Transgene-Assisted Genetic Screen Identifies rsd-6 and Novel Genes as Key Components of Antiviral RNA Interference in Caenorhabditis elegans

Figures

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  FIG 1. Design of a biased genetic screen for the identification of novel C. elegans genes involved in antiviral RNAi. (A) Schematic structure of plasmid constructs to be used to create the reporter transgene array for biased genetic screen. HIP, heat-inducible promoter; protein A, the replicase of Flock house virus; Rz, self-cleaving ribozyme sequence derived from hepatitis D virus, which functions to remove all nonviral sequence at the 3′ end of the FR1gfp primary transcripts; GFP, the coding sequence of enhanced green fluorescence protein; Psur-5, the promoter of the endogenous gene sur-5; UTR, the 3′-end untranslated region of unc-54; Pmyo-2, the myo-2 promoter which directs gene expression in pharyngeal muscles. (B) The workflow for identification of worm genes required for antiviral RNAi.

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  FIG 2. Generation of reporter transgene array ty48. (A) Coinjection of the plasmid constructs shown in Fig. 1A restored antiviral RNAi in rde-1;rde-4 double mutants that contain an FR1gfp transgene. Shown here is the visualization of green fluorescence in the rde-1;rde-4 double mutants that contain extrachromosomal arrays formed by the injected plasmids 48 h after heat induction. The image was produced by merging images recorded under red and green fluorescence using the same exposure. (B) Accumulation of FR1gfp transcripts in single and double mutants corresponding to rde-1 and rde-4 as indicated. An asterisk indicates worms that carry the ty48 transgene array. The FR1gfp transcripts were detected by Northern blotting, for which the probe was prepared using GFP coding sequence; RNA1, FR1gfp genomic RNA; RNA3, FR1gfp subgenomic RNA. Methylene blue-stained rRNA serves as an equal loading control. (C) Northern blot detection of Orsay virus RNA1, ovRNA1, in single and double mutants corresponding to rde-1 and rde-4 as indicated. An asterisk indicates worms that carry the ty48 transgene array. Orsay virus RNA1 cDNA was used to prepare probe for the detection of Orsay virus RNA1 through…

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  FIG 3. Both drh-1 and rsd-2 transgenes are functional in the ty48 transgene array. (A) Northern blot detection of FR1gfp replication in transgenic worm strains, as indicated, that contain the ty48 transgene array after heat induction. See the legend to Fig. 2B for experimental details. An asterisk indicates worms that were fed with E. coli food expressing drh-1 dsRNA. A double asterisk indicates worms that were fed with E. coli food expressing rsd-2 dsRNA. (B) Accumulation of Orsay virus RNA1 in drh-1 and rsd-2 mutants and wild-type N2 worms that carry the ty48 transgene array. An asterisk indicates worms that were fed with E. coli food expressing drh-1 dsRNA. A double asterisk indicates worms that were fed with E. coli food expressing rsd-2 dsRNA. See the legend to Fig. 2C for experimental details.

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  FIG 4. Antiviral RNAi remains defective in rrf-1 and drh-3 mutants containing the ty48 transgene array. (A) Northern blot detection of FR1gfp replication in rrf-1 and drh-3 mutants, as indicated, that contain the ty48 transgene array. See the legend to Fig. 2B for experimental details. (B) Visualization of green fluorescence in rrf-1 and drh-3 mutants, as indicated, carrying the ty48 transgene array 48 h after heat induction. These images were produced by merging images recorded using the same exposure and at the same time. (C) Accumulation of Orsay virus RNA1 in rrf-1 and drh-3 mutants that carry the ty48 transgene array.

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  FIG 5. Identification of rrf-1 and rde-3 alleles that are defective in antiviral RNAi. (A) Identification of point mutations in rrf-1 coding sequence that disrupt rrf-1 function in antiviral RNAi. Shown here is the rrf-1 cDNA sequencing results for rrf-1 alleles 1025a and 1026e. The rrf-1 coding sequence in wild-type worm N2 was sequenced as a reference. (B) Point mutations in rde-3 allele 1031b that disrupt antiviral RNAi. The rde-3 coding sequence in wild-type worm N2 was sequenced as a reference. (C) Ectopic expression of wild-type rde-3 restored antiviral RNAi in worm mutants containing the 1031b allele. (Upper) Structure of plasmid construct that expresses wild-type rde-3. (Lower) Visualization of antiviral RNAi in worms containing the 1031b allele. Worms marked with red fluorescence in the head region contain a transgene that expresses wild-type rde-3.

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  FIG 6. Function characterization of the candidate genes. (A) Experimental design for the introduction of candidate alleles into worm strain LR11. m, loss-of-function alleles identified in the biased genetic screen. Pmyo-3, myo-3 promoter that is constitutively active in body wall muscle. (B) Northern blot detection of FR1gfp replication in LR11 worms that contain genetic alleles corresponding to asd-1, asd-5, and rde-3. The replication of FR1gfp was also detected in wild-type N2 worms and drh-1 and rde-4 mutants as references. (C) Northern blot detection of FR1gfp replication in LR11 worms that contain genetic alleles corresponding to asd-2, asd-3, asd-4, asd-7, asd-8, asd-9, asd-10, asd-11, and asd-12. (D) Visualization of mCherry fluorescence in LR11 worms that contain genetic alleles corresponding to candidate alleles, as indicated within each set of images. All worms were fed using E. coli food expressing dsRNA derived from mCherry coding sequence. The inset images were taken under white light. All images were taken using the same exposure. (E and F) unc-22 RNAi phenotype observed in LR11 worms containing genetic alleles, as indicated in panels B and C, corresponding to candidate…

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  FIG 7. Schematic presentation of the workflow for the identification of candidate alleles using the mapping-by-sequencing strategy. VCF, variant call format. snpEff, a toolbox for genomic variant annotations and functional effect prediction.

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  FIG 8. Identification of rsd-6 as key component of antiviral RNAi in C. elegans. (A) Schematic gene structures for wild-type rsd-6 and rsd-6 alleles isolated in the genetic screen. Exons are shown as solid boxes, whereas the lines indicate introns. The putative protein sizes from conceptual translation are indicated for each allele. (B) Schematic structure of the plasmid construct that was used for constitutive expression of wild-type rsd-2 gene in transgenic worms. RSD-6, the open reading frame of wild-type rsd-6. (C) Ectopic expression of wild-type rsd-6 restored antiviral RNAi in worm mutants containing the 1026a alleles. Shown here is the visualization of green fluorescence in the 1026a mutants that have been injected with plasmid construct Psur-5::RSD-6. The transgenic worm is marked by red fluorescence in the head region. (D) Constitutive overexpression of wild-type rsd-6 restored antiviral RNAi in worm mutants containing the 1026a alleles. Shown here is the detection of FR1gfp genomic and subgenomic RNA accumulation after heat induction. An asterisk indicates 1026a mutants that contain integrated transgene derived from the plasmid construct Psur-5::RSD-6. (E) Same as panel D,…

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  FIG 9. Both asd-2 and asd-9 play important roles in antiviral RNAi targeting Orsay virus. (A) Northern blot detection of Orsay virus replication in LR11 worms that carry representative alleles corresponding to each of the candidate genes as indicated. (B) Northern blot detection of Orsay virus replication in candidate mutants isolated from the generic screen. ovRNA1, Orsay virus RNA1.

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  FIG 10. asd-9 and rsd-6, but not asd-2, are required for biogenesis or stability of primary viral siRNAs. (A) Accumulation of FR1gfp genomic and subgenomic RNAs in asd-2 (1029e), asd-9 (1026d), and rsd-6 (1026a) after heat induction. The replication of FR1gfp in wild-type N2 worms and drh-1, rde-1, and rde-4 mutants was also detected as references. (B) Northern blot detection of FR1gfp-derived primary siRNAs in asd-2 (1029e), asd-9 (1026d), rsd-6 (1026a), and control worm strains. The accumulation of miR-58 was also detected as an equal loading control.