Germ Granules Govern Small RNA Inheritance

Abstract

In C. elegans nematodes, components of liquid-like germ granules were shown to be required for transgenerational small RNA inheritance. Surprisingly, we show here that mutants with defective germ granules can nevertheless inherit potent small RNA-based silencing responses, but some of the mutants lose this ability after many generations of homozygosity. Animals mutated in pptr-1, which is required for stabilization of P granules in the early embryo, display extraordinarily strong heritable RNAi responses, lasting for tens of generations. Intriguingly, the RNAi capacity of descendants derived from mutants defective in the core germ granule proteins MEG-3 and MEG-4 is determined by the genotype of the ancestors and changes transgenerationally. Further, whether the meg-3/4 mutant alleles were present in the paternal or maternal lineages leads to different transgenerational consequences. Small RNA inheritance, rather than maternal contribution of the germ granules themselves, mediates the transgenerational defects in RNAi of meg-3/4 mutants and their progeny. Accordingly, germ granule defects lead to heritable genome-wide mis-expression of endogenous small RNAs. Upon disruption of germ granules, hrde-1 mutants can inherit RNAi, although HRDE-1 was previously thought to be absolutely required for RNAi inheritance. We propose that germ granules sort and shape the RNA pool, and that small RNA inheritance maintains this activity for multiple generations.

Keywords: P granules; RNAi; endogenous siRNAs; germ granules; hrde-1; meg-3/4; non-Mendelian inheritance; pptr-1; small RNAs; transgenerational inheritance.

Graphical abstract

Figure 1

Germ Granule Mutants Exhibit Enhanced RNAi Inheritance and Display Severe Mis-expression of Endogenous Small RNAs in the Germline (A and B) Worms of the indicated genotypes containing a transgene expressing GFP in the germline (Pmex-5::gfp) were exposed to dsRNA complementary to gfp, to initiate gene silencing via RNAi. The percentage of worms expressing GFP (y axis) was assessed over generations (x axis). Shown are mean ± SD from three independent experiments. Mutant meg-3/4 worms (B) expressing Pmex-5::gfp were generated by crossing Pmex-5::gfp hermaphrodites with meg-3/4 males, and exposed to anti-gfp dsRNA two generations after homozygosity. Mutant pptr-1 worms (A) expressing Pmex-5::gfp were generated by crossing Pmex-5::gfp males with pptr-1 hermaphrodites, and exposed to anti-gfp dsRNA multiple generations after homozygosity. p values were determined via two-way ANOVA. ^∗∗∗∗p < 10⁻⁴. (C) Representative pictures of the germline in the descendants of mutated worms treated with gfp RNAi (up) or empty vector (down). The genotype and generation after RNAi initiation are indicated above the pictures. The number of gfp-expressing worms out of all examined worms is indicted in white. Scale bar, 80 μm. (D) Representative pictures of embryos expressing pgl-1::rfp (red) and wago-4::gfp (green), and their corresponding binarized representations further used for analysis (bottom). The genotypes of the embryos are indicated above the pictures. Scale bar, 5 μm. (E) Expression of small RNAs in the germline of pptr-1 and meg-3/4 mutants (y axis) compared to wild-type animals (x axis). Shown are the average expression values (log2 of RPM) of small RNAs targeting all genomic elements (each dot represents one annotation). Red dots, genomic elements with differential expression values of targeting small RNAs (analyzed via DESeq2, adjusted p < 0.1). (F) X-fold enrichment and depletion values (log2, bar graphs and color-coded) for protein coding genes that displayed upregulated (left) or downregulated (right) expression levels of small RNA targeting them. Shown are results for genes with differential levels in both pptr-1 and meg-3/4 mutants compared to wild-type (see also Figure S3). We tested the enrichment for the list of genes differentially targeted by small RNAs against lists of genes known to be targeted by endogenous small RNAs of specific pathways (y axis; see STAR Methods for details). p values for enrichment were calculated using 10,000 random gene sets identical in size to the tested group (see STAR Methods for details). Enrichments were considered significant if p < 0.05. ^∗∗∗∗p < 10⁻⁴, ^∗∗p < 0.01, ^∗p < 0.05; ns, p > 0.05. (G) Global analysis of the various small RNA species in dissected germlines of pptr-1 and meg-3/4 mutants. Shown are the mean normalized total number of reads (RPM) aligned to the denoted small RNA species. See also Figures S1–S3, Data S1, and Videos S1, S2, and S3.

Figure 2

The Ancestral Germ Granule Function Determines the Ability of the Progeny to Inherit RNAi (A) Schematic diagram depicting the crosses performed to determine the ancestral contribution to RNAi inheritance in the descendants. Long-term meg-3/4 mutants (hermaphrodites and males) were outcrossed using wild-type worms of the opposite sex. All worms contained a transgene expressing GFP in the germline (Pmex-5::gfp). (B) Homozygote descendants of the crosses depicted in (A) were exposed to dsRNA complementary to gfp, to initiate gene silencing via RNAi. To test the transgenerational dynamics of the ability to respond to RNAi, naive descendants were exposed to dsRNA at different time points, 2, 8, 16, and 32 generations following homozygosity. The proportion of GFP-expressing worms (y axis, out of 40∼100 worms for each group and time point) was determined over ten generations (x axis) following exposure to RNAi. The colors depict the genotypes and lineages according to the scheme in (A). Shown are mean ± SD from two independent experiments (two independent ancestral crosses). In each row (initiation at different generations following homozygosity), all groups were tested side by side, and therefore the same values for the wild-type control groups are displayed on both left and right panels. Wild-type and meg-3/4(−/−) in the maternal wild-type lineage for the G2 generation (top left panel) are replicates from Figure 1B, for convenience of visualization purposes only. See also Figures S4 and S5.

Figure 3

meg-3/4 Mutants Accumulate Defects in Germline RNAi Initiation and Mis-expression of Endo-siRNAs over Generations (A) The transgenerational dynamics of the ability of meg-3/4 mutants to respond to RNAi targeting endogenous germline genes. Wild-type (gray) and meg-3/4 (red) worm homozygotes for the indicated amount of generations (x axis) were allowed to lay eggs on plates containing dsRNA-producing bacteria targeting the germline-expressed genes pos-1 (left) and mel-26 (right). Shown are the percentages of eggs that did not hatch into larvae (y axis), indicating the penetrance of the RNAi effect. Each dot represents one tested plate (biological replicate) and bars represent the mean. Each group was tested in two independent experiments. (B) Two-way hierarchical clustering of the different examined biological groups and genes targeted by significantly differential levels of small RNAs (compared to wild-type). Small RNA samples were obtained from dissected germlines of meg-3/4 mutants that were homozygote for 2, 15, or >80 generations (x axis). Clustering of small RNAs targeting all protein-coding genes (left panel) and small RNAs targeting genes known to be targeted by small RNAs associated with the HRDE-1 argonaute (right panel [9]) is presented. Each row represents one gene. Only genes targeted by significantly differential levels of small RNAs in at least one sample were included in the analysis (analyzed with DEseq2, adjusted p < 0.1). Each line is color-coded according to the fold change in expression. Lines depicting no significant differential expression appear in gray. (C) An analysis of poly-uridylation of small RNAs in meg-3/4 mutants across generations. The distribution of the average fractions of poly-uridylated small RNAs against individual genes is shown for each of the examined samples. Data for small RNA samples from extracted germlines of wild-type and meg-3/4 mutants that were homozygote for 2, 15, or >80 generations (x axis) are shown. The fraction of poly-uridylated small RNAs was calculated as the number of reads with untemplated 3′Us out of the total aligning reads against a specific gene (only genes with ≥5 RPM in at least one sample were included). ^∗∗∗p < 10⁻³, ^∗∗∗p < 10⁻⁴, Wilcoxon rank-sum test. For the >80 generations group in (B) and (C), data from two replicates were analyzed and a third replicate was left out since it displayed substantially less depth (3 × 10⁵ reads compared to >10⁷ reads in all other samples). See also Figure S6.

Figure 4

The Morphology of the Germ Granules in the Progeny Is Not Affected by the Ancestral Genotype (A) Genotypes and maternal lineages of different tested groups are encoded in colors similarly to Figure 2. (B) Representative pictures of embryos expressing pgl-1::rfp at the 4-cell stage with fluorescent P granules and their corresponding binarized representations further used for analysis (bottom); the P2 cell (germline lineage) is emphasized with a thicker line. Scale bar, 5 μm. (C) Representative pictures of embryos expressing wago-4::gfp at the 4-cell stage with fluorescent Z granules and their corresponding binarized representations further used for analysis (bottom); the P2 cell (germline lineage) is emphasized with a thicker line. Scale bar, 5 μm. (D and E) Characterization of germ granules in embryos at the 4-cell stage. Each dot represents one analyzed embryo. Data shown for P granules (D) and Z granules (E). Top: percentage of granules in the P2 cell out of total granules. Middle: amount of granules per embryo. Bottom: mean size of granules per embryo. Bars represent mean ± SD. p values were determined via two-way ANOVA with Tukey post hoc correction for multiple comparisons. ^∗∗∗∗p < 10⁻⁴, ^∗∗p < 0.01; ns, p > 0.05. See also Figures S1 and S7.

Figure 5

Endogenous Small RNAs Mediate the Transgenerational Defects of Germ Granule Mutants (A) Schematic diagrams depicting the crosses performed to obtain the data appearing in (B). Left: hermaphrodites mutated in the meg-3/4 genes for >80 generations were crossed with hrde-1(−/−) males. Triple mutant F2 progeny of this cross were isolated and maintained for three generations prior to crossing with wild-type males. Wild-type (light yellow) and meg-3/4 (light red) F2 progeny resulting from this cross were isolated and tested for gfp RNAi on the second generation of homozygosity. (B) The percentage of GFP-expressing worms (y axis) over generations (x axis) after exposure to gfp dsRNA in descendants of worms where hrde-1 was erased for three generations. The colors depict the genotype and lineages according to scheme in (A). Both groups as well as uncrossed wild-type controls (gray) were tested side-by-side, and therefore the same wild-type data appear in both panels. Shown are mean ± SD from two independent experiments. Data for the groups with no hrde-1 history (dark yellow and dark red) are replicates from Figure 2B, for convenience of visualization purposes only. Reset versus non-reset groups in both panels display statistically significant differences in silencing (p < 10⁻⁴) based on two-way ANOVA with Bonferroni post hoc correction for multiple analysis. (C) Crosses were performed similarly to the scheme in Figure 2A. (D) Distributions of normalized small RNA counts (y axis) as function of genomic location (x axis) of small RNAs targeting the genes indicated on the right. Exons are represented as gray and black arrows pointing to the direction of transcription. Small RNA libraries were produced from extracted germlines. The genotype and lineage of the tested animals are color-coded according to the scheme in (C). (E) A principal component analysis (PCA) projection of germline small RNA samples based on normalized small RNA counts against annotated genes. Each point represents one independent replicate. Corresponding genotype and lineage are indicated and color-coded. (F) Two-way hierarchical clustering of the different biological groups and genes targeted by significantly differential levels of small RNAs. The clustering is based on the differentially expressed small RNAs compared to wild-type control samples. Each row represents one gene. Only genes targeted by significantly differential levels of small RNAs in at least one sample were included in the analysis (analyzed with DEseq2, adjusted p < 0.1). Each line is color-coded by the fold change in expression, where cells depicting no significant differential expression appear in gray. Biological groups are color-coded according to the scheme in (C) and appear above the columns.

Figure 6

Mutants with Defective Germ Granules Inherit RNAi in the Absence of HRDE-1 GFP fluorescence (y axis) of adult worms with the indicated genotype over generations (x axis), following exposure to gfp dsRNA. Fluorescence in each group was normalized to the mean fluorescence value of the corresponding isogenic control worms originally exposed to empty vector. Shown are mean ± SD of two independent pptr-1 experiments (left) and three independent meg-3/4 experiments (right), in which 25∼90 animals were analyzed for each group and generation. Tested strains derived from crosses of hrde-1 hermaphrodites with pptr-1 or meg-3/4 males (all with a Pmex-5::gfp transgene in the background) and the experiments were performed up to 6 generations since homozygosity. p values were determined via two-way ANOVA with Bonferroni post hoc correction for multiple comparisons to hrde-1. ^∗∗∗p < 0.001, ^∗p < 0.05.

Figure 7

A Schematic Model Describing the Roles of Germ Granules in Small RNA Inheritance Our results suggest that germ granules shape the heritable small RNA pool. Mutants in the meg-3/4 core germ granule proteins accumulate small RNA defects over generations, resulting in abnormal processing of exogenous RNAi responses. The RNAi defects are inherited from meg-3/4 (−/−) maternal lineages to the progeny via endogenous HRDE-1-dependent small RNAs, and not by contribution of the germ granules themselves. In addition, exogenous RNAi-derived small RNAs can be inherited transgenerationally through alternative routes in meg-3/4 mutants (independent of HRDE-1). The scale represents the balance between different small RNA populations. The circle in the middle of the scales represents intact germ granules (the P, Z, and M granules) that “keep the scale balanced.” The genotype of the animal is shown below each corresponding scale. The presence of a non-interrupted black arrow originating from dsRNA (represented in red) signifies that the animal can respond to exogenous RNAi. Dotted lines that crossed out by an “X” symbol indicate that this animal lost its ability to respond to exogenous RNAi. The arrows between scales represent transitions between generations. Red and blue single-stranded RNA molecules depict exogenous RNAi-derived siRNAs and endogenous siRNAs, respectively.