The History of RNAiJohn Doench

Insight and discovery are functionally separable. The one precedes the other. Insight can happen every day. Discovery does not. Insight takes more intelligence, but it is discovery that is rewarded...

Francis Crick

What genes allow a worm to wiggle?QuickTime™ and aGIF decompressorare needed to see this picture.

mutate genes at random

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Classical (Forward) Genetics

• Allows identification of genes involved in a given phenotype.

• Works great in yeast, worms, flies– small organisms– fast generation time

• Very important (necessary) genes can’t be found

Reverse Genetics

• Have a gene in hand (genome sequence, for example), and want to know what it does.

• Potentially applicable to all organisms: no breeding necessary.

DNA: homologous recombination (yeast, mice)

chromosome chromosomegene

marker

protein: eliminate its functionality with an antibody

cell cell

chromosome chromosomemarker

Kill the messenger!

cap AAAA...mRNA:

in vitro transcribe an antisense RNA:

cap AAAA...

mRNA can no longer be translated into a protein

prediction: sense RNA shouldn’t do anything...

Insight• Antisense technology was used in worms...

• Difficult to explain: sense and antisense RNA preparations are each sufficient to cause interference.

• Perhaps, the interfering RNA populations include some molecules with double-stranded character.

inject worms with dsRNAcorresponding to a geneinvolved in wiggling (unc-22)

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Discovery

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conclusion: dsRNA triggers potent and specific gene silencing

inject worms with dsRNAcorresponding to a geneinvolved in wiggling (unc-22)

Discovery

dsRNA Hypothesis explains other phenomena

• Attempts to overexpress a gene (chalcone synthase) by inserting multiple copies of that gene into the plant’s genome.

• Purple plants should become purpler...

small dsRNAs -- appearance of both sense and antisense -- correlated with gene silencing (disappearance of mRNA)

dsRNA hypothesis explained this plant phenomenon

Re-examine Silencing in Plants

Biochemistry to Examine Structure

short interfering RNA: siRNA

21 nt long, dsRNA:P

P

The Genomic Age

• Unusual structure of siRNA implicated RNAse III type enzyme, which had been characterized in E. coli.

• Search through the fly genome for proteins containing signature RNAse III motifs.

• Found a few candidates, and tested them biochemically for the ability to produce siRNA. Identified the protein, and named it Dicer.

RNA interference (RNAi)dsRNA

siRNA

Dicer

RISC

AAAAcapmRNA

AAAAcapmRNA

• dsRNA has been used for genome-wide screens in worms.

• siRNA have been used in mammalian tissue culture and live mice.

Discoveries to Tools

Medline Citations

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1998 1999 2000 2001 2002

RNAi

siRNA

Timeline

1998

1999

2000

2001

2002

1990 cosuppression of purple color in plants

dsRNA injection in worms

short RNAs identified in plants

RNAi shown in vitro

RISC activity partially purified

siRNAs identified Dicer identified

RNAi used against HIVgenome-wide RNAi screens begin