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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?. - PowerPoint PPT Presentation
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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
What is RNAi?What is RNAi?
A cellular mechanism to regulate the expression of genes, mutant gene products and the replication of viruses
(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
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
Gratuitous RNAi MovieGratuitous RNAi Movie
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
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
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
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
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
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
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
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?
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
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?
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
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
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?