RNA Interference (RNAi) (see also sirna, micrna, shrna, etc.)

Transcription

1 Biochemistry 412 RNA Interference (RNAi) (see also sirna, micrna, shrna, etc.) April 3, 2007

2 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.

3 And eight years after that. Zamore (2006) Cell 127, 1083.

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

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

6 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.

7 Zamore & Haley (2005) Science 309, 1519.

8 A model for RNA-induced silencing complex (RISC) assembly Note: the proteins that combine with the sirna/mirnas are members of the Argonaute family. Note: slight sequence mismatch Note: perfect basepairing Schwarz et al (2003) Cell 115, 199.

9 The RNAi silencing phenomenon may be part of an ancient cellular anti-virus defense mechanism, since many viruses generate virus-specific dsrna that could trigger the response (see also interferon). Consistent with this is the fact that RNAi has been found in a large range of metazoan organisms from plants to fruit flies to worms to humans. On the other hand, RNAi silencing may stem from an even more ancient mechanism of RNAs regulating RNAs, the machinery of which was later recruited (via evolution and selection) to take on an additional antivirus role.

10 Some data regarding endogenous mirnas A. thaliana 20 conserved families 90 genes 72 genes known to have a role in plant development C. elegans 65 conserved families genes found (so far) H. sapiens Approximately 130 gene families > 400 known genes Source: D. Bartel, Oct. 2005

11 The human genome is highly transcribed, even where there are no known genes What s with all this extra RNA? To provide the raw material for si/mirnas?? Kapranov et al (2002) Science 296, 916.

12 Empirical rules for designing sirnas with proper specificity and efficacy Mittal (2004) Nature Rev. Genetics 5, 355.

13 RISC-associated helicase can unwind less well from this end because of higher stability. RISC-associated helicase can unwind better from this end because of lower stability. Mittal (2004) Nature Rev. Genetics 5, 355.

14 Transitive RNAi - Spreading of Silencing Note: this provides a means of amplifying the RNAi effect. Hannon (2002) Nature 418, 244. Due to the action of an RNA-directed RNA polymerase (RdRP)? Yes! (see next slide)

15 Secondary RNAi triggered by primary RNAi (note: has only been observed in C. elegans so far) Miska & Ahringer (2007) Nature Biotechnology 25, 302.

16 An RNAi movie: [Note: see also the RNA Silencing poster (downloaded as a pdf file from Science 309, 1518 (2005)]

17 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.

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19 Construction of the hairpin expression vector

20 Endogenously-expressed hairpin RNA actually sometimes works better than injected dsrnai

21 RNAi Knockdown Experiments are Very Useful for Studying the Functions of Genes In Vivo Hannon (2002) Nature 418, 244.

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23 Carpenter & Sabatini (2004)

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25 Stevenson (2003) Nature Reviews 3, 851.

26 But how to deliver the RNAi for therapeutic effect??

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28 A lentivirus vector Note: hairpins should be <30bp long.

29 Also can deliver synthetic sirnas directly, just like an other medicine. (An advantage of this approach is that the sirnas can be modified with chemical bells and whistles to increase their stability, enhance their bioavailability, etc.)

30 Nature 432, (2004)

31 In vivo use of sirna conjugated with cholesterol at the 3 -end of the sense strand Soutschek et al (2004) Nature 432, 173.

32 Dorsett & Tuschl (2004) Nature Rev. Drug Disc. 3, 318.

33 Alternatives for gene silencing in vivo: gene knockouts vs. various RNAi approaches Dorsett & Tuschl (2004) Nature Rev. Drug Disc. 3, 318.