Natural RNA interference directs a heritable response to the environment

Abstract

RNA interference can induce heritable gene silencing, but it remains unexplored whether similar mechanisms play a general role in responses to cues that occur in the wild. We show that transient, mild heat stress in the nematode Caenorhabditis elegans results in changes in messenger RNA levels that last for more than one generation. The affected transcripts are enriched for genes targeted by germline siRNAs downstream of the piRNA pathway, and worms defective for germline RNAi are defective for these heritable effects. Our results demonstrate that a specific siRNA pathway transmits information about variable environmental conditions between generations.

Figure 1. Inheritance of a response to environmental conditions.

Single-channel microarray analysis of 4-cell stage embryo mRNAs shows inheritance of temperature responses. Scatterplot of 15,208 genes: x-axis, generation 2 (G2); y-axis, generation 3 (G3). Values are ratios of geometric mean signal (25°C treatment/constant 20°C, six 50-embryo replicates each condition). Numbers in each quadrant count genes whose mRNA levels differ between treatment and control in both generations (>2×, and ANOVA/Student's T P < 0.01, independently in each generation, larger dots; note this figure uses the statistics as a filter, not as a test). Pale dots are the lowest-signal 1/6 of genes and any genes with >4× the average inter-replicate variance. At right margin, G3 replicate RT-QPCR of additional experiments.

Figure 2. RT-QPCR of multigenerational experiments.

Values are mRNA abundances relative to constant 20° control (dotted line) for multiple generations after one (black) or two (magenta) generations growth at 25°, geometric mean ± SEM. Two-tailed, paired T-test for difference with 20° control (shown for last two generations only), *P < 0.01 **P < 0.0001. To eliminate males and for more accurate staging, we transferred L4 hermaphrodite larvae from plate to plate (by platinum pick) instead of embryos. See Supplementary Table S5 for statistical details.

Figure 3. Temperature-responsive early embryonic mRNAs are highly enriched for targets of endogenous 22G RNAs.

Circles labeled “G2” and “G3” represent genes whose mRNA levels in each generation differ >2× between treatment and control and ANOVA/Student's T P < 0.01 (see Figure 1). All P values are from cumulative hypergeometric distributions. Unless noted otherwise, P values represent the probabilities of the observed or greater overlap between the genes highlighted in Figure 1 and the sRNA set, relative to all genes on the array that are also represented among the annotations used by the published studies (15,197 and 15,12036). The circle representing the set of all genes is omitted despite being required for a complete Venn diagram. The P values shown in parentheses below are for enrichment relative to the “G2” set alone, restricting analysis to only the genes identified as differing in G2. See Supplementary Notes for calculations. (a) Overlap with oocyte antisense sRNAs occurring at >50 (left), >100 (center) and >200 (right) reads per million. P = 6 × 10⁻⁸ (7 × 10⁻⁶), 7 × 10⁻¹⁰ (4 × 10⁻⁶) and 7 × 10⁻⁹ (4 × 10⁻⁵), respectively. (b) Overlap with sRNAs reported to be depleted >2× in all three of a mut-2 strain, a mut-7 strain, and a strain with twelve argonaute genes disrupted. P = 8 × 10⁻⁹ (7 × 10⁻⁵). (c) Overlap with sRNAs occurring at >10 reads per million in wild-type and depleted >20× in a mut-16 mutant. P = 1 × 10⁻¹⁰ (1 × 10⁻⁵).

Figure 4. WAGO class 22G RNAs but not CSR-1 class 22G RNAs are associated with transcripts showing heritable temperature effects.

(a) Among transcripts targeted by 22G RNAs isolated from oocytes, most are targeted by either WAGO-9 binding or CSR-1 binding 22G RNAs, as previously reported. (b) CSR-1 binding 22G RNAs tend to target transcripts that are highly expressed in 4-cell stage embryos. Histograms are distributions of fluorescence values on 20°C control microarray hybridizations. Dotted lines are median values for each group of genes indicated. (c) Failure of the temperature-responsive mRNAs highlighted in Figure 1 to overlap with 22G RNAs that co-immunoprecipitate with the germline argonaute CSR-1 (26% of the genes represented on our microarray). P(no overlap) = 1 × 10⁻³ (P = 2 × 10⁻⁴ if analysis is restricted to only transcripts targeted by oocyte 22G RNAs). (d) By contrast, overlap with 22G RNAs that co-immunoprecipitate with the germline argonaute WAGO-9. P(equal or greater overlap) = 2 × 10⁻⁵ (P = 0.03 if analysis is restricted to only transcripts targeted by oocyte 22G RNAs).

Figure 5. Targets of PRG-1 dependent 22G RNAs are more likely than other transcripts to show effects in the following generation.

(a) Among transcripts differing in G2, likely PRG-1 targets are more likely to show a difference in G3, P = 1 × 10⁻⁸. Circle labeled “G2” represents genes whose mRNA levels differ >3× between treatment and control (one-tailed Student's T P < 0.001; this results in an estimated false positive rate for G2 of approximately 1 gene out of 15,208 based on swap tests). Circle labeled “targets of prg-1 dependent 22G RNAs” represents genes for which antisense 22G RNAs are consistently depleted in five comparisons of N2 (wild type) with prg-1 mutants (see Methods). (b) Same analysis as in (a), looking only at targets of oocyte 22G RNAs. Among transcripts differing in G2, likely PRG-1 targets are more likely to show a difference in G3, P = 4 × 10⁻³. (c) Among targets of oocyte 22G RNAs, predicted 21U RNA targets tend to be WAGO targets and not CSR-1 targets, as previously reported.

Figure 6. B0286.1 and K10B3.5 transcripts are targets of temperature-responsive endogenous RNAi.

(a) Response of 22 nt RNAs (geometric mean ± SEM) to temperature. (b) Effect of inactivating germline RNAi on B0286.1 and K10B3.5 transcripts. WT, values for N2 (wild type) duplicated from Figure 2; mut-2, values for WM30 mut-2(ne298); GFP and mut-16, feeding RNAi of GFP::unc-22 and mut-16, respectively, from G1 to end of experiment. * & , two-way ANOVA effect & interaction, respectively P < 10⁻³. See Supplementary Table S6 for statistical details.

Figure 7. Female line transmission of the heritable effect on B0286.1 transcript levels.

We either exposed worm cultures to 25°C or kept them at 20°C, and crossed male HC67 progeny with hermaphrodite N2 progeny, using the GFP transgene from HC67 to identify cross-progeny. All four possible crosses are shown. Values are B0286.1 mRNA abundances in the second generation after the crosses, geometric mean ± SEM, relative to the self-fertilized N2 20°C control from Figure 2 (dotted line). In two-way ANOVA (n = 7 cultures per cross), **P = 3 × 10⁻⁷ for the effect of the female line; P = 0.2 for the effect of the male line; and P = 0.8 for the interaction effect.

Figure 8. A single-mechanism numerical model recapitulates divergent response to temperature changes.

Graphic results from a simple continuous function model (Model 2 in Supplementary Table S3). The assumptions for this numerical model are that a new temperature-dependent silencing signal is generated at the silencing temperature and that this signal is maintained at the non-silencing temperature by limited, incomplete amplification resulting in dilution of the signal with each new generation.