Biotechnology & Biotechnological Equipment

ISSN: 1310-2818 (Print) 1314-3530 (Online) Journal homepage: https://www.tandfonline.com/loi/tbeq20

RNA Interference

G. Russev

To cite this article: G. Russev (2007) RNA Interference, Biotechnology & Biotechnological
Equipment, 21:3, 283-285, DOI: 10.1080/13102818.2007.10817462
To link to this article: https://doi.org/10.1080/13102818.2007.10817462

Published online: 15 Apr 2014.

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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, the cytoplasm, where it, in its turn serves as template in the
gene regulation, gene silencing, antiviral treatment; translation, the process of protein synthesis on the ribosomes.
RNA interference acts between the steps of transcription and
Introduction translation.
RNA interference (RNAi) is a highly evolutionally conserved It has long been known that introduction of RNA into cells
process of post-transcriptional gene silencing (PTGS) by interferes with the function of the genes (3, 5). These effects
which double stranded RNA (dsRNA) causes sequence- have been proposed to result from the so called “antisense”
specific degradation of homologous mRNA sequences, when mechanism that depends on hybridization between the
introduced into cells. There is general agreement that it is very exogenous RNA and endogenous messenger RNA transcripts
strange indeed that such a fundamental cellular regulatory and thus blocking the translation of the latter into proteins. However,
defense pathway has remained unnoticed for so long. First antisense RNA alone was marginally effective in silencing the
R. Jorgensen and colleagues (4), while trying to improve the targeted genes and the reason for this poor performance was not
coloration of petunias by introducing extra copies of genes clear. Fire and Mello set themselves to study the requirements
responsible for pigmentation, noticed that in some cases for structure and delivery of the interfering RNA (1). As a
instead of intensification, colors were entirely or partially lost. model organism they used the nematode C. elegance and as
They correctly concluded that the treatment has switched the a model gene - the so called ung-22 gene, which encodes a
respective genes off. Later on the same results were obtained non-essential myofilament protein. The expression of this
with other organisms (2, 6, 8) and it soon became evident that gene is easily detected and monitored since the decrease, or
the phenomenon represents a major and universal regulatory the absence of the respective protein produced obvious and
pathway in the eukaryotic cells. However, the mechanism of specific phenotypic characteristic – twitching of the worms.
this process remained elusive until 1998 when Andrew Fire, After injection of purified single strand or double strand
Craig Mello and colleagues published their fundamental paper RNAs, complementary to regions of the unc-22 gene, Fire
”Potent and specific genetic interference by double-stranded and Mello found out that dsRNA was much more effective in
RNA in Caenorhabditis elegans” in Nature (1). In this paper the silencing of the gene than either of the strands. In another
they described the mechanism of the so called gene silencing series of experiments they used specific reporter gene – the
and used for the first time the popular name by which it is gene for the green fluorescent protein (GFP) and in this case
known since then – RNA interference. In the following years again the dsRNA targeted to the gene was much more effective
RNAi became a major tool in the life science experiments in blocking the expression of the GFP than either of the strands
and a promising therapeutic opportunity. Its impact on the alone. Thus they came to the conclusion that dsRNA is much
development of the biological sciences was so deep and more potent and specific inhibitor of the gene expression
profound that it came as no surprise when in 2006 the Nobel then single strand RNA. Secondly they noticed that even if
committee awarded the Nobel prize for physiology or medicine only a few copies of the respective dsRNA were present in
to Fire and Mello for discovering the RNA interference. the cell, they can completely prevent translation of highly
abundant RNAs, which indicates that the process of RNA
Mechanism of RNAi interference occurs by the involvement of a complex, but at
The first step in protein synthesis, transcription, takes place in that time totally unknown cellular catalytic mechanism and
the cell’s nucleus, where the DNA template is used to make not by a conventional antisense mechanism in which sense and
single strand mRNA. The RNA then exits the nucleus and enters antisense molecules hybridize in 1:1 ratio. A major step in
BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/3 283
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 Fig. 1. RNA interference. (A) On entering the cell, long ds RNA act as a
executes a gene-silencing effect using cell’s own enzymatic trigger of RNAi process. (B) It is first processed by the RNAse III enzyme
activities and protein complexes, shows that the process of RNA 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
interference is a normal cellular pathway evolved to perform synthesized outside the cell and then introduced into a cell. (D) The siRNAs are
specific tasks. These include antiviral defense, regulation of incorporated into the RNA-inducing silencing complex (RISC) which consists
the gene expression and protection of the genome. of the RNAse Argonaute as one of its main components. At this point one of
• Antiviral defense mechanism 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 sense-
Prokaryotic cells have developed the system of restriction antisense hybridization. (F) mRNA is cut by RISC and RISC itself is released
enzymes, which cut any foreign DNA that eventually appears for interaction with another molecule mRNA
in the cells. The higher organisms also possess effective
ways to fight foreign DNA, the most common being the virus exists in eukaryotic cells. However, only lately it has been
induced apoptosis. To avoid these defense mechanisms many discovered that they play the role to control the amount of
eukaryotic viruses have evolved into RNA viruses and many different mRNAs. They are transcribed from regions of the
plant, animal and human viruses such as HIV for instance, are genome with specific sequence symmetry and after transcription
dsRNA viruses. Eukaryotic cells have reacted to this challenge the resulting RNAs fold back to form a hairpin structures with
by developing the RNA interference. By this pathway any viral double-strand stem and a loop. These RNAs are called hairpin
dsRNA is chopped by the cellular Dicers and then incorporated RNAs. They are recognized by the Dicers and chopped to
into the cellular RISCs to degrade the viral mRNAs. Thus give small hairpin RNAs (shRNA). They have homology to
RNAi seems to serve as an antiviral immune system evolved different mRNAs and could degrade the latter thus maintaining
to protect eukaryotic cells from invasion of foreign genetic the mRNA populations within certain limits. Over 30% of the
(RNA) material. genes are fine regulated by this mechanism.
• Gene expression regulation • Genome stability maintenance
Since RNAi degrades mRNAs and is conveniently The RNAi is also used to control the spread of transposons.
positioned between transcription and translation, it can regulate Transposons are like the viruses that are inserted into the
the gene expression, i.e. the rate of the respective gene product genomes. A good example are the Alu elements which
synthesis, without changing the gene activity. Cells are using represent a few hundred bp DNA sequence repeated over 105
this way to achieve “fine tuning” of the expression without times in the human genome. Transposons can arrange their own
interfering with transcription. It has long been known that a transcription to produce RNA copies. These RNA copies are
class of short dsRNA molecules called micro RNA (miRNA) then used as templates to synthesize complementary DNA by a
284 BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/3
process known as reverse transcription. Then the DNA strand knock down would inhibit viral replication or its ability to
is replicated by the normal cellular replication machinery to infect cells. The same result could also be achieved if host
produce dsDNA copies of the original Alu element, which cells are treated with dsRNA against mRNA for cell surfice
are inserted randomly in the genome. If left unchecked this receptors specific for the virus. In this case the virus will not
process would lead to ever increasing number of Alu elements. infect the cells since it will not find the receptors on the cell
Apart from increasing the so called “junk” DNA this process surface.
represents a treat to the integrity of the genome since the Alu Most of the neurodegenerative diseases are result of minor
elements could be inserted in exons or other important regions nonessential mutations in some of the genes coding for specific
and to interfere with their normal function. To avoid this cells class of proteins called amyloids. These mutations occur only
recruit the RNAi mechanism. Certain DNA regions contain in one of the alleles and produce altered proteins which tend
sequences which after transcription give rise to shRNAs with to aggregate. The peculiar thing is that this aggregation can
homology to the transposon sequences. They are cut by the involve the normal protein as well thus causing the aggregation
Dicers and used by the RISCs to degrade the initial transposone of essentially all protein molecules, both mutated and normal,
RNA transcripts thus maintaining the genome stability. to form extracellular deposits of amyloid filaments. The
RNAi application obvious approach to treat such a condition is to silence the
mutated gene thus allowing the normal gene to express the
RNAi holds many promises as antiviral treatment and for
normal protein that would not aggregate alone. In both cases
controlling gene expression in eukaryotic cells. However, for
preliminary experiments on animal models with siRNAs have
the time being it is only used as experimental tool. There is
shown very promising results. However, in these and all other
hardly any molecular biology or molecular genetics lab in the
cases the problem is not the siRNA itself, but its delivery into
world that is not using RNAi to knock down different genes to
the target cells. To achieve the desired therapeutic effect it is
study their functioning. The procedure is simple, and the “knock
not enough siRNA to reach and enter few cells. For successful
down” is reversible which represent clear advantages over the
therapy the siRNA has to be introduced in all cells of the
so called “knock out” procedures in which the gene is deleted
affected tissue which makes the things difficult. Relatively
or irreversibly damaged. As for its application in the clinics
simple are the situations when these tissues are easily accessible
– it still seems several years in the future, although a few trials
and siRNA can be applied directly. Such are the cases with
on patients have been reported. In one of them the company
macular degeneration treated by direct injection of siRNA in
Acuity Pharmaceuticals in Philadelphia has completed safety
the eye, the RSV infection, when the drug is administered by
tests on RNAi treatment for macular degeneration, the leading
inhalation, and of certain vaginal and anal viral infections when
cause for blindness in the elderly. This disease is triggered by
the drug is directly applied as ungventum. In all these cases to
the loss of control on the expression of a protein called VEGF
help siRNA to get into the cells it was complexed with lipids
(Vascular Endothelial Growth Factor) which is responsible
or chemically modified to minimize its charge. On the other
for the formation of the blood vessels. Normally this protein
hand, for most of the diseases expected to treat with siRNA,
is not expressed in the retina which stays transparent and
the direct application is not an option. To this end specific and
clear. However, when it is expressed, extensive blood vessel
at the same time harmless for the host organism vectors have
formation occurs there and the retina losses its transparency
to be constructed. They should be able to infect cells with high
thus causing blindness. In the trial a group of about 24 people
efficiency and to integrate in their genomes and should carry
have undergone direct injection of dsRNA against VEGF
cloned DNA sequences that after transcription would give the
mRNA in the eye. Two months after being injected with the
therapeutic shRNAs.
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
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