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Distinct Roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA Silencing Pathways

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?.

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Distinct Roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA Silencing Pathways

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  1. 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

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

  3. (some) History of RNAi 1984 1990 1995 1998 2003 • 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

  4. 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

  5. Gratuitous RNAi Movie

  6. 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

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

  8. 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

  9. Engineered Drosophila strain to express an eye-specific hairpin dsRNA corresponding to an exon of White Red eyes to pale orange Screen for RNAi mutants • 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

  10. 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 Dicer-2 functions uptstream & downstream of siRNA production

  11. 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

  12. 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

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

  14. 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

  15. Perfect basepairing? High abundance? DExH (helicase domain) vs. PAZ domain? 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?

  16. 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

  17. 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

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

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