The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression

Abstract

Organisms can develop adaptive sequence-specific immunity by reexpressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piwi-interacting RNA (piRNA) pathway recruits RNA-dependent RNA polymerase (RdRP) to foreign sequences to amplify a transgenerational small-RNA-induced epigenetic silencing signal (termed RNAe). Here, we provide evidence that, in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self-mRNAs. We refer to this mechanism, which can prevent or reverse RNAe, as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a transgenerational CSR-1 memory that recognizes and protects self-mRNAs, allowing piRNAs to recognize foreign sequences innately, without the need for prior exposure

Figure 1. CSR-1 is required for RNAa

(A and D) Schematic diagrams of crosses between silenced (RNAe) and licensed (RNAa) GFP transgenic strains as indicated. B, C, E–I, Epifluorescence images of representative germlines (outlined with dashes) in first (F1) and subsequent (F2, F3, F5) generations. The cytoplasmic fluorescence signal is OMA-1::GFP; the nuclear signal is GFP::CDK-1. The percentages indicate the number of animals exhibiting the shown phenotype in this and the subsequent figures. See also Figure S1.

Figure 2. CSR-1-associated small RNAs targeting GFP in neSi22 oma-1::gfp(RNAa)

(A) Schematic of oma-1::gfp transgene. The exon-intron structure is indicated with boxes and lines, respectively. (B–F) Plots showing the density of antisense small RNAs mapping along oma-1::gfp in wild-type (B) and mutant strains rde-3 (C) and csr-1(D). In (E and F) the histograms show read densities of small RNAs obtained from the same lysate before (Input) and after FLAG::CSR-1 Immunoprecipitation (IP). The height of each peak corresponds to the number of RNA reads that begin at that position per million total reads. See also Figure S2.

Figure 3. RNAa counteracts Piwi-dependent silencing and acts over multiple generations to establish an active epigenetic gene-expression state

A–H, Genetic crosses with corresponding epifluorescence images showing representative germlines of resulting progeny. The percentages of animals expressing gfp::cdk-1 (nuclear GFP signal) at each generation and the number of animals scored ‘n’ are indicated. A–D, Analysis of RNAa exposure on the durability of gene activation in wild-type animals. Newly trans-activated F2 double transgenic animals (A), were outcrossed to wild-type (WT), either immediately or after propagating as a double transgenic strain for 30 generations (F30), to obtain gfp::cdk-1 “single transgenic” animals shown in B and C, respectively. Siblings of animals shown in (C) were allowed to produce self progeny (D) for multiple generations, and GFP fluorescence was scored in each generation as indicated. E–H, Analysis of the genetic influence of Piwi (prg-1) on transactivation. RNAa and RNAe transgenes established in a wild-type background were crossed into prg-1 prior to conducting the trans-activation assay shown in (E). After one generation, oma-1::gfp was segregated away to yield gfp::cdk-1 single transgenic animals assayed in (F). Siblings of animals shown in (F) were allowed to produce self progeny for multiple generations and GFP fluorescence was scored in each generation (G) as indicated. In (H) gfp::cdk-1was outcrossed from the prg-1(tm872) mutant background and the animals were scored for GFP in subsequent generations as indicated.

Figure 4. Model for transactivation by CSR-1

See Discussion for details.