Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline - PubMed (original) (raw)

. 2009 Oct 23;36(2):231-44.

doi: 10.1016/j.molcel.2009.09.020. Epub 2009 Oct 1.

Masaki Shirayama, Darryl Conte Jr, Jessica Vasale, Pedro J Batista, Julie M Claycomb, James J Moresco, Elaine M Youngman, Jennifer Keys, Matthew J Stoltz, Chun-Chieh G Chen, Daniel A Chaves, Shenghua Duan, Kristin D Kasschau, Noah Fahlgren, John R Yates 3rd, Shohei Mitani, James C Carrington, Craig C Mello

Affiliations

Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline

Weifeng Gu et al. Mol Cell. 2009.

Abstract

Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the Dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that, in the germline, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage structures called P granules. WAGO-1 silences certain genes, transposons, pseudogenes, and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest additional regulatory functions for small RNAs.

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Figures

Figure 1

Figure 1. Hypomorphic alleles of drh-3 are RNAi deficient and temperature sensitive

A) Schematic of the drh-3 gene structure. Top panel: conserved DExH and HELICc domains and drh-3 lesions; bottom panel: four missense alleles (indicated) map to the HELICc domain. B) RNAi deficient phenotypes of drh-3 mutants. Fraction of viable embryos produced by animals exposed to pos-1 RNAi food, or viability of animals reared on let-2 RNAi food at 20°C. C and D) Brood size and Him phenotypes of drh-3 mutants at 20°C (dark bars) and 25°C (light bars). Mean brood size (top panel) of ≥10 hermaphrodites was determined by counting the number of embryos produced. Frequency of males among viable offspring was determined (bottom panel).

Figure 2

Figure 2. DRH-3 is required for the biogenesis of 22G-RNAs

A) Ethidium bromide staining of small RNAs. 22nt position is denoted by bracket. Note persistence of ~21nt band in all samples. 5.8S rRNA was used as loading control. B) Thin-layer chromatography analysis of first nucleotide of gel-purified 22 and 21nt RNA, as indicated, from wild type and drh-3 mutant samples. Bars (right), position of each nucleotide; “+”, treated with Terminator exonuclease; “−”, untreated. C) Length and first nucleotide distribution of genome matching reads from wild type and drh-3 mutant small RNA libraries. Sense structural RNAs were excluded in the analysis. D) Distribution of reads that match indicated genome annotations sequenced in wild type and drh-3 mutant small RNA libraries. E) Examples of small RNA distribution within rrf-1 and ama-1. Density of antisense reads indicated by vertical bars above (wild-type) or below (drh-3) gene structure. F) Genome-wide analysis of small RNA distribution within genes. Total antisense reads (y-axis) plotted according to the relative position (%) within all genes (x-axis; from 5′ to 3′).

Figure 3

Figure 3. Distinct requirements and genetic redundancy in 22G-RNA pathways

A) Northern blots of 22G-RNA expression in somatic gene Y47H10A.5, and germline targets F37D6.3 and Tc1 transposon in RNAi mutants. Loading controls: SL1 precursor and mir-66. B) Northern blots of germline 22G-RNAs in RdRP mutants. DA1316 is a congenic wild type control for the ego-1 and rrf-1 ego-1 mutants. Loading controls: SL1 precursor and mir-66. C) Coimmunoprecipitation analyses of DRH-3 from wild type and drh-3 lysates. Total lysate (left panels) and DRH-3 IPs (right panels) were analyzed by Western blot for candidate interacting proteins (indicated at right). The drh-3(0): the deletion allele (tm1217) control. Some DRH-3 protein detected in the drh-3(0) sample likely represents persistence of maternal product.

Figure 4

Figure 4. WAGO-1 and highly redundant WAGOs required for germline 22G-RNA biogenesis

A) Phylogenetic representation of WAGO proteins (see Supplemental Methods). WAGOs deleted in MAGO12 indicated in red. B), C) Northern blots of F37D6.3 22G-RNAs in multiple WAGO mutants. Loading control: SL1 precursor (B) and tRNA staining (C). D), E) Fluorescence microscopy of GFP::WAGO-1 and RFP::PGL-1, which colocalize to Pgranules in the germline.

Figure 5

Figure 5. Deep-sequence analyses identified at least two distinct 22G-RNA pathways in the germline

A) Graph depicting change in reads matching indicated genome annotations between input and WAGO-1 IP samples. B) Change of 22G-RNAs derived from genes (red) or transposons (blue) in each mutant. Relative enrichment calculated as ratio of mutant / (mutant + wild type) for ‘n’ genes or transposons. WAGO-1 IP enrichment calculated as WAGO-1IP / (WAGO-1IP + IP input). C) Venn comparison of genes depleted of 22G-RNAs (≥ 2-fold) in indicated mutants (loci below the lower dashed line in (B)). D) Frequency of reversion of dpy-5::Tc5 as indicated (Ketting et al., 1999). E) Quantitative RT-PCR analysis of 22G-RNA target expression in drh-3 mutant (red) relative to wild-type (blue). WAGO and CSR-1 (gray) targets are indicated.

Figure 6

Figure 6. WAGO-associated 22G-RNAs define a surveillance system

A) Enrichment or depletion of 22G-RNAs derived from ‘n’ annotated pseudogenes. B) Quantitative RT-PCR analysis of pseudogenes and cryptic loci targeted by WAGO pathway in drh-3 mutant (red) relative to wild-type (blue). C) Northern blots of 22G-RNAs in smg mutants grown at 20°C. Similar results obtained with mutants grown at 25°C (not shown). Loading control: SL1 precursor. D) Venn comparison of genes depleted of 22G-RNAs (≥ 2-fold) in mutants.

Figure 7

Figure 7

Model of germline 22G-RNA pathways required for genome surveillance in C. elegans.

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