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, 7 (11), e1002369

The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification Pathway That Targets Recent Gene Duplications

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The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification Pathway That Targets Recent Gene Duplications

Sylvia E J Fischer et al. PLoS Genet.

Abstract

Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7-dependent siRNAs require Dicer. We identify that the eri-6/7-dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3-4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7-dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA-associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. eri-6/7 is required for the accumulation of distinct classes of endogenous siRNAs.
(A) Northern blot analysis of endogenous siRNAs derived from K02E2.6. tRNA is shown as a loading control. (B) Ratio of two oocyte/embryo-enriched 26G siRNAs in eri mutant embryos as determined by qRT-PCR (wild type = 1.0). (C) Ratio of two sperm-enriched 26G siRNAs in eri mutant L4/young adult animals as determined by qRT-PCR (wild type = 1.0).
Figure 2
Figure 2. eri-6/7 is required for accumulation of 26G siRNA in embryos and for a subset of 22G siRNAs in adults.
(A) Small RNA length and 5′ nucleotide distribution in wild type and eri class mutants. (B) Scatter plots displaying each feature indicated as the number of siRNA reads in wild type versus eri class mutants. (C) Ratio of small RNA reads in eri-7 to wild type embryos and adults after log2 transformation. Total small RNA reads within 5 kb interval were plotted across each chromosome in 1 kb increments. Bars are color coded according to the most abundant size class of small RNAs within each interval. (D) Ratio of 26G siRNA reads in eri class mutants to wild type embryos (wild type = 1.0). (E) Ratio of ERGO-1 and ALG-3/4 class 22G siRNA reads in eri-7 mutants to wild type adults (wild type = 1.0). (F) Scatter plot displaying siRNA reads derived from ERGO-1 target genes in wild type versus eri-7 mutant adults. (G) Scatter plot displaying siRNA reads derived from ALG-3/4 target genes in wild type versus eri-7 mutant adults.
Figure 3
Figure 3. Genes targeted by eri-6/7-dependent endogenous siRNAs share regions of high sequence identity.
(A) Overlap between genes targeted by eri-6/7-dependent siRNAs identified by deep sequencing of embryo small RNA and of adult small RNA. (B, C) Two groups of target genes that share sequence identity. Indicated are stretches with high identity and stretches of 100% identity. (D) Target genes show sequence identity in regions with siRNAs. siRNA reads in wild type embryo libraries were plotted against four target gene regions. Unique siRNAs and non-unique siRNAs are color-coded. Gene structures of different splice variants are shown with genes on the Watson strand pointing to the right, genes on the Crick strand pointing to the left. Regions of high sequence identity are indicated by arrows, color-coded and marked by a letter. (E) Homology between the adult eri-6/7-dependent siRNA target genes C36A4.11 and K06B9.6. Unique and non-unique siRNAs are color-coded. The grey arrow indicates the region of identity.
Figure 4
Figure 4. eri-6/7-dependent siRNAs associate with NRDE-3 to direct cotranscriptional gene silencing in the nucleus.
(A) Ratio of soma-enriched siRNAs in eri class mutants to wild adults (wild type = 1.0). (B) RDE-3 localization in wild type and eri-6 mutants. The insets show single hypodermal cells with either mostly cytoplasmic (eri-1 and eri-6 mutants) or nuclear (in wild type) GFP::NRDE-3 expression. (C) Ratio of NRDE-3-associated siRNA reads in eri class mutants to wild type adults (wild type = 0). (D) Ratio of mRNA levels of two genes that yield eri-6/7-dependent siRNAs in eri-7 mutants to wild type adults, as determined by qRT-PCR (wild type = 1.0).
Figure 5
Figure 5. 26G siRNAs are derived from non-canonical siRNA duplexes.
(A) 26G siRNA distribution (indicated are 5′ positions) across the E01G4.7 locus. (B) siRNA distribution across the fbxb-37 locus. (C) The proportions of 5′ and 3′ ends of the most abundant sequences overlapping and antisense to each 26G siRNA sequence are shown. Inset displays the consensus 26G siRNA duplex. (D) Ratio of 26G siRNA passenger strand reads in eri mutants to wild type (wild type = 1.0).

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