Gene silencing in Caenorhabditis elegans by transitive RNA interference

Abstract

When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). Here, we provide evidence that dsRNA is amplified in Caenorhabditis elegans to ensure a robust RNAi response. Our data suggest a model in which mRNA targeted by RNAi functions as a template for 5' to 3' synthesis of new dsRNA (termed transitive RNAi). Strikingly, the effect is nonautonomous: dsRNA targeted to a gene expressed in one cell type can lead to transitive RNAi-mediated silencing of a second gene expressed in a distinct cell type. These data suggest dsRNA synthesized in vivo can mediate systemic RNAi.

FIGURE 1.

Genetic evidence for dsRNA amplification in vivo. (A) If target mRNA functions as a template for synthesis of new dsRNA, then dsRNA directed against a translational fusion between GFP (green) and a C. elegans gene (red) will lead to the production of new dsRNA that can target the endogenous C. elegans gene (red lines). This phenomenon is called transitive RNAi. Here and throughout, target mRNAs are depicted as boxes and dsRNA as lines. (B) Injection of full-length GFP dsRNA (GFP–FL) leads to killing of pha-4::GFP and tlf-1::GFP worms but not pGFP or wild-type animals (top four rows). Injection of full-length unc-60B dsRNA kills wild-type worms but not unc-60B mutants (bottom two rows). (n′) The number of mothers whose progeny were scored except for unc-60B where mothers were typically scored. A total of 83% of wild-type animals injected with unc-60B dsRNA died and, for the surviving mothers, none of their progeny survived. (C) unc-60 produces two mRNAs, A and B, which share a common first exon (hatched box). RNAi is initiated using unc-60B dsRNA and progeny animals scored for lethal (unc-60A+B) or uncoordinated (unc-60B) phenotypes. (FL) Full-length 453-bp dsRNA for either unc-60A or unc-60B. (5′) 358-bp dsRNA located 2 nucleotides from the exon 1/2 boundary. (3′) 360-bp dsRNA located 96 nucleotides from the exon 1/2 boundary.

FIGURE 1.

Genetic evidence for dsRNA amplification in vivo. (A) If target mRNA functions as a template for synthesis of new dsRNA, then dsRNA directed against a translational fusion between GFP (green) and a C. elegans gene (red) will lead to the production of new dsRNA that can target the endogenous C. elegans gene (red lines). This phenomenon is called transitive RNAi. Here and throughout, target mRNAs are depicted as boxes and dsRNA as lines. (B) Injection of full-length GFP dsRNA (GFP–FL) leads to killing of pha-4::GFP and tlf-1::GFP worms but not pGFP or wild-type animals (top four rows). Injection of full-length unc-60B dsRNA kills wild-type worms but not unc-60B mutants (bottom two rows). (n′) The number of mothers whose progeny were scored except for unc-60B where mothers were typically scored. A total of 83% of wild-type animals injected with unc-60B dsRNA died and, for the surviving mothers, none of their progeny survived. (C) unc-60 produces two mRNAs, A and B, which share a common first exon (hatched box). RNAi is initiated using unc-60B dsRNA and progeny animals scored for lethal (unc-60A+B) or uncoordinated (unc-60B) phenotypes. (FL) Full-length 453-bp dsRNA for either unc-60A or unc-60B. (5′) 358-bp dsRNA located 2 nucleotides from the exon 1/2 boundary. (3′) 360-bp dsRNA located 96 nucleotides from the exon 1/2 boundary.

FIGURE 1.

Genetic evidence for dsRNA amplification in vivo. (A) If target mRNA functions as a template for synthesis of new dsRNA, then dsRNA directed against a translational fusion between GFP (green) and a C. elegans gene (red) will lead to the production of new dsRNA that can target the endogenous C. elegans gene (red lines). This phenomenon is called transitive RNAi. Here and throughout, target mRNAs are depicted as boxes and dsRNA as lines. (B) Injection of full-length GFP dsRNA (GFP–FL) leads to killing of pha-4::GFP and tlf-1::GFP worms but not pGFP or wild-type animals (top four rows). Injection of full-length unc-60B dsRNA kills wild-type worms but not unc-60B mutants (bottom two rows). (n′) The number of mothers whose progeny were scored except for unc-60B where mothers were typically scored. A total of 83% of wild-type animals injected with unc-60B dsRNA died and, for the surviving mothers, none of their progeny survived. (C) unc-60 produces two mRNAs, A and B, which share a common first exon (hatched box). RNAi is initiated using unc-60B dsRNA and progeny animals scored for lethal (unc-60A+B) or uncoordinated (unc-60B) phenotypes. (FL) Full-length 453-bp dsRNA for either unc-60A or unc-60B. (5′) 358-bp dsRNA located 2 nucleotides from the exon 1/2 boundary. (3′) 360-bp dsRNA located 96 nucleotides from the exon 1/2 boundary.

FIGURE 2.

Phenotype of tlf-1::GFP transgenic animals treated with GFP dsRNA. (Top) Wild-type worms treated with no dsRNA (WT) or tlf-1 dsRNA (tlf-1 RNAi). (Bottom) Two examples of arrested embryos from tlf-1::GFP transgenic worms treated with GFP dsRNA (GFP RNAi).

FIGURE 3.

Transitive RNAi moves 5′ to 3′. dsRNA homologous to full-length, 5′, 3′, or middle of GFP was used to initiate RNAi in worms carrying one of five transgenes: PG txg, GPtxg, pha-4::GFP, tlf-1::GFP, or pGFP. (*) Effects of GFP-FL for pha-4::GFP, tlf-1::GFP, and pGFP worms are from Figure 1 ▶.

FIGURE 4.

Two models for RNAi with loop sequences of a dsRNA hairpin. (A) GFPhp is a dsRNA hairpin with GFP sense RNA(s) on the 5′ end of the stem and GFP antisense RNA (as) on the 3′ end of the stem; unc-22 sequences are located in the loop (Winston et al. 2002). The hairpin is predicted to undergo processing to form siRNAs, with the last fragment of dsRNA bearing the unc-22 loop. Association of siRNAs with the GFP mRNA will induce endonucleolytic cleavage of the target. Cleaved GFP mRNA may function as a primer to synthesize a complementary strand of unc-22 RNA using the loop-containing siRNA as template (1, left). Alternatively, siRNAs generated during GFP RNAi may prime dsRNA synthesis with a new GFPhp (2, right). (B) Silencing of GFP and unc-22. (*) Percentage of animals with GFP completely off. (‡) Percentage of uncoordinated animals. (+++) Animals that were paralyzed on the plate; (+) animals that were sluggish or egg-laying defective. pGFP is not silenced effectively by GFPhp feeding, but presumably some RNAi is occuring since this strain was tested concurrently with pha-4::GFP.

FIGURE 4.

Two models for RNAi with loop sequences of a dsRNA hairpin. (A) GFPhp is a dsRNA hairpin with GFP sense RNA(s) on the 5′ end of the stem and GFP antisense RNA (as) on the 3′ end of the stem; unc-22 sequences are located in the loop (Winston et al. 2002). The hairpin is predicted to undergo processing to form siRNAs, with the last fragment of dsRNA bearing the unc-22 loop. Association of siRNAs with the GFP mRNA will induce endonucleolytic cleavage of the target. Cleaved GFP mRNA may function as a primer to synthesize a complementary strand of unc-22 RNA using the loop-containing siRNA as template (1, left). Alternatively, siRNAs generated during GFP RNAi may prime dsRNA synthesis with a new GFPhp (2, right). (B) Silencing of GFP and unc-22. (*) Percentage of animals with GFP completely off. (‡) Percentage of uncoordinated animals. (+++) Animals that were paralyzed on the plate; (+) animals that were sluggish or egg-laying defective. pGFP is not silenced effectively by GFPhp feeding, but presumably some RNAi is occuring since this strain was tested concurrently with pha-4::GFP.

FIGURE 5.

Phenotypes of worms exposed to dsRNA hairpins. (A) Motility of wild-type (WT), pha-4::GFP, or pGFP transgenic worms after RNAi induced with GFP- unc-22 hairpin dsRNA (GFPhp). Three F1 animals were allowed to move freely for 11 min and photographed in the absence (no treat) or presence of GFP–unc-22 hairpin dsRNA. (B) GFP fluorescence for pha-4::GFP transgenic worms treated with no dsRNA (no treat), or GFP–unc-22 hairpin dsRNA (GFPhp).

FIGURE 5.

Phenotypes of worms exposed to dsRNA hairpins. (A) Motility of wild-type (WT), pha-4::GFP, or pGFP transgenic worms after RNAi induced with GFP- unc-22 hairpin dsRNA (GFPhp). Three F1 animals were allowed to move freely for 11 min and photographed in the absence (no treat) or presence of GFP–unc-22 hairpin dsRNA. (B) GFP fluorescence for pha-4::GFP transgenic worms treated with no dsRNA (no treat), or GFP–unc-22 hairpin dsRNA (GFPhp).