Biochemical Analysis of the Drosophila RNAi Pathway
RNA interference is post-transcriptional gene silencing mediated by (21-26 nt) miRNAs and siRNAs. In Drosophila, the RNase III enzymes Dicer-1 and Dicer-2 generate miRNAs and siRNAs, respectively. Nascent miRNA and siRNA duplexes are assembled into distinct RNA induced silencing complexes termed miRISC and siRISC, of which AGO1 and AGO2 are the respective catalytic subunits. My dissertation project is focused on identifying new RNAi components and understanding mechanisms of RISC assembly by biochemical reconstitution. Our group previously identified a novel dsRNA-binding protein named R2D2 which functioned in complex with Dicer-2 to process dsRNA into siRNA. Only the Dicer-2/R2D2 complex, but neither Dicer-2 nor R2D2 alone, efficiently interact with duplex siRNA. Furthermore, the tandem dsRNA binding domains of R2D2 are required for siRNA binding. Therefore, although R2D2 is dispensable for siRNA production, it is required for incorporating siRNA onto the siRISC complex. Generation of recombinant AGO2 protein is essential for in vitro reconstitution of the RNAi pathway. We believe that the unique poly glutamine repeat region of fly AGO2 may be problematic for expression. Thus, a series of truncated AGO2 baculoviruses that remove some or all polyQ repeats of AGO2 were generated. Co-expression with AGO1 increases the expression level of AGO2 by at least 10 fold. Affinity purified full length and one truncated form of AGO2 show minimal RISC activity, i.e. could be programmed with single stranded siRNA and perform sequence specific cleavage of mRNA. Most interestingly, adding purified recombinant Dicer-2/R2D2 complex to recombinant Ago2 generated dsRNA and siRNA initiated RISC activity. Catalytic mutant of Ago2 is unable to reconstitute RISC activity with recombinant Dicer-2/R2D2 complex, showing that the RISC activity is specific. Therefore, the three component system, Dicer-2, R2D2, and Ago2, can reconstitute the RNAi pathway of Drosophila. By a bioinformatics approach, a novel protein named Loquacious (Loqs) was identified with considerable sequence homology to R2D2. Loqs and Dicer-1 interact with each other by co-immunoprecipitation in S2 cell extract. Recombinant Loqs could enhance miRNA production by Dicer-1 by increasing its affinity for the pre-miRNA substrate. Furthermore, depleting Loqs or Dicer-1 by dsRNA knockdown resulted in reduction of the miRNA-generating activity and accumulation of pre-miRNA in S2 cells. To study the physiological function of loqs in flies, we obtained a piggyback (PB) fly strain in which the PB transposon was inserted into the first exon and before the translation start site of loqs gene. Pre-miRNAs accumulate in the loqs PB flies, indicating they are defective for miRNA biogenesis. However, while both siRISC and miRISC activities are greatly reduced in dcr-1 null extract, these activities are not affected in loqs null extract, indicating that loqs is not essential for miRISC assembly. To test whether the known components are sufficient to reconstitute the miRNA pathway, recombinant AGO1 protein was expressed using the insect cell expression system. It is generally believed that siRISC slices, whereas miRISC represses translation of cognate mRNA in animals. However, recombinant AGO1 can be programmed by single stranded miRNA into a minimal miRISC and sequence specifically cleaves complementary mRNA in vitro. Furthermore, the catalytic activity of AGO1 is dependent on the consensus catalytic "DDH" motif. My present studies suggest that recombinant Dicer-1, Loqs and AGO1 are not sufficient to reconstitute the miRNA pathway, indicating that there are other unknown components to be discovered.