1 / 17

The History of RNAi John Doench

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. mutate genes at random.

phyre
Download Presentation

The History of RNAi John Doench

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

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

  3. 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

  4. 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.

  5. DNA: homologous recombination (yeast, mice) chromosome gene chromosome marker chromosome marker chromosome protein: eliminate its functionality with an antibody cell cell

  6. 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...

  7. 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.

  8. Discovery inject worms with dsRNA corresponding to a gene involved in wiggling (unc-22)

  9. Discovery inject worms with dsRNA corresponding to a gene involved in wiggling (unc-22) conclusion: dsRNA triggers potent and specific gene silencing

  10. 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...

  11. 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

  12. P 21 nt long, dsRNA: P Biochemistry to Examine Structure short interfering RNA: siRNA

  13. 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.

  14. dsRNA siRNA RISC cap cap mRNA mRNA AAAA AAAA RNA interference (RNAi) Dicer

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

  16. short RNAs identified in plants RNAi shown in vitro siRNAs identified Dicer identified genome-wide RNAi screens begin RNAi used against HIV Timeline 1990 cosuppression of purple color in plants dsRNA injection in worms 1998 1999 RISC activity partially purified 2000 2001 2002

More Related