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, 11 (3), e1005078
eCollection

Tertiary siRNAs Mediate Paramutation in C. Elegans

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Tertiary siRNAs Mediate Paramutation in C. Elegans

Alexandra Sapetschnig et al. PLoS Genet.

Abstract

In the nematode Caenorhabditis elegans, different small RNA-dependent gene silencing mechanisms act in the germline to initiate transgenerational gene silencing. Piwi-interacting RNAs (piRNAs) can initiate transposon and gene silencing by acting upstream of endogenous short interfering RNAs (siRNAs), which engage a nuclear RNA interference (RNAi) pathway to trigger transcriptional gene silencing. Once gene silencing has been established, it can be stably maintained over multiple generations without the requirement of the initial trigger and is also referred to as RNAe or paramutation. This heritable silencing depends on the integrity of the nuclear RNAi pathway. However, the exact mechanism by which silencing is maintained across generations is not understood. Here we demonstrate that silencing of piRNA targets involves the production of two distinct classes of small RNAs with different genetic requirements. The first class, secondary siRNAs, are localized close to the direct target site for piRNAs. Nuclear import of the secondary siRNAs by the Argonaute HRDE-1 leads to the production of a distinct class of small RNAs that map throughout the transcript, which we term tertiary siRNAs. Both classes of small RNAs are necessary for full repression of the target gene and can be maintained independently of the initial piRNA trigger. Consistently, we observed a form of paramutation associated with tertiary siRNAs. Once paramutated, a tertiary siRNA generating allele confers dominant silencing in the progeny regardless of its own transmission, suggesting germline-transmitted siRNAs are sufficient for multigenerational silencing. This work uncovers a multi-step siRNA amplification pathway that promotes germline integrity via epigenetic silencing of endogenous and invading genetic elements. In addition, the same pathway can be engaged in environmentally induced heritable gene silencing and could therefore promote the inheritance of acquired traits.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. 22G-RNAs distal to piRNA target sites require the nuclear RNAi pathway.
A) Small RNA high-throughput sequencing reads with unique matches antisense to the piRNA sensor from wild type and various mutant animals as indicated. The values of the y-axes correspond to reads matching the piRNA sensor normalised to reads matching Histone 2B (his-58). The x-axes represent the relative position of reads in the piRNA sensor transgene with numbers representing nucleotides from the start codon (set as 0). The transgene structure is schematically represented at the bottom. Colour code: green = GFP, grey = Histone 2B (his-58), dark blue = 21UR-1 target site plus/minus 50 bp, light blue = tbb-2 3′UTR. B) Left panel: Enrichment of small RNA high-throughput sequencing reads with matches antisense to the piRNA sensor in HRDE-1 Immunoprecipitation (IP). Displayed is the fold enrichment of reads found in anti-HRDE-1 IP from wild type versus hrde-1 mutant animals. X-axis, schematic representation of the transgene and colour code as in A). Right panel: Western blot of anti-HRDE-1 Immunoprecipitation from wild type (left 3 lanes) and hrde-1 mutant (right 3 lanes) animals. Antibodies used for western blot are anti-HRDE-1 and anti-PRG-1 as loading control. Inp. = 1% input, sup. = supernatant, IP = Immuoprecipitate. On the left, the relative migration of the 130 and 100 kDa bands of the PAGE Ruler Plus (MBI Fermentas) marker are indicated.
Fig 2
Fig 2. An endogenous target shows 3′ to 5′ spreading of 22G-RNAs.
Small RNA high-throughput sequencing reads with unique matches antisense to Y48G1B8M.5 from wild type and mutant animals as indicated. The values of the y-axes are antisense 22G siRNA reads per million of total reads. The x-axes represent the relative position of reads in the target gene with numbers representing nucleotides from the start codon (set as 0). The transcript structure is schematically depicted at the bottom with light grey and dark grey boxes representing alternating exons.
Fig 3
Fig 3. Endogenous 22G-RNA targets show evidence of nrde-dependent 3′-5′ spreading of 22G-RNAs.
A) Genes were divided into 7 clusters based on the pattern of 22G-RNAs along the gene in nrde-4 mutants (see methods). The positions of 22G-RNAs relative to the normalized gene length are on the x-axes (see methods). y-axes represent the average abundance of 22G-RNAs relative to transcript position between nrde-4 and wild type (WT) for each cluster. Cluster 6 showing clear reduction at the 5′ end of the gene in nrde-4 relative to wild type is shown in red. B) Log2 enrichment of predicted piRNA target sites at the 3′ half relative to the 5′ half of endogenous 22G-RNA target transcripts found in clusters 1–7. Cluster 6 is shown in red. C and D) 22G-RNA levels for each gene in total RNA from prg-1 mutant animals (C) and anti-HRDE-1 IPs (D) relative to 22G-RNA levels in total RNA from wild type animals. Each box corresponds to one of the clusters 1–7. Boxes show the interquartile range, whiskers show the most extreme point that is no less than 1.5 times the interquartile range and outliers are shown as circles. Cluster 6 is shown in red.
Fig 4
Fig 4. Nuclear RNAi is necessary to initiate synthesis of tertiary siRNAs.
A) The principle of the operon transgene expression. Straight line represents the DNA locus (bottom), curved lines pre-mRNA and mRNA (before and after nuclear export). Diamonds and snowflakes are mCherry protein (red) and GFP protein (green), respectively (top). B) Schematic representation of the transgenes in the operon; piRNA sensor strain. The piRNA sensor (bottom) generates 22G-RNAs (black lines) against GFP that can confer trans-silencing of the operon-derived GFP. In case a spreading of 22G-RNAs occurs in the nucleus, silencing of mCherry would be expected. C) Representative fluorescence images of somatic and germline GFP (top row) and mCherry (middle row) expression and DIC images (bottom row) of the parental operon strain (left), silenced wild type operon; piRNA sensor animals (middle) and de-silenced operon; piRNA sensor; hrde-1 mutant (right) animals. D) Small RNA high-throughput sequencing reads with unique matches antisense to the operon from animals as indicated in C). The values of the y-axes correspond to reads matching the operon normalised to reads matching Histone 2B (his-58). The x-axes represent the relative position of reads in the operon transgene with numbers representing nucleotides from the start codon (set as 0). The transgene structure is schematically represented at the bottom. Colour code: red = mCherry, dark grey = gpd-2 trans-splicing linker (L), green = GFP, light grey = par-5 3′UTR.
Fig 5
Fig 5. Tertiary 22G-RNAs mediate paramutation.
A) Representative fluorescence images of somatic and germline GFP (top row) and mCherry (middle row) expression and DIC images (bottom row) of the parental operon strain (left) and the outcrossed, silenced wild type operon animals (right). B) Small RNA high-throughput sequencing reads with unique matches antisense to the operon from outcrossed operon animals. The values of the y-axes correspond to reads matching the operon normalised to reads matching Histone 2B (his-58). The x-axes represent the relative position of reads in the operon transgene with numbers representing nucleotides from the start codon (set as 0). The transgene structure is schematically represented at the bottom. Colour code: red = mCherry, dark grey = gpd-2 trans-splicing linker (L), green = GFP, light grey = par-5 3′UTR. C) Fluorescence images of germline mCherry expression (top panels) and DIC images (bottom panels) of the parental mCherry::H2A (left) and the outcrossed, trans-silenced wild type mCherry::H2A strains (right). D) Small RNA high-throughput sequencing reads with unique matches antisense to the mCherry transgene from animals as indicated in C). The values of the y-axes correspond to reads matching the mCherry sequence normalised to reads matching Histone 2B (his-58). The x-axes represent the relative position of reads in the mCherry::H2A transgene with numbers representing nucleotides from the start codon (set as 0). E) Crossing schemes (top) to generate trans-silenced mCherry::H2A animals as indicated above the representative fluorescence (GFP top, mCherry middle) and DIC images (bottom). Left panels are from a control cross using the parental non-silenced operon strain. Trans-silencing occurs with or without transmission of a silenced operon transgene copy.
Fig 6
Fig 6. Model of multigenerational target gene silencing by piRNAs and downstream 22G-RNAs.
Left: piRNAs against target sites (blue) initiate localized 22G-RNA (blue) production that involves RNA-dependent RNA Polymerases (RdRPs) and Mutator proteins (Muts). 3′ to 5′ spreading of 22G-RNAs along the target gene (e.g. GFP, green) requires the nuclear RNAi pathway. This induces gene silencing that can be maintained over subsequent generations. Middle: Tertiary 22G-RNAs against a target (e.g. GFP, green) are able to silence genes with sequence similarity in trans. This leads to further generation of tertiary 22G-RNAs along the trans-silenced target (e.g. the operon/mCherry, red) by nuclear RNAi factors. Silencing by tertiary 22G-RNAs can become trigger-independent. Right: Paramutation by tertiary 22G-RNAs against mCherry (red) can be stably maintained in the absence of the original trigger(s).

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