PowerPoint Download

Transcript
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. elegans
genes, 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 sense
and 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 in
eukaryotic 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
>>> Note that the inhibition of expression is exerted by binding at the 3’-UTR!?
Grosshans & Slack (2002). J. Cell Biol. 156, 17.
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.
The human genome is highly transcribed, even where there are no known genes…
What’s with all this extra RNA??
Kapranov et al (2002) Science 296, 916.
Problem:
How to make stable RNAi-mediated gene
knockdowns?
and
…also would be nice to be able to do tissueand/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)