SID-4/NCK-1 is important for dsRNA import in Caenorhabditis elegans

Abstract

RNA interference (RNAi) is sequence-specific gene silencing triggered by double-stranded (ds)RNA. When dsRNA is expressed or introduced into one cell and is transported to and initiates RNAi in other cells, it is called systemic RNAi. Systemic RNAi is very efficient in C. elegans and genetic screens for systemic RNAi defective (Sid) mutants have identified RNA transporters (SID-1, SID-2 and SID-5) and a signaling protein (SID-3). Here we report that SID-4 is nck-1, a C. elegans NCK-like adaptor protein. sid-4 null mutations cause a weak, dosesensitive, systemic RNAi defect and can be effectively rescued by SID-4 expression in target tissues only, implying a role in dsRNA import. SID-4 and SID-3 (ACK-1 kinase) homologs interact in mammals and insects, suggesting they may function in a common signaling pathway, however, a sid-3; sid-4 double mutants showed additive resistance to RNAi, suggesting that these proteins likely interact with other signaling pathways as well. A bioinformatic screen coupled to RNAi sensitivity tests identified 23 additional signaling components with weak RNAi defective phenotypes. These observations suggest that environmental conditions may modulate systemic RNAi efficacy, and indeed, sid-3 and sid-4 are required for growth temperature effects on systemic RNAi silencing efficiency.* Partial Dpy's, Supplemental Figures Figure S1. Lineal relatedness of cells that fuse to form syncytial hypodermal cell hyp7.A) Lineage tree showing the cell divisions (vertical lines) leading to the seven early blastomeres that give rise to embryonic (E) or postembryonic (PE) cells that fuse with hyp7. The blue bar show the relative contributions to hyp7 and the green circles show the origins of pharyngeal muscle cells that express sid-4::gfp. B, C) Examples of extrachromosomal array transmission patterns that could generate the patterns observed in bli-1 (RNAi) resistant sid-4::gfp positive mosaic animals ( Figure 6). B) At the first division the array segregates to P1 but not to AB and at the second division segregates to EMS but not to P2. The resulting animal expresses sid-4::gfp in the intestine and cells of the posterior pharynx, but lacks sid-4::gfp in all hyp7 nuclei. C) At the first division the array segregates to AB but not to P1, at the second division segregates to ABa but not to ABp, and at a subsequent division segregates to ABar, ABarp, or ABarpp. The resulting animal expresses sid-4::gfp in cells of the anterior pharynx, but lacks sid-4::gfp expression in all or most hpy7 nuclei.

Integrated Regulation of Dopaminergic and Epigenetic Effectors of Neuroprotection in Parkinson’s Disease Models

“…We speculate that SID-3 may bind to DAT and SID-1 through alternative motifs and, when it is bound to SID-1, the activated NEDD4/WWP-1 E3 ligase is unable to interact with TNK2/SID-3 to target it for degradation. This is supported by evidence that the NCK-1/SID-4 protein, a SH2/SH3 domain-containing adaptor protein, is also required for SID-3-dependent RNAi silencing (49). A putative complex between SID-3 and SID-4 may represent a steric hinderance to NEDD4/WWP-1 interaction.…”

Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference

“…(8)) or in inhibition of silencing by extracellular dsRNA (18), the mechanism(s) of silencing disrupted in mutants from these screens are unclear. Nevertheless, these screens isolated genes required for import of dsRNA ( sid-1 (13), sid-2 (75), sid-3 (76), sid-4 (77), sid-5 (78)), or for silencing within target cells ( rde-10 (61,62), rde-11 (61,62)). We have constructed a screenable worm that could be used to isolate genes required for the biogenesis and/or export of dsRNA without confounding effects from repetitive transgenes expressed in target cells (Figure 4).…”