The History of RNAi - Whitehead Institute

The History of RNAi - Whitehead Institute

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

mutate genes at random QuickTime and aGIF decompressorare needed to see this picture. QuickTime and aGIF decompressorare needed to see this picture. QuickTime and aGIF decompressorare needed to see this picture. QuickTime and aGIF decompressorare needed to see this picture. QuickTime and aGIF decompressorare needed to see this picture. 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 cant 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 shouldnt 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) QuickTime and aGIF decompressorare needed to see this picture. Discovery inject worms with dsRNA

corresponding to a gene involved in wiggling (unc-22) QuickTime and aGIF decompressorare needed to see this picture. 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 plants 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) dsRNA

Dicer 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

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