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REVIEW
RNA INTERFERENCE
G. Russev
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Correspondence to: George Russev
Email: [email protected]
ABSTRACT
RNA interference (RNAi) refers to the ability of double-stranded RNA molecules to cause sequence-specific degradation of single
stranded RNAs such as messenger RNAs and viral RNAs in vivo. RNAi is an ancient mechanism of gene regulation, genome
maintenance and antiviral defense found in all eukaryotes. The effector molecules are 21 to 23 nucleotides small interfering
RNAs (siRNAs) that are anticipated to serve as novel therapeutic agents in the battle against cancer, AIDS and neurodegenerative
diseases. In the laboratory, RNAi is used to investigate complex biological phenomena such as development, cell signaling and
infection, and thanks to its application many important breakthroughs have been made in these and related areas during the last
years. On the other hand its therapeutic application in the clinic may be few years away. The main obstacle in this field is not the
RNAi itself, but rather problems connected with the targeting and delivery of the therapeutic siRNAs.
Keywords: RNA interference, RNAi, small interfering RNA,
gene regulation, gene silencing, antiviral treatment;
Introduction
RNA interference (RNAi) is a highly evolutionally conserved
process of post-transcriptional gene silencing (PTGS) by
which double stranded RNA (dsRNA) causes sequencespecific degradation of homologous mRNA sequences, when
introduced into cells. There is general agreement that it is very
strange indeed that such a fundamental cellular regulatory and
defense pathway has remained unnoticed for so long. First
R. Jorgensen and colleagues (4), while trying to improve the
coloration of petunias by introducing extra copies of genes
responsible for pigmentation, noticed that in some cases
instead of intensification, colors were entirely or partially lost.
They correctly concluded that the treatment has switched the
respective genes off. Later on the same results were obtained
with other organisms (2, 6, 8) and it soon became evident that
the phenomenon represents a major and universal regulatory
pathway in the eukaryotic cells. However, the mechanism of
this process remained elusive until 1998 when Andrew Fire,
Craig Mello and colleagues published their fundamental paper
”Potent and specific genetic interference by double-stranded
RNA in Caenorhabditis elegans” in Nature (1). In this paper
they described the mechanism of the so called gene silencing
and used for the first time the popular name by which it is
known since then – RNA interference. In the following years
RNAi became a major tool in the life science experiments
and a promising therapeutic opportunity. Its impact on the
development of the biological sciences was so deep and
profound that it came as no surprise when in 2006 the Nobel
committee awarded the Nobel prize for physiology or medicine
to Fire and Mello for discovering the RNA interference.
Mechanism of RNAi
The first step in protein synthesis, transcription, takes place in
the cell’s nucleus, where the DNA template is used to make
single strand mRNA. The RNA then exits the nucleus and enters
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the cytoplasm, where it, in its turn serves as template in the
translation, the process of protein synthesis on the ribosomes.
RNA interference acts between the steps of transcription and
translation.
It has long been known that introduction of RNA into cells
interferes with the function of the genes (3, 5). These effects
have been proposed to result from the so called “antisense”
mechanism that depends on hybridization between the
exogenous RNA and endogenous messenger RNA transcripts
thus blocking the translation of the latter into proteins. However,
antisense RNA alone was marginally effective in silencing the
targeted genes and the reason for this poor performance was not
clear. Fire and Mello set themselves to study the requirements
for structure and delivery of the interfering RNA (1). As a
model organism they used the nematode C. elegance and as
a model gene - the so called ung-22 gene, which encodes a
non-essential myofilament protein. The expression of this
gene is easily detected and monitored since the decrease, or
the absence of the respective protein produced obvious and
specific phenotypic characteristic – twitching of the worms.
After injection of purified single strand or double strand
RNAs, complementary to regions of the unc-22 gene, Fire
and Mello found out that dsRNA was much more effective in
the silencing of the gene than either of the strands. In another
series of experiments they used specific reporter gene – the
gene for the green fluorescent protein (GFP) and in this case
again the dsRNA targeted to the gene was much more effective
in blocking the expression of the GFP than either of the strands
alone. Thus they came to the conclusion that dsRNA is much
more potent and specific inhibitor of the gene expression
then single strand RNA. Secondly they noticed that even if
only a few copies of the respective dsRNA were present in
the cell, they can completely prevent translation of highly
abundant RNAs, which indicates that the process of RNA
interference occurs by the involvement of a complex, but at
that time totally unknown cellular catalytic mechanism and
not by a conventional antisense mechanism in which sense and
antisense molecules hybridize in 1:1 ratio. A major step in
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the deciphering of this mechanism was made in 2001 when
G. Hannon (5) reported the discovery of 2 specific enzyme
complexes with ribonuclease activity. The first one called
Dicer cuts dsRNA into small uniform double-stranded pieces
21-23nt in length called small interfering RNAs (siRNA).
Recent works have shown that Dicer acts as a molecular ruler
measuring pieces of dsRNA in the proper 23nt range and cutting
them off. Dicer contains a conserved dsRNA binding domain
called PAZ and 65 angstrom (which corresponds to 25 nt) apart
a RNA cutting domain containing 2 RNAse III sites that cut
across the dsRNA. The second enzyme, called RISC (RNA
Induced Silencing Complex) cuts the target mRNA. RISC is
the conglomeration of several proteins including certain RNA
unwinding proteins, and a protein called Argonaute which is
central for the RNA cutting endonuclease activity of RISC.
First Argonaute degrades one of the strands of the siRNA
called “passenger” strand. The strand selection is carried out on
the basis of the thermodynamic stability of the siRNA duplex
termini, the strand whose 5’-terminus has higher base-pairing
stability being degraded. Thus in its final form RISC is loaded
with only one of the siRNA strands, called “guide strand” that
forms a temporary complex with the target mRNA on the basis
of sense-antisense complementation, and cuts it. The RISC
complex with the guide RNA strand is quite stable and can
successively degrade many mRNA molecules thus acting as a
catalyst (7). The whole process of RNA interference is shown
in (Fig. 1).
Role of RNAi in eukaryotic cells
The finding that dsRNA introduced into eukaryotic cells
executes a gene-silencing effect using cell’s own enzymatic
activities and protein complexes, shows that the process of RNA
interference is a normal cellular pathway evolved to perform
specific tasks. These include antiviral defense, regulation of
the gene expression and protection of the genome.
•
Antiviral defense mechanism
Prokaryotic cells have developed the system of restriction
enzymes, which cut any foreign DNA that eventually appears
in the cells. The higher organisms also possess effective
ways to fight foreign DNA, the most common being the virus
induced apoptosis. To avoid these defense mechanisms many
eukaryotic viruses have evolved into RNA viruses and many
plant, animal and human viruses such as HIV for instance, are
dsRNA viruses. Eukaryotic cells have reacted to this challenge
by developing the RNA interference. By this pathway any viral
dsRNA is chopped by the cellular Dicers and then incorporated
into the cellular RISCs to degrade the viral mRNAs. Thus
RNAi seems to serve as an antiviral immune system evolved
to protect eukaryotic cells from invasion of foreign genetic
(RNA) material.
•
Gene expression regulation
Since RNAi degrades mRNAs and is conveniently
positioned between transcription and translation, it can regulate
the gene expression, i.e. the rate of the respective gene product
synthesis, without changing the gene activity. Cells are using
this way to achieve “fine tuning” of the expression without
interfering with transcription. It has long been known that a
class of short dsRNA molecules called micro RNA (miRNA)
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Fig. 1. RNA interference. (A) On entering the cell, long ds RNA act as a
trigger of RNAi process. (B) It is first processed by the RNAse III enzyme
Dicer in an ATP-dependent reaction. (C) Dicer processed dsRNA into 21-23
nt short interfering RNA (siRNA) with 2 nt 3’-overhangs. SiRNA can also be
synthesized outside the cell and then introduced into a cell. (D) The siRNAs are
incorporated into the RNA-inducing silencing complex (RISC) which consists
of the RNAse Argonaute as one of its main components. At this point one of
the RNA strands of the dsRNA (passenger strand) is degraded. (E) RISC’s
guide siRNA strand is complexed with the target mRNA on the basis of senseantisense hybridization. (F) mRNA is cut by RISC and RISC itself is released
for interaction with another molecule mRNA
exists in eukaryotic cells. However, only lately it has been
discovered that they play the role to control the amount of
different mRNAs. They are transcribed from regions of the
genome with specific sequence symmetry and after transcription
the resulting RNAs fold back to form a hairpin structures with
double-strand stem and a loop. These RNAs are called hairpin
RNAs. They are recognized by the Dicers and chopped to
give small hairpin RNAs (shRNA). They have homology to
different mRNAs and could degrade the latter thus maintaining
the mRNA populations within certain limits. Over 30% of the
genes are fine regulated by this mechanism.
•
Genome stability maintenance
The RNAi is also used to control the spread of transposons.
Transposons are like the viruses that are inserted into the
genomes. A good example are the Alu elements which
represent a few hundred bp DNA sequence repeated over 105
times in the human genome. Transposons can arrange their own
transcription to produce RNA copies. These RNA copies are
then used as templates to synthesize complementary DNA by a
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process known as reverse transcription. Then the DNA strand
is replicated by the normal cellular replication machinery to
produce dsDNA copies of the original Alu element, which
are inserted randomly in the genome. If left unchecked this
process would lead to ever increasing number of Alu elements.
Apart from increasing the so called “junk” DNA this process
represents a treat to the integrity of the genome since the Alu
elements could be inserted in exons or other important regions
and to interfere with their normal function. To avoid this cells
recruit the RNAi mechanism. Certain DNA regions contain
sequences which after transcription give rise to shRNAs with
homology to the transposon sequences. They are cut by the
Dicers and used by the RISCs to degrade the initial transposone
RNA transcripts thus maintaining the genome stability.
RNAi application
RNAi holds many promises as antiviral treatment and for
controlling gene expression in eukaryotic cells. However, for
the time being it is only used as experimental tool. There is
hardly any molecular biology or molecular genetics lab in the
world that is not using RNAi to knock down different genes to
study their functioning. The procedure is simple, and the “knock
down” is reversible which represent clear advantages over the
so called “knock out” procedures in which the gene is deleted
or irreversibly damaged. As for its application in the clinics
– it still seems several years in the future, although a few trials
on patients have been reported. In one of them the company
Acuity Pharmaceuticals in Philadelphia has completed safety
tests on RNAi treatment for macular degeneration, the leading
cause for blindness in the elderly. This disease is triggered by
the loss of control on the expression of a protein called VEGF
(Vascular Endothelial Growth Factor) which is responsible
for the formation of the blood vessels. Normally this protein
is not expressed in the retina which stays transparent and
clear. However, when it is expressed, extensive blood vessel
formation occurs there and the retina losses its transparency
thus causing blindness. In the trial a group of about 24 people
have undergone direct injection of dsRNA against VEGF
mRNA in the eye. Two months after being injected with the
siRNA, a quarter of the patients had significantly clearer vision
and the other patients’ vision has stabilized. Another clinical
trial of siRNA based drug was carried out in Cambridge, MA
by the pharmaceutical company Alnylam. In this case patients
were treated with siRNA against RSV gene by inhalation. RSV
(Respiratorial Syncythial Virus) infects almost all children
by the age of two. Infection typically leads to cold-like
symptoms, but in many cases have far serious consequences
such as pneumonia and respiratory failure. The treatment led
to reduction of the viral loads.
Prospects and problems
Although in its infancy, RNAi therapy is expected to cure
almost everything from common flu to cancer. However, the
hopes are especially high in two fields - those of RNA virus
caused diseases such as AIDS and Hepatitis B and C, and of
neurological disorders such as Alzheimer’s, Jacob-Croizfeld’s
and Hutchington’s diseases. The antiviral strategies aim to use
dsRNA to target mRNA for an important viral gene, whose
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knock down would inhibit viral replication or its ability to
infect cells. The same result could also be achieved if host
cells are treated with dsRNA against mRNA for cell surfice
receptors specific for the virus. In this case the virus will not
infect the cells since it will not find the receptors on the cell
surface.
Most of the neurodegenerative diseases are result of minor
nonessential mutations in some of the genes coding for specific
class of proteins called amyloids. These mutations occur only
in one of the alleles and produce altered proteins which tend
to aggregate. The peculiar thing is that this aggregation can
involve the normal protein as well thus causing the aggregation
of essentially all protein molecules, both mutated and normal,
to form extracellular deposits of amyloid filaments. The
obvious approach to treat such a condition is to silence the
mutated gene thus allowing the normal gene to express the
normal protein that would not aggregate alone. In both cases
preliminary experiments on animal models with siRNAs have
shown very promising results. However, in these and all other
cases the problem is not the siRNA itself, but its delivery into
the target cells. To achieve the desired therapeutic effect it is
not enough siRNA to reach and enter few cells. For successful
therapy the siRNA has to be introduced in all cells of the
affected tissue which makes the things difficult. Relatively
simple are the situations when these tissues are easily accessible
and siRNA can be applied directly. Such are the cases with
macular degeneration treated by direct injection of siRNA in
the eye, the RSV infection, when the drug is administered by
inhalation, and of certain vaginal and anal viral infections when
the drug is directly applied as ungventum. In all these cases to
help siRNA to get into the cells it was complexed with lipids
or chemically modified to minimize its charge. On the other
hand, for most of the diseases expected to treat with siRNA,
the direct application is not an option. To this end specific and
at the same time harmless for the host organism vectors have
to be constructed. They should be able to infect cells with high
efficiency and to integrate in their genomes and should carry
cloned DNA sequences that after transcription would give the
therapeutic shRNAs.
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