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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
Example
What effects are caused by the Staphylococcus aureus -toxin alone?
If we cannot create a knock-out, then we reduce expression of the gene instead using RNAi
Lecture 5 [Page 1]
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.
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.
Lecture 5 [Page 2]
Molecular Biology II Nucleic Acids: RNA Interference
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
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)
Lecture 5 [Page 3]
Molecular Biology II Nucleic Acids: RNA Interference
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.
Lecture 5 [Page 4]
Molecular Biology II Nucleic Acids: RNA Interference
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
Lecture 5 [Page 5]