Transcript Distinct Roles for Drosophila Dicer-1 and Dicer

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 RNAinduced 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 stemloop 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 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
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
Implies dcr-2 is required
for siRNA dependent
mRNA degradation
• Rapid degradation of bicoid
mRNA in wt, but not in dcr-2
mutants
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 bicoidmeasured 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
What role does dcr-1 and dcr-2
play in the formation of a
functional siRISC complex?
Dicer
R2D2
Dicer
R2D2
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
Imperfect basepairing? Low abundance? PAZ
domain vs. DExH domain?
• Dicer-2 processes dsRNA
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?