RNA interference: RISC assessment

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Dcr-1 and Dcr-2 have distinct but overlapping roles in the siRNA and miRNA silencing pathways.

The RNase III Dicer enzyme processes RNAs into small interfering (si)RNAs and micro (mi)RNAs. These small RNAs then function as guides for RISC (RNA-induced silencing complex) to cleave messenger RNA (siRNA) or block its translation (miRNA). New insights into RISC formation, and the role of Dicer in this process, now come from two studies published in Cell.

In the first paper, the groups of Sontheimer and Carthew used a sophisticated genetic screen to identify Drosophila eye mutants with either enhanced or reduced gene silencing — or RNA interference (RNAi) — activity. And among the mutated loci were dicer-1 (dcr-1) and dicer-2 (dcr-2), which encode the two Dicer enzymes that are present in Drosophila.

dcr-2 mutants had reduced siRNA levels and a complete RNAi defect in the eye and in the female germline, which indicates that Dcr-2 is important for dsRNA processing into siRNAs.

When the authors injected Drosophila eggs with a pre-processed siRNA that corresponded to the bicoid gene, the level of bicoid transcript was reduced in wild-type eggs, but did not change significantly in mutant dcr-2 eggs due to a defective RNAi response. This means that Dcr-2 must also function downstream of siRNA production in the RNAi pathway.

The dcr-1 RNAi phenotype was less severe than that of dcr-2 and siRNA levels were normal, which is consistent with Dcr-2 being the major dsRNA-processing enzyme. So how can one explain the partial RNAi defect? dcr-1-mutant eggs injected with pre-processed siRNA complementary to bicoid transcripts contained sixfold more bicoid mRNA compared with wild-type eggs. This shows that Dcr-1 also has a downstream function in siRNA-dependent RNAi.

dcr-1 mutants also showed some developmental defects, which hints at a role for Dcr-1 in the miRNA-mediated silencing pathway. Dcr-1 is indeed essential for miRNA production, as miRNA levels in dcr-1-mutant eggs were undetectable. And, using a genetic assay for miRNA-mediated gene silencing, the authors showed that functional dcr-1, but not dcr-2, is necessary for this process. So Dcr-1 functions in both siRNA- and miRNA-mediated gene silencing.

Reporting in their second paper, the groups identified an assembly process incorporating siRNAs into active RISC. This assembly involved formation of three distinct complexes — R1, R2 and R3. These complexes assemble in the presence of siRNAs in extracts derived from wild-type flies, but not from dcr-2-null mutants.

Partially purified fractions that could form the R1 complex have dsRNA-processing, but not mRNA-cleavage, activity. In a pulse-chase experiment, R1 complexes were identified in R2 and R3 complexes, which suggests that R1 might be an initiating complex in the RISC-assembly pathway. And, as shown in UV-crosslinking studies, Dcr-2 associates directly with siRNAs in R1, R2 and R3.

The R2 complex remains to be characterized but is thought to be an intermediate complex that forms the R3 complex. mRNA-cleavage activity coincided precisely with R3, which suggests that this is a functional RISC. Indeed, in the presence of siRNA, R3 binds specifically to complementary target mRNAs but not to unrelated mRNAs. Other known RISC-associated factors were also present in the R3 fractions, and co-purified as a partially preformed 'holo-RISC' in the absence of siRNAs.

Given that Dcr-2 was present in the active R3 complex, and both studies showed that siRNAs were unable to associate with RISC in the presence of dcr-2-mutant-embryo lysate, this indicates that Dcr-2 is necessary for the siRNA-specific assembly of functional RISC. In the presence of dcr-1-mutant lysate, R1 was formed, but R2 and R3 could not be detected — so Dcr-1 might be necessary for later steps in the assembly pathway.

Dcr-1 and Dcr-2 have distinct but overlapping roles in the siRNA and miRNA silencing pathways. Dicers are required for functional RISC assembly and, in Drosophila, Dcr-1 and Dcr-2 might direct the assembly of functionally different RISCs.

Arianne Heinrichs

References

Lee, Y. S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69–81 (2004). | Article | PubMed |
Pham, J. W. et al. A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila. Cell 117, 83–94 (2004). | Article | PubMed |