Molecular Biology II Nucleic Acids: RNA Interference

Illustrating genes:
Red – Function known through experimentation such as assays
Yellow – Very good sequence homology with another gene of known function
Orange – Contains a conserved domain
Green – No functional homologues and no similar domain elsewhere
 These genes make up 50% of the genome

Tools to assign function to a gene:

Bioinformatics
Biochemical assays
Genetic analysis
 Forward – Use phenotype to track back to gene (important when genetic exchange occurs)
In organism that do not have sex, forward genetics is not valuable.
 Reverse – Take a gene, change expression, monitor results

Gene ablation (reduced expression)

Gene knockout (RNA interference)

Example
What effects are caused by the Staphylococcus aureus -toxin alone?

S. aureus is a pathogenic bacterium

The -toxin, as well as the //-toxins will all cause tissue damage on their own.
To study the -toxin we can create a ‘gene knock-out’ bacterium
 We create a mutant that lacks the -toxin gene
 This is easy in bacteria (transformation) but very difficult in higher organisms
 Essential genes cannot be knocked-out (death), unless resucued by another gene copy

If we cannot create a knock-out, then we reduce expression of the gene instead using RNAi

RNAi is an endogenous mechanism in a variety of cell tyes

RNA Interference: (Discovered 1998, Nobel Prize 2006)

Caenorhabditis elegans (C. elegans) is useful organism to study:

 Non-parasitic worm, a multicellular eukaryote with cellular differentiation
 All studies are carried out on identical worms
 Easy to genetically manipulate

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Molecular Biology II Nucleic Acids: RNA Interference

 Can be cryopreserved
 Experimentally tractable (easy to control)
 Easy and cheap to maintain in a lab
 Complete cell lineage has been determined

It was initially believe that RNAi was occurring via an antisense mechanism
This was an already know mechanism:
1. mRNA is exported from the nucleus
2. Complimentary antisense RNA hybridises with the mRNA (sense)
3. This hybrid cannot be read by a ribosome, so transcription does not occur
 This will result in reduced gene expression
 Scientists added antisense RNA and noticed reduced gene expression

Later studied showed that adding both sense + antisense RNA would cause reduced gene
expression. Sense and anti-sense RNAs were equally efficient at blocking expression
(interference). This interference was passed onto the next generation even though transcripts are
easily degraded. It was also later revealed that in the initial experiments, there had been some
contamination of dsRNA to the sample of antisense added.

To test this, researchers injected cells with the following:

 Sense RNA
 Anti-sense RNA
 Sense + Anti-sense RNA
 No RNA

The sense RNA was homologous with that of a gene Sense Wild-type
product. The gene chosen was Unc22, as it is non- Anti-sense Wild-type
Sense + Anti-sense Severe twitching
essential, and has a visible phenotype when No RNA Wild-type
expression falls (twitching). It codes for a myofilament
protein found in abundance in muscle cells. Another method was to add the gene that codes for
GFP as an artificial gene to the worm. Use sense RNA for the mRNA transcript for GFP then view
results: Sense + Anti sense will not-fluoresce, whereas the others all will as GFP is expressed.

Therefore: Introduction of dsRNA reduces specific gene expression

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Molecular Biology II Nucleic Acids: RNA Interference

Mechanism of RNA Interference

1. dsRNA is introduced to, or formed within a cell. It is around 200

nucleotides long.

2. The RNA is chopped into fragments (~20 nucleotides long)

Fragments are known as siRNAs (small interfering RNAs)
Each fragment has a 2nt overhang at the 3’ end (important for
directing the RISC). Reaction is carried out by dicer – an ATP
dependent RNase. A member of the RNase III family of
ds-specific RNA endonucleases.
3. Each siRNA is transferred to a multi-subunit RISC complex
(RNA-induced silencing complex). The siRNA binds to Argonaute
proteins within the RISC complex. Argonaute contains an
endonuclease that is distinct from dicer
4. RISC includes a ribonuclease called slicer (a type of
argonaute). dsRNA is unwound by the slicer, passenger RNA
released
5. The antisense strand of the siRNA guides the RISC complex to
a complementary mRNA. The argonaute within RISC cleaves the
mRNA. The mRNA is subsequently cleaved further in the cell by
other endonucleases. Translation is prevented.

RNAi Function:
Occurs naturally. A form of genomic defence against viruses (invading nucleic acids)
Studies of RNAi defective mutants show that they have increased rates of transposition and have
a greater susceptibility to viral infection.
In a number of organisms it is used to control gene expression.
Can be used to control developmental timing

Endogenous RNAi Mechanism:

1. Copies of a primary miRNA (micro RNA) transcript are made in the nucleus
2. Drosha? Converts this to an miRNA precursor (pre-miRNA)
3. Exportin-5 transports this into the cytoplasm via a nuclear pore complex (NPC)

LOOK UP THE ENDOGENOUS MECHANISM!!!!

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RNAi Delivery:

C. elegans
 in vitro synthesised dsRNA is microinjected into the germline via the gonads. Progeny are
then screened for RNAi induced effects
 Feed with E. coli that express the dsRNA. Worms eat them and will ingest the dsRNA
Other methods
 Naked / modified ds/siRNA (physical means)
 Liposomes complexed with siRNA
 Nanoparticles encapsulating siRNA
 Cholesterol conjugated with siRNA
 Aptamer siRNA chimeras
 Antibody bound (protamine) siRNA
 Viral vectors – RNA or DNA based (lentivirus)
It is important for the DNA to be protected from degradation, but it still has to be able to cross
membranes.

RNAi Limitations:
 Competition with endogenous RNAs
 Stimulation of innate immune responses
 Suppression of off-target RNAs

Disease Treatment:
Genetic disease (e.g. degenerate neurological disorders)
Viral disease (e.g. Hepatitis C)
Cancer (some miRNAs are natural oncogenes or tumour suppressors). Some are in clinical trial
already:
 Macular degeneration (blindness)
 Diabetic retinopathy (eye disease)

Oral delivery has been proven to work. This has huge therapeutic value for humans (better than
injections). No clinical trial just yet.

However it is tough to find good targets, there have been no clinical trials on effective routes of
delivery and it is important to minimise toxicity.

Other Applications of RNAi:

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RNAi in plants = co-suppression / PTGS (Post Transcriptional Gene Silencing)

If extra copies of a transgene (for pigmentation in Petunias) are introduced then a decreased
level of pigment is seen. There is reduced expression of the transgene and endogenous genes.

Viruses can be used to infect plants – is any of the viral RNA/DNA is homologous to a plant gene
then it could suppress expression of that gene.
RNAi has been used to naturally decaffeinate coffee plants.

The ability to switch of the expression of a gene is very useful for study.
siRNAs can be introduced by transfection using lipid carriers and result in up to 90%
downregulation of genes.
Can be used on a genomic scale to study gene function
 Create an E. coli gene library containing dsRNAs for all C. elegans genes
 Feed worms the E. coli clones
 Watch the result (change in phenotype)
These RNAi screens have also been carried out on Drosophila melanogaster

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