2016 Feb 25
The RNase PARN-1 Trims piRNA 3' Ends to Promote Transcriptome Surveillance in C. Elegans
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The RNase PARN-1 Trims piRNA 3' Ends to Promote Transcriptome Surveillance in C. Elegans
Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and are essential for fertility in diverse organisms. An interesting feature of piRNAs is that, while piRNA lengths are stereotypical within a species, they can differ widely between species. For example, piRNAs are mainly 29 and 30 nucleotides in humans, 24 to 30 nucleotides in D. melanogaster, and uniformly 21 nucleotides in C. elegans. However, how piRNA length is determined and whether length impacts function remains unknown. Here, we show that C. elegans deficient for PARN-1, a conserved RNase, accumulate untrimmed piRNAs with 3' extensions. Surprisingly, these longer piRNAs are stable and associate with the Piwi protein PRG-1 but fail to robustly recruit downstream silencing factors. Our findings identify PARN-1 as a key regulator of piRNA length in C. elegans and suggest that length is regulated to promote efficient transcriptome surveillance.
Copyright © 2016 Elsevier Inc. All rights reserved.
Figure 1. PARN-1 is required for 3′ trimming of piRNAs
(A) Northern blot analysis of 21U-RNA-1 and miR-66 from total RNA prepared from wild type, parn-1, parn-2, prg-1, and parn-1; prg-1 double mutant strains. (B) The expression profile for the bulk population of piRNAs as determined by small RNA sequencing. Plotted for each library is the percent of reads that represented piRNAs after normalized to non-structural RNAs. For wild-type and parn-1 samples, the average of three replicates is shown, error bars represent standard error of mean (SEM). One set of small RNA libraries from prg-1 and prg-1; parn-1 mutants is shown. n.s.=not significant (Student's t-test). (C, D) Length distribution (C) and first nucleotide distribution (D) of piRNA reads from wild type and parn-1 mutant small RNA libraries. The average of three experiments is shown and error bars represent SEM. (E) A browser view of representative piRNA loci. Small RNA reads mapped the 21U-RNA-3772 and 21U-RNA-1390 genomic loci. See also Figure S1.
Figure 2. Untrimmed piRNAs are loaded onto PRG-1 and possess 2′–O–methylation
(A) Bar plots showing the change in small RNA reads matching indicated genome annotations between input and PRG-1 IP samples prepared from wild type and parn-1 strains. (B) Correlation analysis of the piRNA level. Libraries were prepared from PRG-1 IP samples from WT and parn-1 strains. Data from reads for each piRNA were normalized to total reads in the same sample. For a perfect correlation, the Spearman rank correlation coefficient (r) = 1 or −1, and for no correlation, r = 0. (C) Oxidation and β–elimination followed by Northern blot analysis of RNA prepared from WT, parn-1 and henn-1 strains. RNA samples were not treated (−) or β–elimination treated (+), and probed for 21UR-1949. Probing for miRNA-66 served as controls for loading and β–elimination reactions. (D) Fold enrichment of piRNA reads was calculated by comparing β–eliminated and non-treated samples. Small RNA libraries were generated from β–eliminated and untreated RNAs prepared from WT, parn-1, henn-1 strains. See also Figure S2.
Figure 3. PARN-1 is expressed in the germline and localizes in P granules
(A) Quantitative RT-PCR of parn-1 mRNA from total RNA isolated from synchronized wild-type populations at the indicated developmental stages and germline-deficient glp-4 (bn2) mutants at the adult stage. Expression of act-3 served as the internal control. Data were collected from three independent biological replicates. Error bars represent standard deviation. (B) Fluorescence micrographs showing PARN-1::GFP expression (green) from an adult hermaphrodite. Expression RFP::PGL-1(red) served the P granule marker. The dashed lines outline the position of germline, (C) Immunostaining of PRG-1 (red) and PARN-1::GFP (green) in dissected gonad arms from the parn-1 ::gfp rescue line. See also Figure S3.
Figure 4. Recombinant PARN-1 possesses 3′-to-5′ exonuclease activity in vitro
(A) Schematic of the parn-1 genes showing exons (boxes) and intron (lines) with the catalytic ribonuclease domain shaded in dark orange. The deletion allele ( tm869) is highlighted in blue. The alignment shows human, mouse, C. elegans, B. mori, and S. pombe PARN and poly(A)-specific ribonuclease-like domain containing (PNLDC) protein with conserved catalytic residues (asterisk). (B) Electrophoresis analysis on 21nt and 14nt 5′ Fluorescein labeled RNA substrates incubated with purified PARN-1 (WT and D29A) proteins. (C-E) Electrophoresis analysis of 14-mer RNAs containing 3′ hydroxyl (14mer) and phosphorothioate (PS) bonds from positions 10 to 14 (14mer-PS-Terminal) (C), 14-mer RNAs with PS bond between positions 7 to 8 (14mer-PS-Center). Asterisk represents the degradation intermediate (D), and 14-mers containing 3′ hydroxyl and 2′–O–methylated 3′ termini (14mer-OME) (E). (F) Quantification of the ratio between processed and unprocessed products among biological replicates. The average of six replicates for 14mer and the average of three replicates for 14mer-OME are shown. Error bars represent standard deviation. See also Figure S4.
Figure 5. PARN-1 is required for fertility and epigenetic silencing of single-copy transgenes
(A) Brood size counts in WT, prg-1, parn-1, prg-1; parn-1 double, and parn-1 rescue animals at 25 °C. n=Number of parental adults scored. (B, C) Schematics of the gfp::cdk-1 reporter (B), and of the h2b::gfp::21U-RNA-target-site reporter (C), that were injected into wild-type, parn-1 and prg-1 worms to establish single copy transgenic lines. Images of GFP fluorescence signals in the resulting strains are shown. Oocyte nuclei that either express, or fail to express the construct are indicated by arrowheads. The dashed lines outline the position of germline. Bright signals outside of the germline are from gut granule autofluorescence. The gfp::h2b::21U-RNA-target-site reporter established in wild-type and parn-1 animals was crossed to the rde-3 mutant strain. n=number of independent transgenic lines exhibiting the pattern of expression shown.
Figure 6. Trimming of piRNAs is important for 22G-RNAs production
(A) Venn diagram summarizing the overlap in the number of genes whose 22G-RNAs levels decrease by two fold when compared to wild type. Gene sets from prg-1, parn-1, and prg-1; parn-1 double mutants are plotted (p-values < 0.0001; Hypergenomic Test). (B) Box and whisker plot showing fold change of 22G-RNAs at predicted target sites in wild type and parn-1 mutants as compared to prg-1 mutants. Asterisks indicate statistical significance (p-value = 6.26 × e −227, Wilcoxon Rank-Sum Test). (C) Density of 22G-RNAs in a 100 nt interval around predicted piRNA target sites in the wild type, parn-1, and prg-1 mutants. The plots are centered on piRNAs. (D) Model illustrating the role of PARN-1 in piRNA biogenesis and functions. See also Figure S5, Tables S2 and S3.
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Research Support, Non-U.S. Gov't
Argonaute Proteins / metabolism
Caenorhabditis elegans / metabolism
Caenorhabditis elegans Proteins / metabolism
Exoribonucleases / chemistry
Exoribonucleases / metabolism
Metabolic Networks and Pathways
RNA Processing, Post-Transcriptional
RNA, Small Interfering / genetics
RNA, Small Interfering / metabolism
Caenorhabditis elegans Proteins
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