Figure 4. RDE-12 Binds Target mRNA during RNAi and Localizes to Cytoplasmic and Perinuclear Foci(A) RDE-12 associates with target mRNA during RNAi. Bar graph showing qRT-PCR analysis of
sel-1 mRNA recovered from rabbit immunoglobulin G orRDE-12 IP from wild-type or
rde-1(
ne300) mutant lysates after control (cont) or
sel-1(RNAi) (as indicated). Error bars indicate the SD calculated from three technical replicates.(B) Fluorescence micrograph showing GFP::RDE-12 (green) is visible throughout the cytoplasm and in cytoplasmic and perinuclear foci, while PGL-1::mRFP(red) [29] is localized exclusively in the germline blastomere P4 (indicated). Colocalization in P4 appears (yellow) in this merged image.(C and D) Confocal images of GFP::RDE-12 (green) and nuclear pore complexes (NPC; red) stained with GFP and nucleoporin-specific antibody mAb414, respectively. A single confocal section of an
w50-cell-stage embryo (C) shows cytoplasmic and perinuclear foci of GFP::RDE-12 in somatic cells and enrichedin the two primordial germ cells (Z2 and Z3, indicated). Arrowheads show examples of perinuclear localization of GFP::RDE-12 in somatic and germ cells. A 3D projection of confocal images from a 24-cell-stage embryo is shown in (D).(E) GFP::RDE-12 fails to colocalize with PATR-1. A 3D projection of confocal images from a 36-cell-embryo showing GFP::RDE-12 (green), mCherry::PATR-1(red), and DNA (blue).(F) Schematic model illustrating potential functions and interactions for RDE-12 during RNAi. A nuclear envelope and pore (NPC) are drawn at the base of the diagram with a P granule (light-blue sphere) docked above. A mutator focus (dark-blue sphere) is located in the top part of P granule, where secondary siRNAs are produced. RDE-12 is shown engaged at the pore through its FG domains. Dashed arrows indicate potential functions for RDE-12.Scale bars represent 10 mm (B-E). See also Figure S1 and Movies S1 and S2.