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RNA interference is a process whereby a gene is silenced by double stranded RNA, as a result of sequence specific degradation of messenger RNA. Gene silencing in plants takes place at both transcriptional and posttranscriptional levels but only posttranscriptional RNAi occurs in animals. A distinguishing feature of RNAi in both plants and animals is the presence of 22 nucleotides long RNAs which are homologous to the gene being silenced. A multicomponent complex, RISC, is an effector nuclease in posttranscriptional gene silencing. With the guidance of the 22 nucleotide sequences processed from dsRNAs, RISC specifically targets and degrades mRNA. This study describes the identification of a distinct enzyme, DICER which can produce putative guide RNAs.

Extracts prepared from Drosophila embryos and Drosophila S2 cells were used to produce guide sequences during in vitro RNAi. Biochemical fractionation was performed to test whether RISC and RNAi-initiating enzyme activities were separate. RISC was isolated from Drosophila S2 cells which were transfected with dsRNA to initiate in vivo RNAi. After high-speed centrifugation, supernatant remained the activity that produced 22-nt guide sequences. RISC, on the other hand, was mostly removed from extracts, suggesting that these two enzyme activities were distinct.

The dsRNA specific RNase III family members have several types according to genome analysis of Drosophila and Caenorhabditis elegans. Class I has a single RNase III domain and a single DSRNA-binding domain. Drosha, a class 2 example, has two RNAse domains (RIIIa and RIIIb) and a single dsRBD. A third class consisted of two RNase III domains and an amino-terminal helicase domain (CG4792 and CG6493 in Drosophila).

S2 cells were transfected with dsRNA and lysed in immunoprecipitation buffer. Following centrifugation, supernatant that contained T7 epitope tagged Drosha and a class III nuclease (CG4792), was added to T7 antibody-agarose beads, and Drosha and CG4792 were purified from the extract. The treatment of dsRNA with immunoprecipitate of CG4792 resulted in the production of ~22-nt fragments. Treatment with Drosha and with immunoprecipitate of homeless gene (which does not contain any RNase III domains) showed negative results.

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