Figure 1: RSD-3 is required for systemic RNAi not only in germ cells but also in somatic cells. | Scientific Reports

Figure 1: RSD-3 is required for systemic RNAi not only in germ cells but also in somatic cells.

From: Endomembrane-associated RSD-3 is important for RNAi induced by extracellular silencing RNA in both somatic and germ cells of Caenorhabditis elegans

Figure 1

(a) Upper: genomic structure of rsd-3. Black boxes indicate coding exons. Deletion regions of tm9006, tm6416 and tm6420, which were isolated in this study, and the Tc1 insertion site in pk2013 are indicated. Lower: Schematic representation of human epsinR and RSD-3 proteins. Protein domains and motifs are indicated. γ-ear/GAE indicates ear domain of the γ-subunit of AP-1 (adaptor protein-1) and the γ-adaptin ear (GAE) domain of GGA (Golgi-localized, Gamma-ear-containing, Arf-binding protein). AP-1 and GGA are both clathrin adaptors. Corresponding deletion regions of tm9006, tm6416 and tm6420 are indicated. (b) Newly isolated rsd-3 mutants show resistance to germline RNAi induced by pos-1 dsRNA feeding as rsd-3(pk2013). Bars represent the percentage of hatched progeny. (c,d) rsd-3 mutants show resistance to bli-3 feeding RNAi. (c) L1 larvae were placed on plates seeded with bacteria without dsRNA (mock) or bacteria expressing bli-3 dsRNA, and 48 hr later photographed. Wild type animals that were fed on food with bli-3 dsRNA showed severe molting defects and resulted in larval arrest, whereas almost all rsd-3(tm9006) worms reached adulthood. A portion of rsd-3(tm9006) worms showed mild cuticle blister formation (inset, arrowhead). Scale bars, 500 μm. (d) Bars represent the percentage of animals reached adulthood. Expression of RSD-3::GFP under the control of the rsd-3 promoter (see Fig. 2a for a schematic representation of the construct) rescues the phenotype of rsd-3(tm9006). (e) rsd-3 mutants show resistance to unc-22 feeding RNAi. Bars represent the percentage of twitching animals. (f) rsd-3 mutants show resistance to elt-2 feeding RNAi. Bars represent the percentage of animals reached adulthood. (g) rsd-3(tm9006) worms show resistance to transgene-induced systemic RNAi. Expression of gfp hairpin RNA and mCherry in the neurons induces silencing of GFP signal (LET-858::GFP) in most cells of wild type animals, but not in rsd-3(tm9006) background (upper panels). snb-1p::mCherry indicates the neurons that express gfp hairpin RNA (lower panels). Scale bars, 100 μm. Data are shown as mean ± SEM of three separate experiments. *P < 0.001 (Student’s t-test, two-tailed).

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