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

Abstract

RNA interference is sequence-specific gene silencing triggered by double-stranded RNA. Systemic RNA interference is where double-stranded RNA, expressed or introduced into 1 cell, is transported to and initiates RNA interference in other cells. Systemic RNA interference is very efficient in Caenorhabditis elegans and genetic screens for systemic RNA interference-defective 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, dose-sensitive, systemic RNA interference defect and can be effectively rescued by SID-4 expression in target tissues only, implying a role in double-stranded RNA import. SID-4 and SID-3 (ACK-1 kinase) homologs interact in mammals and insects, suggesting that they may function in a common signaling pathway; however, a sid-3; sid-4 double mutants showed additive resistance to RNA interference, suggesting that these proteins likely interact with other signaling pathways as well. A bioinformatic screen coupled to RNA interference sensitivity tests identified 23 additional signaling components with weak RNA interference-defective phenotypes. These observations suggest that environmental conditions may modulate systemic RNA interference efficacy, and indeed, sid-3 and sid-4 are required for growth temperature effects on systemic RNA interference silencing efficiency.

Keywords: C. elegans; NCK-1; RNAi; dsRNA.

Fig. 1.

sid-4 encodes NCK-1, a noncatalytic region of tyrosine kinase adaptor protein. Schematic representation of protein-altering SNPs identified in ZK470.5 and their location in SID-4A and SID-4B (adapted from Mohamed and Chin-Sang 2011). sid-4A and sid-4B are produced by independent promoters and trans-spliced to SL1.

Fig. 2.

SID-4 isoform rescue of systemic RNAi. Representative images (top) of variable sur-5::GFP silencing. Quantification (bottom) of rescue of silencing in 3 independent lines for each SID-4 isoform. Scale bar: 0.1 mm

Fig. 3.

sid-4 mutants are partially defective for RNAi. Adult progeny of indicated sid mutant L4 hermaphrodites in the HC57 background (pharyngeal GFP; body wall muscle GFP; pharynx expressed gfp dsRNA) placed on bacteria expressing gfp dsRNA (GFP RNAi) or L440 empty-vector bacteria (control RNAi). All animals approximately 1 mm. sid-4 (ok694) and sid-4 (qt33) progeny showed indistinguishable penetrance and expressivity of systemic RNAi-silencing defects (Supplementary Fig. 1).

Fig. 4.

sid-4 and sid-3 have similar RNAi phenotypes. The adult progeny (F₁) of L4 animals (F₀) placed on RNAi foods were scored for RNAi sensitivity (fraction sensitive on each plate). Each circle represents the plate mean sensitivity for each F₀ (n = 10) with ≥150 F₁’s for each F₀. 100% sensitive (left) to 100% resistant (right).

Fig. 5.

sid-4 is a dose-dependent Sid. Wild-type and sid-4(ok694) young adults were injected with varying pal-1 dsRNA concentrations into 1 (open symbols) or both gonad arms (filled symbols) and then 6 h postinjection singled and allowed to lay eggs for 24 h. The fraction of hatched eggs for each injected adult (n ≥ 10) was scored 3 days later.

Fig. 6.

sid-4 is required in the importing tissue. Progeny of sid-4(ok694); [Ex: sid-4::gfp] animals were scored for bli-1 RNAi sensitivity and SID-4::GFP expression. a) A SID-4::GFP positive bli-1 RNAi-sensitive animal. Scale bar: 0.1 mm. b, b′, and b″) Confocal image of a bli-1 RNAi-sensitive animal showing SID-4 GFP expression in hyp7. In (b), the bright gut GFP obscures dim diffuse GFP in syncytial Hyp7, which (b′) is outlined in the enlarged gray-scale image that excludes the bright intestinal signal. b″) The same image without outlining. Scale bars: 25 and 5 µm. c) Intestine and d) pharyngeal SID-4::GFP expression in bli-1 RNAi-resistant animals.

Fig. 7.

Double Sid weak mutants show enhanced RNAi resistance. The F₁ adult progeny of F₀ L4 animals placed on RNAi foods were scored for RNAi sensitivity (fraction sensitive on each plate). Each circle represents the plate mean sensitivity for each F₀ (n = 10) with ≥40 F₁’s per F₀. 100% sensitive (left) to 100% resistant (right). t-test P-values: *<0.01, **<0.0005.

Fig. 8.

sid-3 and sid-3 temperature-dependent systemic RNAi. F₁ progeny of F₀ animals placed on unc-22 food at the indicated temperature were scored in 3 mM Levamisole for paralysis/twitching (sensitivity). Mean (large filled circle), 95% confidence interval (vertical line), and each F₀ plate mean (small unfilled circle) are shown for each genotype temperature. Distribution of 10 plate means for 2 independent trials is shown to the left and right of each mean.