FIG. 6.

Knockdown of DCR2 by RNAi. (A) Diagram of the DCR2 gene. DEXD helicase and RNase III domains are represented by gray boxes. The regions used for hairpin constructs 1 and 2 are represented by joined parallel lines. The region used to probe for DCR2 mRNA is indicated by the dark line under the gene diagram. (B) Expression of DCR2 hairpins 1 and 2 leads to both induction and degradation of DCR2 mRNA. Cells transformed with DCR2hp1 (1A to 1D) and DCR2hp2 (2A and 2B) were starved briefly and then induced with 0.05 μg/ml Cd for 2 or 4 h. Northern blots were hybridized with a probe for DCR2 to determine mRNA levels and with a probe for SERH3 as a loading control. Degradation products (deg) are visible as smears in the DCR2hp2 samples and as distinct bands in the DCR2hp1 samples. (C) Preinitiation of RNAi of DCR2 in mating cells does not affect subsequent RNAi of a second target. Starved DCR2hp lines (2A-1, 2A-2, 2B-1) or control GRL8hp cells were either left untreated or induced for 2 h and then were washed to remove Cd. These cells were then mixed with SERH3hp cells and allowed to mate for 2 h. Then, the mating cultures were divided, and half were induced with Cd again to initiate RNAi of SERH3, while the other half continued mating untreated. −, matings of untreated cells; M, cells treated only after mating had begun; S (for “starved”), matings of preinduced cells that were not induced a second time; B (for “both”), matings of preinduced cells that received the second induction. Cells were harvested 2 h after the second induction. Northern blots were hybridized with probes to SERH3 and DCR2 to determine message degradation and with probes to GRL8 to show control RNAi was functioning and to RPL21 as a loading control.