Experimental Systems Course: RNA Interference

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/

INDEX to these course notes

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

General Introductory Remarks

RNA inteference, until a couple of years ago a very obscure backwater of molecular biological research, has suddenly developed into one of the most talked about molecular biological techniques currently available. Conferences are devoted to the subject, pharmaceutical companies are throwing research money at it and the pages of Nature and Cell are packed with advertisements trying to convince researchers that the companies that placed them are each ahead of a very competitive game.

For this reason, the course team considers it essential that we cover the essentials of RNA interference, even though the field is so new that there is much still to learn and our best understanding is expected to change quickly.

You will be able to find many excellent guides to RNAi on the web, but be warned: I have been in this field for a few of years now, and one of the most important things I find myself having to say to those entering it is "be critical, and don't beleive the hype...". This is a useful technique, but it is not an answer to all our prayers, however much advertisers try to convey that impression..

1. The basic mechanism of RNAi

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:

This is a cartoon of RNAi action

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.


2. Using RNAi in research

RNAi promises any researcher who can arrange to get dsRNA or siRNA into the correct cell at the correct time a way of knocking out (or at least, down) any gene she wishes to target.. To understand why this is so important, it is important to consider why we want to suppress the action of genes, and the limitations of alternative ways of doing this.

Deleting the expression of a gene has, for a long time, been the most powerful way of testing a hypothesis about the function of that gene. If ruddyconk2 is thought to be the gene that causes reindeer to develop red noses, then knocking out the gene and observing the colour of the noses produced is a powerful way of testing whether this is true (if the nose is still red, ruddyconk2 is not required for making a red nose; if it is not red, then the gene is needed).

There are several methods for removing the function of a gene, all with advantages and disadvantages;
The use of RNAi promises all of the advantages of pharmacology, but with absolutely any protein being targetted, and in principle experiments could be done in mere weeks without requiring animal breeding etc.

In this lecture, I shall concentrate on applying RNAi to mamalian systems, because this is where most effort is being expended (because RNAi may prove a very valuable anti-viral and anti-cancer treatment, as well as being a research tool, and because mammals do not have the clever genetic tricks available in worms and flies so new techniques are especially valuable).

Because they have the interferon response, mammalian cells cannot be treated with long dsRNAs, so have to be treated with siRNAs instead. There are several ways of doing this;
Designing siRNAs is not straightforward, although there are some useful guidelines. Generally, only about 1/4 work well, and people identify these by simple tests in cell lines before going on to do anything complicated.

All siRNA experiments require good controls. These are, typically;
Careful use of controls have demonstrated that there are several surprising effects of dsRNAs that do not act via the pathway outlined in lecture 1. One is induction of the interferon pathway. Another is activation of TLR3 on the outside of cells; this also induces an interferon/IL12 type response and blocks growth of blood vessels, something originally thought to operate via the specific RNAi pathway.

References for lecture 2

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

Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001 Jan 15;15(2):188-200. PMID: 11157775

Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24;411(6836):494-8. PMID: 11373684

Poy MN, Eliasson L, Krutzfeldt J, Kuwajima S, Ma X, Macdonald PE, Pfeffer S, Tuschl T, Rajewsky N, Rorsman P, Stoffel M. (2004) A pancreatic islet-specific microRNA regulates insulin secretion. Nature. 2004 Nov 11;432(7014):226-30. PMID: 15538371

Sledz CA, Williams BR (2004) RNA interference and double-stranded-RNA-activated pathways. Biochem Soc Trans. 2004 Dec;32(Pt 6):952-6. PMID 15506933

Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RH, Fire A. (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell. 2001 Nov 16;107(4):465-76 PMID: 11719187

Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000 Mar 31;101(1):25-33. PMID 10778853


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.

RNAi - RNA interference: this is the general term for the ability of double-stranded RNAs to block the expression of a messenger RNA with which they have a sequence in common (or almost in common).

siRNA - short interfering RNA - the product of dicer cutting of long dsRNA. siRNA is sometimes chemically synthesized for application to cells.

ssRNA - single stranded RNA

Date of this file: 5th November 2012.  Jamie.Davies@ed.ac.uk