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
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
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
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
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
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
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
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
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.
The Problem in Mammals Key: use 21-23 nt ds RNAs RNaseL eIF2 Figure 1 Bass, B.L (2001) Nature 411:428
RNAi in Mammalian Cells Figure 4 Elbashir, SM et al 2001 Nature 411:494
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!
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.
Analysis of a Signaling Pathway in Drosophila S2 Cells Figure 2 Clemmens, J.C. et al (2000) PNAS 97:6499
Analysis of a Signaling Pathway in Drosophila S2 Cells Figure 2 Clemmens, J.C. et al (2000) PNAS 97:6499
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.