Download The History of RNAi

The History of RNAi
John 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
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What genes allow a worm to wiggle?
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
gene
chromosome
marker
chromosome
marker
chromosome
protein: eliminate its functionality with an antibody
cell
cell
Kill the messenger!
mRNA:
cap
AAAA...
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.
Discovery
inject worms with dsRNA
corresponding to a gene
involved in wiggling (unc-22)
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Discovery
inject worms with dsRNA
corresponding to a gene
involved in wiggling (unc-22)
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conclusion: dsRNA triggers potent and specific gene silencing
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...
Re-examine Silencing in Plants
small dsRNAs -- appearance of both sense and antisense -correlated with gene silencing (disappearance of mRNA)
dsRNA hypothesis explained this plant phenomenon
Biochemistry to Examine Structure
21 nt long, dsRNA:
P
P
short interfering RNA: siRNA
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)
Dicer
dsRNA
siRNA
RISC
mRNA
cap
AAAA
mRNA
cap
AAAA
Discoveries to Tools
• dsRNA has been used for genome-wide screens in worms.
• siRNA have been used in mammalian tissue culture and
live mice.
Medline Citations
250
200
150
RNAi
siRNA
100
50
0
1998
1999
2000
2001
2002
Timeline
1990
cosuppression of purple color in plants
1998
dsRNA injection in worms
1999
short RNAs
identified in
plants
2000
RISC activity partially purified
2001
2002
siRNAs identified
RNAi
shown in
vitro
Dicer identified
RNAi used against HIV
genome-wide RNAi
screens begin