Gene-specific requirements and complex redundancy can arise from a single RNA regulatory network.
(A) Schematic (as in Fig. 1) depicting nrde-3 alleles. (B) Feeding RNAi of bli-1 and unc-22. Fractions silenced, numbers scored, comparisons, asterisks, and error bars are as in Fig. 1. Single mutants lacking NRDE-3 (nrde-3(jam205)) fail to silence bli-1 but not unc-22. Double mutants fail to silence both targets. (C and D) Mutual constraints among parameters required for a single RNA regulatory network to support experimental results. (C, left) Model for a single network of interactors that regulate all RNAi targets in somatic cells. All targets require import (SID-1) and processing (RDE-4 and RDE-1) of dsRNA. Branching after 1° siRNA processing results in four distinct paths (Nm, Nr, Om, Or) that together contribute to gene silencing, which could occur through co-transcriptional gene silencing (TGS) and/or post-transcriptional gene silencing (PTGS) mechanisms. (C, right) Representation of simulation workflow. First, random values between 0 and 2 were drawn for each of the four variables (Nm, Nr, Om, Or). Second, constraints were added based on the experimental results in Fig. 2B and Fig. 1D. Third, allowed values that satisfied all experimental conditions were culled. Of 100,000 sets of random values simulated (0 to 2 for Nm, Nr, Om, Or and 0 to 100 for the ratio of thresholds Tbli-1/Tunc-22), 145 were consistent with all observed responses to RNAi. These allowed numbers reveal the domain of parameter values that support the observed range of gene silencing outcomes using feeding RNAi. (D, left) The contribution of NRDE-3 via MUT-16 (Nm) versus that via RDE-10 (Nr) for different ratios of thresholds for bli-1 versus unc-22 silencing (Tbli-1/Tunc-22) are shown. (D, center and right) The relative contributions to silencing that require MUT-16 (Nm + Om, D, center) or NRDE-3 (Nm + Nr, D, right) do not frequently take extreme values and both support a low value for the ratio of thresholds (Tbli-1/Tunc-22 < ∼5.5 despite allowed values of up to 100). (E) Feeding RNAi of unc-22 assayed as in Fig. 1, but using aged plates, resulting in weaker RNAi. Animals that lack MUT-16 (mut-16(jam148)) have the most severe defect, followed by animals lacking RDE-10 (rde-10(jam206)), which is followed by animals lacking NRDE-3 (nrde-3(jam205)). (F) Feeding RNAi of unc-54 or dpy-7. Fractions silenced, numbers scored, comparisons, asterisks, and error bars are as in Fig. 1. Silencing of unc-54 showed a partial dependency on NRDE-3, while silencing of dpy-7 showed a partial dependency on MUT-16, suggesting that the quantitative requirement for a regulator can differ depending on the target.