Distinct Roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA Silencing Pathways Lee, S.Y., Nakahara, K., Phan, J.W., Kim, K., He, Z., Sontheimer, E.J., Carthew, R.W. (2004). Cell 117: 69-81

What is RNAi? A cellular mechanism to regulate the expression of genes, mutant gene products and the replication of viruses

(some) History of RNAi 1984: Stout & Caskey show antisense RNA can be used to silence gene expression in Mammalian tissue cultures 1990: Fire & Moerman show antisense RNA can disrupt myofilament protein encoding genes 1995: Guo & Kemphues accidentally discover that sense RNA can is as effective as antisense RNA in gene silencing 1998: Mello & Fire illustrate that dsRNA is the agent that leads to potent and specific genetic interference…not ssRNA 2003: Ahringer & Kamath unveil the results of a genome-wide RNAi screen

How does it work? Long dsRNA fragments enter the cell & are diced into small (21-23 nt) fragments known as siRNA by Dicer siRNA associate with RNA- induced silencing complex (RISC); become unwound + activated Bind complementary mRNA mRNA is cleaved by RISC

Gratuitous RNAi Movie

Another class of small RNA’s can inhibit gene expression: miRNA Processed from stem- loop RNA precursors Play roles in growth and development Block translation of mRNA into protein Act as a guide for a multiprotein complex, miRISC

Dicer, an Rnase III, processes both dsRNA and pre-miRNA Dicer Dicer makes staggered cuts (3’ overhangs) to form siRNA Processes miRNA Dicer mutants are defective for both transcript degradation and translational repression Implies a dual role for Dicer in miRNA & dsRNA processing

Dicer also required downstream of dsRNA processing depletion of dicer results in reduced effectiveness of injected siRNA Dicer binds to components of RISC (R2D2) & binds tightly to siRNA Role of Dicer in siRISC is not well characterized… The authors took a genetic approach to study Dicer function in Drosophila

Screen for RNAi mutants Engineered Drosophila strain to express an eye-specific hairpin dsRNA corresponding to an exon of White Red eyes to pale orange Screened for enhanced (white) or suppressed (red) phenotypes Identified 15 loci that led to stronger pigmentation phenotype 1 locus had a complementation group of 39 alleles Mapped locus to Dicer-2 Dicer-2 homozygous suppressor

Dicer-2 functions uptstream & downstream of siRNA production Dcr-2 mutants show marked decrease in siRNA levels Implies dcr-2 required for dsRNA processing Injected eggs with dsRNA corresponding to bicoid Rapid degradation of bicoid mRNA in wt, but not in dcr-2 mutants Implies dcr-2 is required for siRNA dependent mRNA degradation

Dcr-1 is required for miRNA & siRNA-dependent gene silencing Dicer-1 mutants have normal levels of wIR siRNAs (dsRNA) Injected dicer-1 mutant eggs with either dsRNA or siRNA complementary to bicoid- measured bicoid expression Dcr-1 eggs showed impaired RNAi response to dsRNA and siRNA

Dcr-1 is required for miRNA & siRNA-dependent gene silencing Dcr-1 is required for the formation of mature miRNAs No detectable mature miRNA Dicer-1 is primarily for miRNA production, whereas Dicer-2 is critical for siRNA production

RNAi Pathway: To date Dicer 2 Dicer R2D2 Dicer R2D2 What role does dcr-1 and dcr-2 play in the formation of a functional siRISC complex?

Dcr-1/2 are required for siRISC formation R1: Dicer-2 and R2D2 proteins bound to siRNAs R2: intermediate complex that links R1 to R3 R3: siRISC complex that is capable of cleaving cognate mRNA *material too large for the gel to resolve

How do the Dicers recognize their targets? Dicer-1 processes pre-miRNA Dicer-2 processes dsRNA Imperfect basepairing? Low abundance? PAZ domain vs. DExH domain? Perfect basepairing? High abundance? DExH (helicase domain) vs. PAZ domain?

Discussion Dicer-1 is not required to form a stable R1 complex, but is required for the intermediate R2 complex synthesis Dicer-2 is required for the formation of a stable siRISC complex Dicer-1 is required for unwinding dsRNA in the siRISC complex (no helicase domain in Dicer-2) Dicer-2 may cleave siRNA/mRNA duplex

Conclusion Dcr-1, not Dcr-2, is required for gene silencing by miRNAs Dcr-2 is required for efficient dsRNA processing (but weak phenotype) Dcr-2 and Dcr-1 may have some redundant properties Loss of Dcr-1 has effects on growth and patterning; derepresses miRNA target genes

Future Directions 1.Make a double mutant 2.Assay mRNA/siRNA cleavage capabilities of Dcr-2 3.Does this complex function analogously in other systems?