It is the late 1990’s and Andrew Fire and Craig Mello are contemplating the control
of genes through RNA molecules.
The current theory around the topic is extremely vague and states that upon the injection of
antisense RNA into organisms, the sequence binds to a complementary mRNA strand and silences
its expression.
Fire and Mello see that the current theory is incomplete and set out to examine some
unanswered questions regarding this largely unknown mechanism.
The two major features in previous studies that seem to trouble Fire and Mello most were
the seeming ability of both sense and antisense RNA strands to individually induce interference,
and the fact that these interferences seemed to perpetuate onto the progeny of the effected
organism.
Fire and Mello insisted that this refuted the current theory and speculated that another
mechanism was at play.
They termed this unknown mechanism RNA interference, or RNAi for short.
Early on, Fire and Mello deduced that the normal use of bacteriophage RNA polymerases
in preparation of RNA populations created some structures that had a double-stranded
rather than a single-stranded appearance.
They inferred that the hybridisation of complementary antisense and sense RNA formed these double-stranded
RNA molecules and that these molecules might have a role to play in RNA interference.
To test this, Fire and Mello used a gene called unc-22, which when knocked out, produces a
twitching phenotype.
By using a DNA template of the unc-22 gene region and bacteriophage RNA polymerase, they
produced single-stranded sense and antisense RNAs.
After being purified, these RNA strands were then individually injected into C. elegans
organisms, while other C. elegans organisms were injected with RNA populations deliberately
prepared to contain double-stranded RNAs.
To their surprise, Fire and Mello saw that the progeny of C. elegans organisms injected
with purified antisense and sense RNAs saw a wild-type phenotype, whereas the progeny
of animals injected with double-stranded RNA saw a twitching phenotype.
This indicated that single strand injections were susceptible to degradation and cannot
induce interference in the absence of its opposite strand.
Additionally, Fire and Mello discovered that the site of injection had minimal effect on
the animal’s phenotype with injections of double-stranded RNA into the gonads, tail
and head showing similar interference with the organism.
This showcased the surprising ability of double-stranded RNA to cross cellular boundaries which provided
a viable explanation to the robustness of RNA interference across generations.
Upon confirming their findings on other genes such as unc-54, they also discovered that
injected double-stranded RNAs that were complementary to target mRNA intron or promoter regions
produced wild-type phenotypes.
When these double-stranded RNAs were targeted to exon regions however, silencing was induced.
From this, they speculated that RNA interference likely only occurs on post-transcriptional,
mature mRNA strands, rather than on nucleic pre-mRNA.
To observe RNA interference on a more cellular level, Fire and Mello injected C. elegans
embryos with a stain targeting an endogenous mex-3 mRNA.
They then injected both purified mex-3 sense, antisense and double-stranded RNA.
They discovered that the embryos that were injected with single-stranded RNA showed substantial
retention of mex-3 mRNA, while the embryos injected with double-stranded RNA showed no
sign of the mex-3.
This indicated that RNA interference silenced genes through the degradation of the respective
mRNA script.
Despite no clear mechanism for RNA interference being described, Fire and Mello received a
Nobel Prize for their advancements in the field as their accomplishments sequentially
paved the way for many more discoveries on the topic.
The current mechanism underlying RNA interference states that exogenous double-stranded RNA
binds to a protein complex called Dicer which liberates the antisense strand from its annealed
state and cleaves it into small fragments called small interfering RNA.
This antisense strand then binds another protein complex called the RNA Induced Silencing Complex
(or RISC).
This antisense strand then guides this complex to degrade its complementary mRNA strand and
silence its expression.
Endogenously occurring double-stranded RNA is expressed in a hairpin-like structure called
pre-microRNA in the cell nucleus.
When introduced into the cytoplasm, this molecule binds to the Dicer complex and then undergoes
a similar process to that of the exogenous pathway.
Organisms such as the C. elegans utilise these double-stranded RNAs to protect them against
virally introduced RNA or to inhibit the synthesis of unwanted proteins.
When used in an exogenous manner, RNA interference has proven to be an extremely valuable research
tool, as it allows researchers to introduce an unknown RNA sequence into an organism and
observe its function according to the genes it silences and the resulting phenotype.
It also opens exciting opportunities in the treatment of diseases by silencing the expression
of harmful proteins via double-stranded RNAs.
Therapies currently in clinical trials include the treatment of neurological diseases such
as Huntington’s and Alzheimer’s disease, cardiovascular diseases, diseases in the liver
such as Hepatitis B and C, multiple types of cancer such as lung cancer and endocrine
diseases such as chronic renal disease.