The double-stranded RNA binding protein RDE-4 can act cell autonomously during feeding RNAi in C. elegans

Abstract

Long double-stranded RNA (dsRNA) can silence genes of matching sequence upon ingestion in many invertebrates and is therefore being developed as a pesticide. Such feeding RNA interference (RNAi) is best understood in the worm Caenorhabditis elegans, where the dsRNA-binding protein RDE-4 initiates silencing by recruiting an endonuclease to process long dsRNA into short dsRNA. These short dsRNAs are thought to move between cells because muscle-specific rescue of rde-4 using repetitive transgenes enables silencing in other tissues. Here, we extend this observation using additional promoters, report an inhibitory effect of repetitive transgenes, and discover conditions for cell-autonomous silencing in animals with tissue-specific rescue of rde-4. While expression of rde-4(+) in intestine, hypodermis, or neurons using a repetitive transgene can enable silencing also in unrescued tissues, silencing can be inhibited wihin tissues that express a repetitive transgene. Single-copy transgenes that express rde-4(+) in body-wall muscles or hypodermis, however, enable silencing selectively in the rescued tissue but not in other tissues. These results suggest that silencing by the movement of short dsRNA between cells is not an obligatory feature of feeding RNAi in C. elegans. We speculate that similar control of dsRNA movement could modulate tissue-specific silencing by feeding RNAi in other invertebrates.

Figure 1.

Expression of any repetitive transgene in a tissue can inhibit silencing by ingested dsRNA within that tissue. (A) Silencing by feeding RNAi of some endogenous genes is reduced in tissues expressing rde-4(+) or rde-1(+) from a repetitive transgene. Wild-type animals, mutant animals (rde-1(–), top or rde-4(–), bottom) or mutant animals with tissue-specific rescues in the body-wall muscles (Ex[Pmyo-3::rde(+)]) were fed dsRNA against unc-22 or unc-54 and the fractions of animals that showed silencing (fraction silenced) were determined. Asterisks indicate P < 0.01 (compared to wild-type animals). (B) Silencing of gfp in body-wall muscles that express RDE-4 from a repetitive transgene is reduced despite potent silencing in other rde-4(–) somatic tissues. Representative images of animals with gfp expression (black) in all somatic cells (Peft-3::gfp) in a wild-type background (left) or rde-4(–) background with rde-4(+) expressed in body-wall muscles (Ex[Pmyo-3::rde-4(+)) (right) that were fed bacteria that express dsRNA against gfp (gfp RNAi) are shown. Tissues that show reduced silencing (pharynx, muscles) are labelled, insets are brightfield images, and scale bar = 50 μm. Also see Supplementary Figure S1B and C and methods for details on generation of the inverted grey-scale images presented in all figures. (C and D) Silencing of a gene by ingested dsRNA within a tissue can be inhibited by the expression of a repetitive transgene of unrelated sequence within that tissue. (C) Silencing of bli-1 and dpy-7 is inhibited by the expression of gfp from a repetitive transgene in the hypodermis. Wild-type animals that express gfp alone in the hypodermis (Ex[Pnas-9::gfp]) from extrachromosomal repetitive DNA (array) were fed dsRNA against hypodermal genes (dpy-7 or bli-1, green). The fractions of animals either with or without the arrays that showed silencing (fraction silenced) were determined. (D) Silencing of unc-22 and unc-54 can be inhibited by the expression of DsRed from a repetitive transgene in body-wall muscles. Wild-type animals expressing DsRed in the body-wall muscle (Ex[Pmyo-3::DsRed]) from extrachromosomal repetitive DNA (array) were fed dsRNA against body-wall muscle genes (unc-22 or unc-54, magenta). Silencing was determined as in (C). Asterisks indicate P < 0.01 (compared to animals without array). (E) Expression of RDE-4 from a single-copy transgene within a tissue does not inhibit feeding RNAi in that tissue. rde-4(-) animals that express RDE-4 in the hypodermis from a single-copy transgene (Si[Pnas-9::rde-4(+)]) or that additionally express gfp in the hypodermis (Ex[Pnas-9::gfp]) from a repetitive transgene were fed dsRNA against dpy-7 or bli-1 and were analyzed as in (C). Asterisks indicate P < 0.01 (compared to animals without Ex[Pnas-9::gfp]). Error bars indicate 95% confidence intervals (CI), n > 23 animals. Also see Supplementary Figures S1 and S2.

Figure 2.

Tissue-specific rescues of RDE-4 from repetitive transgenes can enable silencing of genes in non-rescued somatic tissues. (A) Tissue-specific expression of RDE-4 but not RDE-1 from repetitive transgenes enables silencing of endogenous genes that function in mutant somatic tissues but not in mutant germline. Wild-type animals (gray), mutant animals (rde-1(–) or rde-4(–), white), and mutant animals with tissue-specific rescues (colors within worms) of rde-1 or rde-4 were fed dsRNA against genes expressed in somatic tissues (the body-wall muscles (unc-22, magenta), intestine (act-5, blue), hypodermis (dpy-7, green)), or in the germline (pos-1, par-1, or par-2, gray) and the fractions of animals that showed silencing (fraction silenced) were determined. Wild-type genes (rde-1 or rde-4) were expressed in the body-wall muscles (Ex[Pmyo-3::rde(+)], magenta), in the intestine (Ex[Psid-2::rde(+)], blue), in the hypodermis (Ex[Pwrt-2::rde(+)], green), or in neurons (Ex[Prgef-1::rde(+)], orange). Error bars indicate 95% CI and n > 24 animals. Also see Supplementary Table S1. Asterisks indicate P < 0.01 (compared to wild-type animals). (B) Tissue-specific rescue of rde-4 enables silencing of gfp in rde-4(–) somatic tissue but not in rde-4(–) germline. Representative images of animals with gfp expression (black) in all somatic and germline cells (Pgtbp-1::gtbp-1::gfp) in a wild-type background, rde-4(–) background, or rde-4(–) background with rde-4(+) expressed in body-wall muscles (Ex[Pmyo-3::rde-4(+)) that were fed control RNAi or gfp RNAi are shown. In all cases, 50 L4-staged animals were analysed and the majority phenotype (wild-type—100%, rde-4(–)—100%, rde-4(–);Ex[Pmyo-3::rde-4(+) control RNAi—100%, and rde-4(–);Ex[Pmyo-3::rde-4(+) gfp RNAi—68%) is shown. Insets are brightfield images and scale bar = 50 μm.

Figure 3.

Repetitive transgenes expressing RDE-4 from different promoters enable different spatial patterns of bli-1 silencing in rde-4(–) hypodermis. (A) A null mutation in bli-1 results in blisters that cover the entire body. A representative image of a bli-1 null mutant animal generated by Cas9-based genome editing is shown. Scale bar = 50 μm. (B) Feeding RNAi of bli-1 in wild-type animals results in blisters that cover part of the body. A representative image of a wild-type animal fed dsRNA against bli-1 (bli-1 RNAi). Scale bar = 50 μm. (C) Susceptibility to bli-1 feeding RNAi decreases from anterior to posterior hypodermis in wild-type animals. (top) Schematic of hypodermal sections (a through h) scored for blister formation. (bottom) Consensus relative frequency of blister formation in each hypodermal section of wild-type animals upon bli-1 feeding RNAi. The frequency ranged from 1.0 (black, a) to 0.06 (∼white, h). (D) The patterns of blisters that result from silencing of bli-1 in rde-4(–) hypodermis are different from the consensus blister pattern. Aggregate patterns of blister formation among animals that deviate from the consensus susceptibility order (consensus bli-1 RNAi susceptibility in (C)) for each strain (variant susceptibility, % variants) are shown. All strains being compared were normalized together (black, section with highest frequency of blisters in all strains; white, section with the lowest frequency of blisters in all strains). Schematic of worms indicate locations of variant blisters (thick black shading) on worms with rde-4(+) expressed in neurons (orange) or in body-wall muscles (magenta). Also see Supplementary Figure S4.

Figure 4.

Expression of RDE-4 within a tissue can restrict silencing by feeding RNAi to that tissue. (A) Expected outcomes of test to distinguish movement of short dsRNAs from other possibilities (e.g. misexpression of RDE-4) in animals with tissue-specific rescue of RDE-4 from a repetitive transgene. (Left) If RDE-4 is expressed only in neurons from the rgef-1 promoter (Prgef-1::rde-4(+)) in a rde-4(–); sid-1(–); Ex[Pmyo-3::sid-1(+)] background no silencing is expected in muscles, which can import dsRNA (have SID-1) but not process dsRNA (lack RDE-4). (Right) If RDE-4 is expressed in neurons and in muscles from the rgef-1 promoter (Prgef-1::rde-4(+)) in a rde-4(-); sid-1(–); Ex[Pmyo-3::sid-1(+)] background silencing can occur in muscles, which can import dsRNA (have SID-1) and process dsRNA (have RDE-4). See text for additional possibilities. (B) RDE-4 could be present in muscles when expressed under the rgef-1 promoter from a repetitive transgene. Wild-type animals, mutant animals (rde-4(–), sid-1(–), or rde-4(-);sid-1(–)) or mutant animals with rde-4(+) and/or sid-1(+) expressed from repetitive transgenes (as schematized in (A)) were fed dsRNA against a gene expressed in the body-wall muscle (unc-54) or in the hypodermis (bli-1) and the fractions of animals that showed silencing (fraction Unc or fraction Bli) were determined. Error bars indicate 95% CI, n > 24 animals and asterisks indicate P < 0.01 (compared to wild-type animals). (C) Expression of RDE-4 from a single-copy transgene reveals a requirement for RDE-4 within a tissue for silencing by ingested dsRNA in that tissue. Wild-type animals or rde-4(–) animals that express RDE-4 from a single-copy transgene in the body-wall muscle (Si[Pmyo-3::rde-4(+)]) or in the hypodermis (Si[Pnas-9::rde-4(+)]) were fed dsRNA against unc-22 (magenta), act-5 (blue) or bli-1 (green). Silencing was scored as in Figure 2A. Error bars indicate 95% CI, n > 24 animals and asterisks indicate P < 0.01 (compared to wild-type animals). Also see Supplementary Figure S7. (D) Model: Silencing by entry of ingested long dsRNA into each tissue could account for tissue-restricted silencing in animals with single-copy rescue of RDE-4. See text for details.