Course Lecturer: Prof Jamie Davies; e-mail Jamie.Davies@ed.ac.uk, Tel (6)502999, lab web page http://golgi.ana.ed.ac.uk/Davieslab/

Please note: the most up-to-date version of these notes will be found, while the course is running, on http://golgi.ana.ed.ac.uk/coursenotes/

General Introductory remarks (please read!)
Lecture 1 - Introduction to RNA interference
Lecture 2 - Using RNA interference as an experimental tool.

General Introductory Remarks

RNA interference (RNAi) refers to the ability of double-stranded RNAs to shut down the expression of a messenger RNA with which they have sequence in common.

RNAi was discovered independently in plants, fungi and invertebrates, in response to attempts to genetically engineer these organisms, and was at first given names such as 'gene silencing' and 'co-suppression'. History is probably not the best way into the subject, because the earliest examples of RNAi are not the best understood, so this lecture will jump straight to our current understanding of RNAi and our best guesses about why the system exists at all.

RNAi is triggered by double stranded RNA (dsRNA):  single stranded RNA (ssRNA), such as mRNA, cannot trigger it, although complementarty ssRNAs can obviously come together in  a cell to form a dsRNA that can trigger RNAi.  Cells have a number of protein complexes that can recognize and bind to dsRNA, possibly because these proteins were once part of a defence mechanism against RNA viruses (perhaps they still are). The cells of vertebrates show a very powerful and (almost) sequence-independent reaction to the presence of long dsRNAs, the  'interferon response', and this complicates the story. In what follows, we will concentrate on invertebrate cells and come back to vertebrates later.

Long dsRNAs are recognized by a protein complex called Dicer, which cuts the dsRNA into short fragments of about 22nt, this length being set by the structure of the Dicer complex itself. An elegant experiment in which these short degradation products were placed in Drosophila cells in lieu of the long dsRNAs showed, to everyone's surprise at the time, that these short lengths of RNA were capable of fully activating the gene silencing response that the original dsRNA was. They are therefore called siRNAs, for 'short interfering RNAs'. They have overhanging ends (by 2 nt). When they are made by a cell as a natural part of its genetic programme (rather than in response to experimentally-introduced dsRNA), they are called microRNAs (miRNAs - be careful not to misread this for mRNA).

The siRNAs associate with another complex of proteins, called RISC, which functions as an RNA-dependent RNA endonuclease. Only siRNAs with the overhanging ends and 5' phosphates, as produced by dicer, can recruit RISC. Once bound to siRNA, RISC is activated and unwinds the siRNA to expose its single strands (or one of its strands - this is not yet clear). If the exposed siRNA strand of a siRNA-RISC complex happens to be complementary to part of an mRNA in the cell, it will bind to this mRNA. On binding, RISC cuts up the mRNA, thus preventing its expression.

Here is a cartoon of the basic mechanism of RNA, from TIBS:

RNAi is a catalytic process, because once the RISC complex has cut up one mRNA, it is free to go off and attack another one, so that very small amounts of siRNA can be used to clear a large amount of mRNA. In principle, it is specific to mRNAs that are homologous to the dsRNA sequences, and bystander RNAs are not affected (generally, this is true - we will return to this point in lecture 2).

In animals such as C. elegans and in plants, siRNA seems to be able to spread from cell to cell (in the case of C elegans, via a protein called SID-1), and tiny amounts of siRNA can silence the entire organism, suggesting that amplification occurs.

In C. elegans, taregetting the 3' region of a message with siRNA results in the generation of new siRNAs for regions more 5'. This seems to happen because the animal has an RNA-dependent RNA polymerase that can be primed by the siRNA/mRNA hybrid. The newly synthesized dsRNA will be converted into siRNA by dicer and so the cycle can repeat with more vigour. This also happens in plants, but apparently not in vertebrates.

The effiiency by which RNAi works has been very useful for experimenters (see next lecture), but it leaves the question about why cells are so good at it. Recent discoveries of micro-RNAs that are expressed during normal development raises the possibility thet RNAi is a method of gene control, maybe a major method of gene control, in plants and animals. There is, for example, evidence that they are used even in a system as apparently well-understood as insulin production.

• Dicer - Zamore PD, Tuschl T, Sharp PA, Bartel DP.
• Interferon response - Sledz CA, Williams BR
• That siRNAs can induce the full RNAi response - Elbashir SM, Lendeckel W, Tuschl T
• RNA amplification - Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RH, Fire A.
• Micro-RNAs in insulin production - Poy MN, Eliasson L, Krutzfeldt J, Kuwajima S, Ma X, Macdonald PE, Pfeffer S, Tuschl T, Rajewsky N, Rorsman P, Stoffel M.
• Evolutionary origin of endogenous interfering RNAs - http://www.nature.com/nature/journal/v453/n7196/full/453729a.html
  
• Endogenous micro RNAs and slicer (as an alternative to Dicer) - http://www.nature.com/nature/journal/v453/n7196/abs/nature07015.html
  

• Transgenic knockout: this has the advantage of being absolute, but the disadvanatge that if ruddyconk2 is also needed to make, say, a placenta, then the embryo will not survive for long enough for a researcher to see if it has a red nose.
• Conditional knockout (eg cre-lox): this can arrange for a knockout to work only in some tissues - it is great when drivers are available that are expressed onyl in the tissue of interest (for early development, esepcailly, this is often a big problem) but it is expensive and takes a coupld of years typically.
• Pharmacological inhibition: this is useful in that a drug can be applied at any time, and therefore you can let something develop normally to stage X and then do the knockout, but has the problem that very few proteins are specifically inhibited by drugs, althoug the list keeps growing.
• Antibodies etc: These are fine for inhibiting extracellular proteins (eg growth factors), but are useless for getting inside the cell.

• Synthesize siRNAs chemically, and apply to cells in some suitable membrane-crossing complex
• Make siRNAs from a plasmid in vitro, then treat as above
• Transfect cells with a plasmid that makes siRNA
• Transfect cells with an inducible plasmid that makes siRNA.

• Negative control cells with no siRNA
• Negative cells with an irrelvant siRNA
• The use of several different siRNAs to the same target, to control for unexpected 'friendly fire' incidents on other messages (off-target effects).
• Careful monitoring of protein knockdown (time, extent, % of cells)
• Careful monitoring that other proteins are not knocked down.
• If possible, rescue of the cells with a plasmid endocing the same protein but using a different DNA sequence (codon redundancy).

• Use of siRNA in mammalian cells - Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T.
• Use of siRNA to test later functions of a gene - Davies JA, Ladomery M, Hohenstein P, Michael L, Shafe A, Spraggon L, Hastie N.
• Good descriptions of typical RNAi technology - http://www.ambion.com/techlib/resources/siRNA/index.html
• The TLR3 path - http://www.nature.com/nature/journal/v452/n7187/full/452543a.html (this is a news and views review with refs to the original paper).
• dsRNAs as activators of transcription - http://www.nature.com/nature/journal/v448/n7156/full/448855a.html - I am not going to cover this in the lectures but if you take the trouble to read it you could be very impressive in a variety of exam questions!
• Off-target effects - statistics - http://www.nature.com/nature/journal/v443/n7109/abs/nature05179.html - I am not going to cover this in the lectures but if you take the trouble to read it you could be very impressive in a variety of exam questions!

Reference List

These references serve two purposes, to help anyone I might have confused,by repeating the lecture material in someone else's words (where possible), and to allow everyone to explore the topics in more detail. This material is too new to be in textbooks, but I am aware that you do not have time, with the exams so close, to spend trawling through original papers. If you want more detail, use these references, but if you just want to cram for the exam use your lecture notes and websites such as Ambion's for quick summaries.

Davies JA, Ladomery M, Hohenstein P, Michael L, Shafe A, Spraggon L, Hastie N. (2004) Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation. Hum Mol Genet. 2004 Jan 15;13(2):235-46 PMID: 14645201

Co-suppression - another synonym for RNAi, usually used by plant and fungi folk.

Dicer - the protein complex that binds to long dsRNAs and chops them to siRNAs

dsRNA - double stranded RNA

Gene silencing - synonym for RNAi.

Interferon response - a slightly slang phrase (in that it is used quite loosely) for the non-sequence-specific response of mammalian cells to long (>20bp or so) dsRNAs.

mRNA - messenger RNA

nt - nucleotide (a way of counting length that is equally useful for ssRNA and dsRNA).

RISC - the nuclease complex that assmebles around siRNAs and cuts target RNAs complementary to them.