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?