Function, targets, and evolution of Caenorhabditis elegans piRNAs

Abstract

Piwi-interacting RNAs (piRNAs) are small RNAs required to maintain germline integrity and fertility, but their mechanism of action is poorly understood. Here we demonstrate that Caenorhabditis elegans piRNAs silence transcripts in trans through imperfectly complementary sites. Target silencing is independent of Piwi endonuclease activity or "slicing." Instead, piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are derepressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes and tend to overlap the start and end of transposons in sense and antisense, respectively. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNA interference in C. elegans.

Fig. 1

A single antisense piRNA site is sufficient for target silencing in vivo. (A) Fluorescence microscopy (GFP-H2B) and differential interference contrast (DIC) images of adult hermaphrodites. Scale bar 20 μm. (B) Flow cytometry analysis of control sensor strain (green) and piRNA sensor strain in wild-type (red) or prg-1 (n4357) mutant background (blue) as in (A). (C) Germline GFP-H2B expression of the 21UR-1349 piRNA sensor. (D) Flow cytometry analysis of the 21UR-1349 piRNA sensor strain in wild-type (red) and prg-1 mutant (blue). (E) Profiles of small RNA high-throughput sequencing reads with unique match to the sensor relative to the target site (indicated in grey). Colors correspond to 5′ nucleotides as indicated in the color key in (F). Positive and negative y-axes correspond to antisense and sense reads, respectively. (F) Length and 5′ nucleotide identity of small RNAs antisense to the piRNA sensor in wild-type. (G) Northern blot of total RNA. Probes were against piRNA 21UR-1, a piRNA sensor-specific 22G-RNA and the PRG-1-independent endo-siRNA siR26-263. (H) qRT-PCR of primary piRNA sensor transcript and mRNA. Data were normalized to wild-type transcript levels. Error bars are standard errors of the mean.

Fig. 2

A specific endo-siRNA pathway acts downstream of and is required for piRNA-mediated silencing. (A) piRNA sensor expression in siRNA pathway mutants as in Fig. 1A. (B) A second 21UR-1 piRNA sensor strain (cherrysensor) expressing mCherry-H2B in prg-1 (n4357).

Fig. 3

piRNAs initiate a localized secondary siRNA response against endogenous transcripts. (A) Average profiles of collapsed small RNAs mapping uniquely to imperfect genomic piRNA matches (1-3 mismatches) in wild-type (left) and prg-1 mutant (right). Top and bottom panels to the right of each profile illustrate characteristics of antisense and sense small RNAs, respectively. (B) Number of genomic piRNA matches (left) and percentage of matches with uniquely mapping 22G-RNAs in wild-type (middle) and prg-1 (right). Black and white bars correspond to piRNAs and matched controls, respectively. Bars for control sequences indicate medians, error bars the range of values obtained for 20 cohorts of control sequences. Numbers above bars indicate the fold-difference between piRNAs and controls. (C) Difference in 22G-RNAs mapping uniquely within 20 bp of genomic piRNA matches between prg-1 and wild-type. Shown are boxplots of the difference in 22G-RNA reads after square root transformation (box indicates interquartile range, plot extends from 5th to 95th percentile). Asterisks indicate statistical significance (P < 0.001, two-sided Wilcoxon rank-sum test). (D) As in (C) with genomic piRNA matches grouped according to motif score of complementary piRNA (as proxy for abundance).

Fig. 4

Endogenous piRNA targets and piRNA evolution. (A) Candidate transposon targets ranked by change in 22G-RNA density at target sites between prg-1 and wild-type. Transposons selected for qRT-PCR validation are in red. Antisense 22G-RNA profiles are shown for selected elements with target sites indicated above each profile as explained in the color key. (B) Candidate protein-coding targets as in (A). (C) qRT-PCR analysis of candidate transposon targets with fold-changes normalized to actin. Error bars are standard errors of the mean, asterisks denote P < 0.05 (two-sided t-test). (D) qRT-PCR analysis of candidate protein-coding targets. (E) qRT-PCR analysis of targeted (F54F2.2b) and non-targeted (F54F2.2a) transcripts from the zfp-1 locus. Data were normalized to wild-type. (F) Enrichment and depletion of genomic piRNA matches overlapping features of interest. Red and blue indicate increased or reduced number of matches for piRNAs compared to control sequences, respectively. Asterisks indicate statistical significance. Start and end refer to the first and last 50 bp of the annotated feature, respectively. Pseudogene annotation was only available for C. elegans. (G) piRNA matches against start (left) and end (right) of DNA transposons. Profiles indicate the number of transposon subfamilies with perfect piRNA match in at least one full-length genomic copy. Positive (blue) and negative (red) y-axes correspond to sense and antisense matches, respectively. Dashed lines correspond to maximal allowed distance between an upstream sequence motif and piRNA 3′ end.