Figure 3. RNA silencing at one repeat... | Rockefeller University Press

Figure 3.

$Figure 3. RNA silencing at one repeat locus can be independent of other repetitive loci or homologous single-copy loci. (A) Schematic of experiment to test if a cell that silences one repeat is likely to also silence another. SUR-5::GFP and nuclear-localized DsRed (nls::DsRED) generated from two repetitive transgenes that lack sequence homology are both expected to localize within intestinal nuclei but be expressed from different promoters (Psur-5 and Psid-2). (B and C) A repeat locus can be silenced independent of another within a cell. Intestinal cells with an integrated repetitive transgene that expresses nuclear-localized GFP (SUR-5::GFP) and an extrachromosomal repetitive transgene that expresses nuclear-localized DsRed (nls::DsRED) were analyzed. (B) Bright-field (top left), GFP fluorescence (top right), DsRed fluorescence (bottom left), and a merged fluorescence image (bottom right) of a representative worm are shown. Arrowhead in merged image indicates a cell that shows silencing of GFP, but not of DsRED. Bars, 100 µm. (C) Frequency of cells with different expression states is consistent with independent silencing of two different repetitive loci. Venn diagram showing number of cells with dim GFP (SUR-5::GFP), dim DsRed (nls::DsRed), and dim DsRed as well as dim GFP. The fraction of cells with dim DsRed as well as dim GFP (30/224) is not significantly different from that expected for chance co-occurrence when dim DsRed is independent of dim GFP (23/224; P = 0.27, n = 224 cells). (D) Schematic of experiment to test if silencing of a repeat locus can spread to another homologous single-copy locus within a cell. SUR-5::GFP and cytoplasmic GFP (GFP) generated from a repetitive transgene and a single-copy transgene that share some sequence homology (gfp) are expected to localize to nuclei and the cytoplasm, respectively, within intestinal cells but be expressed from different promoters (Psur-5 and Peft-3). (E) Silencing of a repeat locus can be independent of a homologous single-copy locus in an eri-1(−) background. Representative bright-field (left) and fluorescent images showing GFP expression (black) in intestinal cells (right) of an animal with a repetitive transgene that expresses nuclear-localized GFP (SUR-5::GFP, top), of an animal with a single-copy transgene that expresses cytoplasmic GFP (GFP, middle), and of an animal with both transgenes (SUR-5::GFP; GFP, bottom) are shown. Arrowhead in animal with both transgenes indicates a cell that shows silencing of SUR-5::GFP, but not of GFP. Bars, 100 µm.$

RNA silencing at one repeat locus can be independent of other repetitive loci or homologous single-copy loci. (A) Schematic of experiment to test if a cell that silences one repeat is likely to also silence another. SUR-5::GFP and nuclear-localized DsRed (nls::DsRED) generated from two repetitive transgenes that lack sequence homology are both expected to localize within intestinal nuclei but be expressed from different promoters (Psur-5 and Psid-2). (B and C) A repeat locus can be silenced independent of another within a cell. Intestinal cells with an integrated repetitive transgene that expresses nuclear-localized GFP (SUR-5::GFP) and an extrachromosomal repetitive transgene that expresses nuclear-localized DsRed (nls::DsRED) were analyzed. (B) Bright-field (top left), GFP fluorescence (top right), DsRed fluorescence (bottom left), and a merged fluorescence image (bottom right) of a representative worm are shown. Arrowhead in merged image indicates a cell that shows silencing of GFP, but not of DsRED. Bars, 100 µm. (C) Frequency of cells with different expression states is consistent with independent silencing of two different repetitive loci. Venn diagram showing number of cells with dim GFP (SUR-5::GFP), dim DsRed (nls::DsRed), and dim DsRed as well as dim GFP. The fraction of cells with dim DsRed as well as dim GFP (30/224) is not significantly different from that expected for chance co-occurrence when dim DsRed is independent of dim GFP (23/224; P = 0.27, n = 224 cells). (D) Schematic of experiment to test if silencing of a repeat locus can spread to another homologous single-copy locus within a cell. SUR-5::GFP and cytoplasmic GFP (GFP) generated from a repetitive transgene and a single-copy transgene that share some sequence homology (gfp) are expected to localize to nuclei and the cytoplasm, respectively, within intestinal cells but be expressed from different promoters (Psur-5 and Peft-3). (E) Silencing of a repeat locus can be independent of a homologous single-copy locus in an eri-1(−) background. Representative bright-field (left) and fluorescent images showing GFP expression (black) in intestinal cells (right) of an animal with a repetitive transgene that expresses nuclear-localized GFP (SUR-5::GFP, top), of an animal with a single-copy transgene that expresses cytoplasmic GFP (GFP, middle), and of an animal with both transgenes (SUR-5::GFP; GFP, bottom) are shown. Arrowhead in animal with both transgenes indicates a cell that shows silencing of SUR-5::GFP, but not of GFP. Bars, 100 µm.

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