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.