cytoplasmic foci in germ cells. Fluorescence microscopy of the hermaphrodite germline in adult animals expressing gfp::hrde-2. Arrow shows perinuclear puncta.
Figure 6
Figure 6
HRDE-2 is part of the nuclear RNAi pathway. (A) Brood sizes for animals of indicated genotype were counted as detailed in Materials and Methods. hrde-1;hrde-2 double mutant animals do not show an additive fertility defect at 25° when compared to the single mutant animals, suggesting HRDE-1 and HRDE-2 act in the same pathway (n = 6, ± SD). (B) Fluorescence images of oocytes from wild-type animals expressing a piRNA sensor transgene (mjIs144), which contains a binding site for the piRNA 21U-1, and a control piRNA sensor transgene (mjIs145) which lacks the 21U-1 site [as described in Bagijn et al. (2012)]. Right panel, hrde-2(gg517) animals harboring mjIs145 (top) and mjIs144 (bottom). (C) Animals of the indicated genotypes were exposed to oma-1 dsRNA (+ oma-1 dsRNA) or empty L4440 vector (− oma-1 dsRNA) and subjected to H3K9me3 ChIP. Coprecipitating oma-1 DNA was quantified by qPCR. Data were normalized to coprecipitating eft-3 DNA and expressed as a ratio ±oma-1 RNAi (“1” denotes no change) (n = 3, ± SD). On the x-axis, 0 denotes the predicted start codon of oma-1. (D and E) FLAG-NRDE-2 was precipitated from animals of indicated genotypes with an anti-Flag antibody and NRDE-2 coprecipitating RNA was isolated by TRIzol extraction. RNA was converted to cDNA and quantified by qRT-PCR with primers detecting oma-1 pre-mRNA or bath-45 pre-mRNA as described in Materials and Methods. Comparisons were performed using t-tests (Prism) (E). Data are expressed as fold change ±oma-1 RNAi, or (E) as fold change mutant/control. Mean ± SD are shown (n = 3).
Figure 7
Figure 7
HRDE-2 is required for HRDE-1 to associate with siRNAs. (A) FLAG::HRDE-1- or HA::ppw-3-expressing animals were treated with oma-1 dsRNA (+oma-1 dsRNA) or with empty L4440 vector (-oma-1 dsRNA). HRDE-1 and ppw-3 were immunoprecipitated with anti-Flag and anti-HA antibodies respectively, and coprecipitating RNA was isolated by TRIzol extraction. oma-1 coprecipitating siRNAs were quantified with an oma-1 TaqMan probe set that detected a 22G RNA antisense to the oma-1 mRNA. TaqMan signals shown relative to Taqman signals from non-RNAi-treated control animals, which was defined as one. Relative enrichment following RNAi treatment is shown (n = 3, ± SD). Comparisons were performed using t-tests (Prism). (B) Wild-type and hrde-2(gg517) animals were treated with oma-1 dsRNA. Small RNAs were sequenced as described in Materials and Methods. Small RNAs that mapped to the oma-1 locus are shown. Sense siRNA (red) and antisense siRNA (blue). Data were normalized by the total number of sequenced reads for each genotype, reads per million (rpm). oma-1 siRNAs sequenced from control (non-RNAi) samples are shown in Figure S16 in File S1. A cartoon representing the oma-1 genomic regions is shown below the graphs. The region of the oma-1 locus targeted by dsRNA is demarcated with a red line and a black box.

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