the power of “genetics” loss of function easy in yeast difficult in mammals powerful tool to...
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RNAi and the New Genetics
A. Why loss of function?B. Discovery of ds RNA interferenceC. RNAi MechanismsD. RNAi Mammalian Cells for KnockoutE. Applications in lower organisms
References:
**1. Bass, B. L. 2001. RNA interference. The short answer. Nature (London)411:428-9.2. Bernstein, E., A. A. Caudy, S. M. Hammond, and G. J. Hannon. 2001. Rolefor a bidentate ribonuclease in the initiation step of RNA interference. Nature(London) 409:363-6.3. Clemens, J. C., C. A. Worby, N. Simonson-Leff, M. Muda, T. Maehama, B.A. Hemmings, and J. E. Dixon. 2000. Use of double-stranded RNA interference inDrosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci U SA 97:6499-503.4. Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T.Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference incultured mammalian cells. Nature (London) 411:494-8.5. Hammond, S. M., E. Bernstein, D. Beach, and G. J. Hannon. 2000. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.Nature (London) 404:293-6.6. Ketting, R. F., S. E. Fischer, E. Bernstein, T. Sijen, G. J. Hannon, and R. H.Plasterk. 2001. Dicer functions in RNA interference and in synthesis of small RNAinvolved in developmental timing in C. elegans. Genes Dev 15:2654-9.7. Paddison, P. J., A. A. Caudy, and G. J. Hannon. 2002. Stable suppression ofgene expression by RNAi in mammalian cells. Proc Natl Acad Sci U S A 99:1443-8.
**8. Sharp, P. A. 2001. RNA interference--2001. Genes Dev 15:485-90.**9. Sharp, P. A. 1999. RNAi and double-strand RNA. Genes Dev 13:139-41.10. Shi, Y., and C. Mello. 1998. A CBP/p300 homolog specifies multipledifferentiation pathways in Caenorhabditis elegans. Genes Dev 12:943-55.11. Victor, M., Y. Bei, F. Gay, D. Calvo, C. Mello, and Y. Shi. 2002. HAT activityis essential for CBP-1-dependent transcription and differentiation inCaenorhabditis elegans. EMBO Rep 3:50-5.
**12. Zamore, P. D. 2001. RNA interference: listening to the sound of silence. NatStruct Biol 8:746-50.
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
Discovery of RNAi
•Sequence-specific destruction of mRNA
•Mediated by ds RNA (siRNA)
•Recruitment of conserved machinery
RNAi = RNA interference
siRNA = small interfering RNA
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
•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
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
Approach: Genetic and biochemical dissection
High specificityIsolation of ds RNA (function was previously unknown)Isolation of mutants defective in RNAiIsolation 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 vitroDevelopmental consequence in Dros. & C. Elegans
Figure 2
Hammond, S.M. et al2000 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
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
RISC= RNA Induced Silencing Complex
RNA-protein complex recruited by siRNA and to the mRNAUnknown components.
Triggers mRNA degradation in response to siRNA.
RNA is essential (MNase experiments).
Other components have been defined by genetics, but function is unknownE.g. RNA-dependent RNA polymerase.
RNAi Machinery (Continued)
eIF2RNaseL
The Problem in Mammals
Figure 1Bass, B.L (2001) Nature 411:428
Key: use 21-23 nt ds RNAs
Figure 4
Elbashir, SM et al2001 Nature 411:494
RNAi in 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!
Breakthrough for Mammalian Cells
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 2Clemmens, J.C. et al(2000) PNAS 97:6499
Analysis of a Signaling Pathway in Drosophila S2 Cells
Figure 2Clemmens, 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.