3. 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

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

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

6. 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 Accidental Discoveries (Or, what happened to the control?) 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

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

8. 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. RNAi Machinery 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

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

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

11. 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. RNAi Machinery (Continued)

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

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

14. 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! Breakthrough for Mammalian Cells

15. 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.

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

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

18. 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.