Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway.

Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). Here we show that R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer alone can initiate RISC assembly, indicating that other factors are dispensable for initiation.

The Making of an siRNA.

An array of gene silencing pathways share a common early step: Dicer cleaves double-stranded RNA (dsRNA) into approximately 20-25 nucleotide fragments that direct the silencing machinery to specific targets. A recent report in Cell reveals how Dicer's two RNase III domains collaborate during dsRNA processing and sets the stage for a deeper understanding of Dicer's roles in later phases of silencing complex assembly.

Thermodynamic and structural characterization of 2'-nitrogen-modified RNA duplexes.

2'-aminonucleosides are commonly used as sites of post-synthetic chemical modification within nucleic acids. As part of a larger cross-linking strategy, we appended alkyl groups onto the N2' position of 2'-amino-modified RNAs via 2'-ureido and 2'-amido linkages. We have characterized the thermodynamics of 2'-amino, 2'-alkylamido and 2'-alkylureido-modified RNA duplexes and show that 2'-ureido-modified RNAs are significantly more stable than analogous 2'-amido-modified RNAs.

A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila.

We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex.

Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways.

The RNase III enzyme Dicer processes RNA into siRNAs and miRNAs, which direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). We have characterized mutations in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of miRNA precursors, whereas dicer-2 mutants are defective for processing siRNA precursors.