Distinct Roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA Silencing Pathways
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Distinct Roles for Distinct Roles for Drosophila Drosophila Dicer-1 and Dicer-2 in the Dicer-1 and Dicer-2 in the siRNA/miRNA Silencing siRNA/miRNA Silencing
PathwaysPathways
Lee, S.Y., Nakahara, K., Phan, J.W., Kim, K., He, Z., Sontheimer, E.J., Carthew, R.W.
(2004). Cell 117: 69-81
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What is RNAi?What is RNAi?
A cellular mechanism to regulate the expression of genes, mutant gene products and the replication of viruses
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(some) History of RNAi(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
19841990
19951998
2003
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How does it work?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
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Gratuitous RNAi MovieGratuitous RNAi Movie
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Another class of small RNA’s can Another class of small RNA’s can inhibit gene expression: miRNAinhibit 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
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Dicer, an Rnase III, processes Dicer, an Rnase III, processes both dsRNA and pre-miRNAboth 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
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Dicer also required downstream of Dicer also required downstream of dsRNA processingdsRNA 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
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Screen for RNAi mutants 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
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Dicer-2 functions uptstream & Dicer-2 functions uptstream & downstream of siRNA productiondownstream 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 mutantsImplies dcr-2 is required
for siRNA dependent mRNA degradation
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Dcr-1 is required for miRNA & Dcr-1 is required for miRNA & siRNA-dependent gene silencingsiRNA-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
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Dcr-1 is required for miRNA & Dcr-1 is required for miRNA & siRNA-dependent gene silencingsiRNA-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
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RNAi Pathway: To dateRNAi Pathway: To date
Dicer 2
DicerR2D2
DicerR2D2
What role does dcr-1 and dcr-2 play in the formation of a functional siRISC complex?
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Dcr-1/2 are required for Dcr-1/2 are required for siRISC formationsiRISC 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
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How do the Dicers recognize their How do the Dicers recognize their targets?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?
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DiscussionDiscussion• 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
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ConclusionConclusion• 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
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Future DirectionsFuture Directions
1. Make a double mutant
2. Assay mRNA/siRNA cleavage capabilities of Dcr-2
3. Does this complex function analogously in other systems?
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