Biochemistry 412

RNA Interference (RNAi)(see also siRNA, micRNA, stRNA, etc.)

8 April 2005 Lecture

The Discovery of the RNA Interference (RNAi) Phenomenon

1. Gene-specific inhibition of expression by anti-sense nucleic acids was discovered in the 1980’s (Inouye, 1988).

2. Guo and Kemphues (1995) showed that, in some cases for C. elegansgenes, the sense strand inhibited just as well as the anti-sense strand (!? - why careful controls are always wise!).

3. Three years later, Mello and Fire (1998) tested whether both the senseand the anti-sense strand together would inhibit or cancel other out. They hit the jackpot: the dsRNA that they inadvertently created inhibited homologous mRNA expression much more strongly than either the sense or the anti-sense strands alone (!!??).

>>> RNAi may be a very ancient defense mechanism that evolved ineukaryotic organisms to protect their cells against viruses.

Tijsterman et al (2002) Annu. Rev. Genet. 36, 489.

Tijsterman et al (2002) Annu. Rev. Genet. 36, 489.

Micro-RNAs (e.g., so-called stRNAs) are also involved in endogenous gene regulation

Grosshans & Slack (2002). J. Cell Biol. 156, 17.>>> Note that the inhibition of expression is exerted by binding at the 3’-UTR!?

Short RNAs regulate gene expression by different mechanisms, but use some of the same cellular machinery (e.g., Dicer) to do it.

Moss (2001) Curr. Biol. 11, R772.

A model for RNA-induced silencing complex (RISC) assembly

Schwarz et al (2003) Cell 115, 199.

Kapranov et al (2002) Science 296, 916.

The human genome is highly transcribed, even where there are no known genes…What’s with all this extra RNA??

Problem:

How to make stable RNAi-mediated gene knockdowns?

and

…also would be nice to be able to do tissue- and/or developmental stage-

specific regulation.

Construction of the hairpin expression vector

Endogenously-expressed hairpin RNA actually sometimes works better than injected dsRNAi

RNAi “Knockdown” Experiments are Very Useful for Studying the Functions of Genes In Vivo

Hannon (2002) Nature 418, 244.

Transitive RNAi - “Spreading” of Silencing

Hannon (2002) Nature 418, 244.

Due to amplification of the effect by an RNA-directed RNA polymerase (RdRP)??

Stevenson (2003) Nature Reviews 3, 851.

But how to deliver the RNAi for therapeutic

effect…??

A lentivirus vector

Note: hairpins should be <30bp long.

Carpenter & Sabatini (2004)