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

What genes allow a worm to wiggle? mutate genes at random

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 marker protein: eliminate its functionality with an antibody cell chromosome marker

Kill the messenger! capAAAA... mRNA: in vitro transcribe an antisense RNA: capAAAA... 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 dsRNA corresponding to a gene involved in wiggling (unc-22) Discovery

conclusion: dsRNA triggers potent and specific gene silencing inject worms with dsRNA corresponding to a gene involved 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 AAAA cap mRNA AAAA cap mRNA

dsRNA has been used for genome-wide screens in worms. siRNA have been used in mammalian tissue culture and live mice. Discoveries to Tools

Timeline cosuppression of purple color in plants dsRNA injection in worms short RNAs identified in plants RNAi shown in vitro RISC activity partially purified siRNAs identifiedDicer identified RNAi used against HIV genome-wide RNAi screens begin