FIG. 5.

RNAi occurs normally in DCR1 knockout cells. (A) Diagram of the DCR1 locus indicating the insertion of the Neo cassette to disrupt gene expression. The letters N and S indicate NcoI and SpeI restriction sites. The Neo3 cassette eliminates a portion of the helicase domain of DCR1. Arrows indicate primers used to amplify the sequence used in the knockout construct and in PCR assays to confirm knockout. (B) PCR assay to confirm DCR1 knockout in parental and hairpin-transformed lines. The primers indicated in panel A were used to amplify genomic DNA from DCR1 knockout cells (DCR1Δ) and their progeny, which were transformed with ATU1 and RPL21 hairpin constructs. The product from wild-type cells (WT) is 2 kb, while the knockout product (KO) is 4 kb. Parental lines contain deletions of the Neo cassette and therefore show products of different sizes (*). PCR on transformed DCR1 knockout progeny lines (A1 and A2, ATU1hp; R1, RPL21hp) produced only the 4-kb knockout product. (C) RNAi occurs normally in DCR1 knockout cells. Wild-type and DCR1 knockout cells transformed with ATU1 and RPL21 hairpin constructs were starved briefly, induced with 0.05 μg/ml Cd for 2 h, and then harvested. Northern blots were hybridized with probes for ATU1 and RPL21 to show mRNA degradation and with a probe for GRL8 as a loading control. ATU1 and RPL21 messages are reduced in both DCR knockout and wild-type transformants treated with Cd, and prominent degradation bands can be seen in the ATU1 samples. (D) DCR1 knockout cells produce small RNAs normally. Wild-type and DCR1 knockout cells transformed with the ATU1 hairpin construct were induced as in panel C, and RNA was harvested for small RNA Northern blotting. Hybridization with oligonucleotide probes complementary to the ATU1 hairpin detected the 23- to 24-nt RNAs in both wild-type and DCR1 knockout samples, indicating that DCR1 is not required for processing the hairpin RNA into small RNAs. 10bp lad., 10-bp ladder.