Coleopteran-specific StaufenC functions like Drosophila melanogaster Loquacious-PD in dsRNA processing.

In Drosophila melanogaster, PD isoform of the double-stranded RNA binding protein (dsRBP) Loquacious (Loqs-PD) facilitates dsRNA cleavage to siRNA by Dicer-2. StaufenC (StauC) was discovered as a coleopteran-specific dsRBP required for dsRNA processing in coleopteran insects. Here, we show that StauC is essential for the high RNAi efficiency observed in coleopterans. Knockdown of StauC but not the homologs of Loqs-PD and R2D2 evoked a long-lasting insensitivity to RNAi in the coleopteran cell line, Ledp-SL1. The dsRNA insensitivity induced by StauC knockdown could not be overcome merely by an increase in dose or time of exposure to dsRNA or expression of Loquacious or R2D2. Furthermore, StauC but not Loqs and R2D2 are required for processing of dsRNA into siRNA. StauC overexpression also partly restored the impaired RNAi caused by the knockdown of Loqs-PD in D. melanogaster Kc cells. However, StauC was unable to compensate for the loss-of-the function of Dcr-2 or R2D2. Overall, these data suggest that StauC functions like Lops-PD in processing dsRNA to siRNA.

Overexpression and purification of Dicer and accessory proteins for biochemical and structural studies.

The Dicer family of ribonucleases plays a key role in small RNA-based regulatory pathways by generating short dsRNA fragments that modulate expression of endogenous genes, or protect the host from invasive nucleic acids. Beginning with its initial discovery, biochemical characterization of Dicer has provided insight about its catalytic properties. However, a comprehensive understanding of how Dicer's domains contribute to substrate-specific recognition and catalysis is lacking. One reason for this void is the lack of high-resolution structural information for a metazoan Dicer in the apo- or substrate-bound state. Both biochemical and structural studies are facilitated by large amounts of highly purified, active protein, and Dicer enzymes have historically been recalcitrant to overexpression and purification. Here we describe optimized procedures for the large-scale expression of Dicer in baculovirus-infected insect cells. We then outline a three-step protocol for the purification of large amounts (3-4mg of Dicer per liter of insect cell culture) of highly purified and active Dicer protein, suitable for biochemical and structural studies. Our methods are general and are extended to enable overexpression, purification and biochemical characterization of accessory dsRNA binding proteins that interact with Dicer and modulate its catalytic activity.