2. The Power of “Genetics”
LOSS OF FUNCTION
Easy in yeast
Difficult in mammals
Powerful tool to address roles in developmental or signaling networks
Gene knockouts have been used to make disease models e.g. for cancer.
Gene knockouts; Dominant-Negatives; Antisense RNA
RNA Interference (RNAi)
Ability to block selective mRNA
Reverse Genetics
??Function of unknown genes in sequenced genomes
Powerful tool in cells and animals

3. Sequence-specific destruction of mRNA Mediated by ds RNA (siRNA)
Discovery of RNAi
RNAi = RNA interference
siRNA = small interfering RNA
Sequence-specific destruction of mRNA
Mediated by ds RNA (siRNA)
Recruitment of conserved machinery

4. RNAi and PTGS RNAi = RNA interference PTGS = Post Transcriptional
Gene Silencing
siRNA = small interfering RNA
Figure 2
Zamore,P.D. (2001)
Nat. Struc. Biol. 9:746

5. Accidental Discoveries (Or , what happened to the control?)
First discovered in C. Elegans
Both antisense and sense RNA interfered with genes
Found small double-stranded RNA (dsRNA)
Unique way to silence genes
Revolutionized C. Elegans Genetics
Transgene expression in plants
Unexpected silencing of target and endogenous gene
Termed Post-Transcriptional Gene Silencing (PTGS)
Thought to be obscure plant phenomenon
A conserved machinery from worms to man

6. RNAi Pathway Dicer RNAi = RNA interference
siRNA = small interfering RNA
siRNP = small interfering Ribonucleoprotein
RISC = RNA Induced Silencing Complex
Figure 1
Zamore,P.D. (2001)
Nat. Struc. Biol. 9:746

7. RNAi Machinery Approach: Genetic and biochemical dissection
High specificity
Isolation of ds RNA (function was previously unknown)
Isolation of mutants defective in RNAi
Isolation of extracts that recapitulate RNAi in vitro.
Dicer
Nuclease that cuts both strands in ds RNA to 21 to 23 nt.
Processive--no larger intermediates.
Found in Drosophila, C.Elegans, Mammals, plants, etc.
Loss of dicer: loss of silencing, processing in vitro
Developmental consequence in Dros. & C. Elegans

8. RNAi Specificity Drosophila S2 cells with ds RNA
Cyclin E or Lac Z ds RNAs
Add indicated RNA substrates
Antisense also degraded
Exquisite specificity
Figure 2
Hammond, S.M. et al
2000 Nature 404:293

9. RNAi Pathway Dicer RNAi = RNA interference
siRNA = small interfering RNA
siRNP = small interfering Ribonucleoprotein
RISC = RNA Induced Silencing Complex
Figure 1
Zamore,P.D. (2001)
Nat. Struc. Biol. 9:746

10. RNAi Machinery (Continued)
RISC= RNA Induced Silencing Complex
RNA-protein complex recruited by siRNA and to the mRNA
Unknown components.
Triggers mRNA degradation in response to siRNA.
RNA is essential (MNase experiments).
Other components have been defined by genetics, but function is unknown
E.g. RNA-dependent RNA polymerase.

11. The Problem in Mammals Key: use 21-23 nt ds RNAs RNaseL eIF2 Figure 1
Bass, B.L (2001)
Nature 411:428

12. RNAi in Mammalian Cells
Figure 4
Elbashir, SM et al
2001 Nature 411:494

13. Breakthrough for Mammalian Cells
Excellent application for cell-based models.
Block PKR to allow more efficient and longer ds RNA.
Dicer also used in mammalian cells.
siRNA to dicer blocks RNAi.
Conserved machinery!

14. Examples of RNAi Applications
C. Elegans and p300 Function
p300 ortholog is cbp-1
RNAi reveals that loss of p300 gives
endoderm and mesoderm developmental defects,
but neuronal differentiation is not affected.
Rescue requires HAT activity.

15. Analysis of a Signaling Pathway in Drosophila S2 Cells
Figure 2
Clemmens, J.C. et al
(2000) PNAS 97:6499

16. Analysis of a Signaling Pathway in Drosophila S2 Cells
Figure 2
Clemmens, J.C. et al
(2000) PNAS 97:6499

17. Conclusions
Begun in worms, flies, and plants--as an accidental observation.
General applications in mammalian cells.
Powerful for analyzing unknown genes in sequenced genomes.
Reverse genetics in cell-based and animal models.