ERA’S JOURNAL OF MEDICAL RESEARCHVOL.2 NO.1
AN INSIGHT TO RNA INTERFERENCE BASED GENE SILENCING
Syed Tasleem Raza
Department of Biochemistry,
Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh India– 226003.
The discovery of RNA interference (RNAi) is among the most signiﬁcant
Address for correspondence
biomedical breakthroughs in recent history. Multiple classes of small
RNA, including small–interfering RNA (siRNA) and micro–RNA
Dr. Syed Tasleem Raza
(miRNA) play important roles in many fundamental biological and
(Department of Biochemistry)
disease processes. RNA interference, triggered by double–stranded RNA
Era's Lucknow Medical College and
molecules, was initially recognized as a handy tool to reduce gene
expression but now it is recognized as a mechanism for cellular protection
and cleansing. It defends the genome against molecular parasites such as
Ph +91 522 2408122, 2408123
viruses and transposons, while removing abundant but aberrant
Fax: +91 5222407824
nonfunctional messenger RNAs. Nonetheless, these new pools of
knowledge have opened up avenues for unraveling the ﬁner details of the
small RNA mediated pathways. In this paper, we discuss the molecular
aspects in biomedical research of RNA interference and its applications. .
Key words: RNA interference, gene silencing, miRNA, siRNA, mRNA targeting.
RNA degradation process called post transcriptional
The studies on RNA led to the discovery of different
gene silencing (PTGS/RNAi) (1). This phenomenon
classes of RNA's like, messenger RNA (mRNA), transfer
was ﬁrst observed by Fire and Mello in the year 1998
RNA (tRNA), ribosomal RNA (rRNA) and small nuclear
for which they were awarded Nobel Prize in 2006. The
RNA (snRNA). Each of the different types of RNA are
central role in this process is mediated by two types of
encoded by their own speciﬁc gene and they having
small RNA molecules miRNA and siRNA. These
various important roles in our body such as mRNA
double stranded molecules trigger suppression of gene
encodes amino acid sequence of various protein
activity in a homology–dependent manner. This
molecules, tRNA helps in bringing different amino acids
phenomenon is seen in many eukaryotes and is initiated
to the ribosomes during the process of translation, rRNA
by an endonuclease DICER. The natural functions of
along with various ribosomal proteins form ribosomes,
RNAi have been found to be mainly in cellular defense
snRNA which are only found in eukaryotes and help in
against viruses, genomic containment of
RNA processing. Apart from these, the discovery of an
retrotransposons, and post–transcriptional regulation of
independent novel class of RNA molecules (miRNA and
gene expression. Infact, RNAi is proving to be of
siRNA) was made which were comparatively small
immense importance in the present scenario for
(~19–30 nts), and were initially thought to be the
treatment of various diseases in plants/animals, drug
degradation products of larger RNA molecules. It is these
designing, crop improvement, increasing the quality of
small RNAs which are now considered to regulate many
food, pest/weed control and will most likely lead to
cellular processes in the eukaryotes like replication,
novel medical applications in the future.
transcription, translation, chromosome structure, RNA
editing. RNA interference (RNAi) is a novel gene
COMPONENTS OF RNAi MACHINERY
regulatory mechanism that regulates a number of
Analysis of mutants defective in RNAi revealed a
processes within living cells. It limits the transcript level
number of proteins and enzymes essential to this
by either suppressing transcription, transcriptional gene
process. Some of the components identiﬁed serve as
silencing (TGS) or by activating a sequence speciﬁc
initiators and others as effectors, ampliﬁers and
42 ERA’S JOURNAL OF MEDICAL RESEARCHJan.–June.2015VOL.2 NO.1
dsRNAs that are ﬁnally the targets for sequence–
transmitters of gene silencing process.
speciﬁc RNA degradation (15,16).
Small interfering RNAs (siRNAs)
The enzyme was ﬁrst discovered in Drosophila (2). It
It was in 1999 when Hamilton and Baulcombe reported
belongs to the RNase III–family, which show speciﬁcity
the presence of 21–25 nt. long dsRNA complimentary
for dsRNA and cleave them with 3' overhangs of 2 to 3
to both strands of silenced genes in Arabidopsis
nucleotides and 5'–phosphate and 3'–hydroxyl termini
undergoing PTGS called siRNA's. They contain two
(3).They can cleave long dsRNAs and stem–loop
nucleotide 3'overhangs (17). These RNA were also
precursors into siRNAs and miRNAs in an ATP–
seen to silence expression in naïve Drosophila S2 cells
dependent manner, respectively (4). Dicer contains
and embryo extracts (17,18).
several characteristic domains, a N–terminal helicase
domain, PAZ domain (found in Piwi/Argonaute/Zwille
Four types of siRNAs have been identiﬁed
proteins), dual RNase III domains, and a double stranded
depending on the biogenesis of dsRNA precursor
RNA–binding domain (2).Animals generally encode a
and its source.
single type of dicer with exceptions of Drosophila and C.
elegans which encode two dicers (5). Unlike animals,
·Trans–acting short interfering RNAs (tasiRNA):
plants usually encode multiple dicers. In Arabidopsis,
They are ~21 nt. long and closely related to
DCL 2, 3, 4 are involved in the generation of different
miRNAs in size and function. They require
siRNA species while DCL1 solely helps in miRNA
endogenous transcript as template,(19,20) and
synthesis. Each dicer produces siRNAs of characteristic
RdRP, AGO–7 and DCL–4 for their production
length, e.g. DCL2, DCL3 and DCL4 generate 22, 24 and
from template transcript (21). Organisms which
21 nt long siRNA species, respectively (6–8).
lack RdRP like humans and ﬂies, are devoid of
RNA–induced silencing complex (RISC) and
·Repeat–associated short interfering RNAs
(rasiRNAs): These are ~24–26 nts. long and
RNA interference is initiated in cell's cytoplasm by short
require DCL3 and RdRP for their synthesis. They
double stranded RNA molecules where they interact with
function in gametogenesis in ﬂies and in silencing
RISC located in ribosome .The RISC proteins have been
of viral transcripts.
identiﬁed by mass spectrometry as Argonaute 2,(9)VIG,
Dfxr (10) and TSN. The duplex siRNAs are unwound by
·Scan RNAs (scnRNAs): They are much longer,
helicase activity of Argonaute. Argonaute has three
~29 nts. long. It was ﬁrst discovered in a protozoan
domais, PAZ (near N–terminal), MID and PIWI (near C–
Tetrahymena thermophila, where it produced
terminal) domains (11). Animals and plants encode
modiﬁed newer versions genome from the existing
multiple Argonaute homologs like AGO1, AGO2, AGO6
etc.(12). AGO6 is involved in DNA methylation and
· Long siRNAs (lsiRNAs)– They are ~30–40 nt. long
transcriptional gene silencing(13) while AGO7 has been
and require AGO–7, DCL–1 and DCL–4 for their
shown to participate in both tasi–RNA and long siRNAs
biogenesis. They cause decapping of target
transcripts and ﬁnally their degradation via. an
They are a diverse array of proteins speciﬁc for each
The target speciﬁcity of siRNA require a “seed region”
organism seems to carry out aberrant RNA elimination
which occurs at the 5' end of siRNA encompassing
surveillance in most eukaryotes. The A. thaliana mutant
ribonucleotides between 2 to 7 position and confer
SDE3, defective in production of RNA helicase is unable
target speciﬁcity to siRNA. The siRNA containing a
to carry out RNAi process. The SMG–2 homolog from
single mismatch with their substrate fail to repress the
C. elegans have ATPase, RNA binding and helicase
target mRNA and don't simply shift their regulatory
activities and contain conserved cysteine–rich region
mode to translation inhibition.
near the C–terminus.
RNA–dependent RNA polymerase (RdRP)
The miRNA were discovered in 1993. Lee and
The major function of this protein is to generate
colleagues in 2004 elucidated the function of a non–
secondary siRNAs in a primer–dependent and
coding transcript in C. elegans, whose expression
independent fashion and in amplifying silencing effect.
varied spatio–temporally and the mutants showed
The RdRP enzymes recognize the aberrant RNAs as
developmental abnormalities (5). MicroRNA genes
templates and synthesize antisense RNAs to form
43 AN INSIGHT TO RNA INTERFERENCE BASED GENE SILENCING
RISC associated proteins with cap or poly–A tail
constitute ~1% of the total coding genes and form the
associated proteins (25,37).miRNA–repressed
largest class of regulatory molecules (23,24). They show
transcripts in animals are engulfed into dynamic
high tissue–speciﬁc and temporal expression and are
vesicles called P (Processing)–bodies (GW1 or
believed to have evolved to take intensive care of
cytoplasmic bodies) that carry out active mRNA
developmental pathways that can be achieved by
degradation via nonsense mediated decay and gene
translation suppression (occurring mainly in animals) or
silencing (38,39). Target mRNA, via interaction
target cleavage (occurring mainly in plants) (25,26).
between one of the RISC members (AGO1) and the P–
They have been named variously i.e., miRNAs which
body proteins (GW1 and AIN1) gain entry into these
mediate spatial development are referred to as sdRNAs,
structures (40). These bodies act as storage sites for
while cell cycle miRNAs are referred to as ccRNAs, etc.
translationally suppressed mRNA that are released
Their biogenesis is believed to be operative by more than
when required and can actively translate (41). P–bodies
one pathway, as described below:
have been shown to be associated with various
components of translation machinery (except
1. Canonical miRNA pathway: Micro–RNAs are ~19–
ribosomes) and RNAi components like AGO1,
23 nucleotide long single–stranded RNAs. Prior to
GW182, miRNAs and CCR4 (41). These are yet to be
maturity, they ﬁrst undergo extensive post–
discovered in plants but some similar nuclear foci
transcriptional modiﬁcation. They are expressed from a
called Cajal bodies have recently been reported from
much longer RNA–coding primary transcript known as a
pri–miRNA which is processed, in the cell nucleus, to a
70–nucleotide stem–loop structure called a pre–miRNA
by the microprocessor complex. This complex consists
The process of RNA interference can be divided into
of an RNase III enzyme called Drosha and a dsRNA–
four stages, which are as follows:
binding protein DGCR8. The dsRNA portion of this pre–
miRNA is bound and cleaved by Dicer to produce the
1. Double stranded RNA cleavage: The ﬁrst step
mature miRNA molecule that can be integrated into the
includes ATP–dependent, processive dsRNA cleavage
RISC complex. Maturation of miRNA begins in the
into double–stranded fragments 21 to 25 nucleotides
nucleus and terminates in the cytoplasm.
long containing 5' phosphate and 3' hydroxyl termini,
and two additional overhanging nucleotides on their
3.6.2 Mirtrons:Animals have been shown to follow yet
3'ends (17,43). The length of the cleaved RNA
another mode of miRNA biogenesis where intron
maximizes target–gene speciﬁcity and minimizes non–
sequences can produce miRNAs. These miRNAs
speciﬁc effects. Exogenous dsRNA is detected and
originating from introns were termed mirtrons (27,28).
bound by an effector protein, known as RDE–4 in C.
Fourteen mirtrons from Drosophila and four mirtrons
elegans and R2D2 in Drosophila that stimulates dicer
from C. elegans have been identiﬁed so far. Mirtrons
activity. In C. elegans, this initiation response is
have not yet been identiﬁed in plants and other
ampliﬁed through the synthesis of a population of
'secondary' siRNAs by an RNA–dependent RNA
The miRNAs are thought to direct RISC–related
polymerase (RdRP) during which the dicer–produced
complexes to their targets on the basis of sequence
initiating or 'primary' siRNAs are used as templates
complimentarity. In contrast to this, most animal
miRNAs appear to recognize their targets by imperfect
pairing. For example, the interaction between let–7 and
2. Activation of silencing complex: In the second step
its target lin–14 involves multiple binding sites, each of
siRNAs are incorporated into RISC (RNA–induced
which pairs imperfectly with the small RNA. This
silencing complex) which undergoes activation in the
problem doesnot exist in plants where almost all
presence of ATP so that the antisense component of the
miRNAs are extensively complimentary to their
unwound siRNA becomes exposed and allows the
targets,(29,30) and act as siRNAs to direct
RISC to perform the downstream RNAi reaction. This
endonucleolytic cleavage of target mRNAs (29,31,32).
siRNA duplex containing ribonucleoprotein complex
is called siRNP (44). This formation requires ATP
Animal miRNA, as a general rule, binds to the 3′ UTR
which reﬂects energy need for unwinding step and
region of the target,(33,34) while in caseof plants,
other conformational requirements. Members of
binding occurs in the coding region (35). The seed region
Argonaute family probably help in stabilization of
determines the target sequence and helps in miRNA
siRNA, RISC formation and mRNA targeting. siRNA,
editing (36). In animals, studies suggest that miRNA
RISC formation and mRNA targeting.
binding promotes either deadenylation or decapping of
3. Unwinding of the siRNA duplex: The third step
the target which is probably achieved by interaction of
44 ERA’S JOURNAL OF MEDICAL RESEARCHJan.–June.2015VOL.2 NO.1
generalized response to pathogens that downregulates
involves unwinding of the siRNA duplex and
any metabolic processes in the host that aid the
remodelling of the complex to create an active form of
infection process. In both juvenile and adult
RISC. Only one of the two strands, which is known as the
Drosophila, RNA interference is important in antiviral
guide strand, binds the argonaute protein and directs gene
innate immunity and is active against pathogens such as
silencing. The other anti–guide strand or passenger strand
Drosophila X virus. A similar role in immunity may
is degraded. This process is ATP–independent and
operate in C. elegans, as argonaute proteins are
performed directly by the protein components of RISC.
upregulated in response to viruses and worms that over
The strand selected as the guide tends to be the one whose
express components of the RNAi pathway are resistant
5' end is less stably paired to its complement. The R2D2
to viral infection.
protein may serve as the differentiating factor by binding
the more–stable 5' end of the passenger strand. The
Down and up–regulation of genes
phosphorylated 5' end of the RNA strand enters a
Endogenously expressed miRNAs, including both
conserved basic surface pocket and makes contacts
intronic and intergenic miRNAs, are most important in
through a divalent cation and by aromatic stacking
translational repression.The roles of endogenously
between the 5' nucleotide in the siRNA and a conserved
expressed miRNA in downregulating gene expression
were ﬁrst described in C. elegans in 1993. In plants, the
majority of genes regulated by miRNAs are
4. Cleavage of mRNA: The ﬁnal step includes the
transcription factors thus miRNA activity is
recognition and cleavage of mRNA complementary to
particularly wide–ranging and regulates entire gene
the siRNA strand present in RISC (guide strand of
networks during development by modulating the
siRNA),(44,45) by exoribonucleases. In some organisms
expression of key regulatory genes, including
(C. elegans, Arabidopsis thaliana) an additional step in
transcription factors as well as F–box proteins. RNA
the RNAi pathway has been described involving a
sequences (siRNA and miRNA) that are
population of secondary siRNAs derived from the action
complementary to parts of a promoter can increase
of a cellular RNA–dependent RNA polymerase (RdRp).
gene transcription, a phenomenon dubbed RNA
Eukaryotic cells possess two major means for regulating
activation. Here, dicer and argonaute play major role,
the turnover of mRNAs. In one pathway they utilize the
possibly via histone demethylation. They have also
3'–5' exonuclease, exosome, to degrade the message in
been proposed to upregulate their target genes upon cell
the cytoplasm (46). The other pathway is thought to
occur in specialized centers known as processing (P)
bodies/cytoplasmic bodies/mRNA decay centers
(38,39). These centers contain decapping enzymes Dcp1
Mutations in dcr–1 and ego–1 lead to complete
and 2 and the 5'–3' exonucleases Xrn1(39). It is thought
sterility,(50,52) indicating importance of RNAi in
that transcripts are transported to these centers to be
germline development. In plants also RNAi–like
degraded by the 5'–3' exonuclease, Xrn1. Ago2 was
processes have a crucial role in development (53,54). It
found to be localized to the cytoplasm, with most of the
is seen to be involved in maturation of endogenously
Ago2 concentrated in discrete cytoplasmic bodies, the
encoded miRNAs,(51,55) some of which are involved
mammalian equivalent of yeast P–bodies (25,40).
in development, but most of which have no known
function at present.
FUNCTIONAL ASPECTS AND APPLICATIONS
Gene function analysis
Double–stranded RNA is artiﬁcially synthesized,
RNA interference acts as a defense mechanism against
viruses and other foreign genetic material, especially in
complementary to a gene of interest and introduced into
plants. It may also prevent the self–propagation of
a cell where it is recognized as exogenous genetic
transposons. Apart from plants there is signiﬁcant debate
material and activates the RNAi pathway. Using this
over the ability of siRNAs and longer dsRNAs to induce
mechanism, researchers can cause a drastic decrease in
innate immune response (47,48). In mammalian cells
the expression of a targeted gene which can show the
molecules less than 30 bp in length are generally believed
physiological role of the gene product. Functional
to avoid induction of interferon pathways (47–49). Long
analysis of almost all the ~19,000 genes of C. elegans
(27–29 bp) dsRNAs and shRNAs provide more efﬁcient
gene silencing than shorter, Dicer–independent
has been carried out with the siRNA–directed
substrates (48,49). Some plant genomes also express
knockdown approach (56).
endogenous siRNAs in response to infection by speciﬁc
types of bacteria. These effects may be part of a
45 AN INSIGHT TO RNA INTERFERENCE BASED GENE SILENCING
RNA INTERFERENCE AS A THERAPEUTIC
To determine gene function
·RNAi is sequence–speciﬁc and thus can be
RNAi is having a lot of applications in biomedical
targeted, requiring only a few transformants per
research and health care and has begun to produce a
paradigm shift in the process of drug discovery (60).
·RNAi is dominant, so phenotypes can be
Presently, many dsRNA molecules are being designed
observed in the T1 generation.
for silencing speciﬁc genes in humans and animals as
shown in Table 1. It may be possible to exploit RNA
·RNAi often leads to partial knockdown and thus
interference in therapy. Although it is difﬁcult to
to a range of phenotypes of differing severity;
introduce long dsRNA strands into mammalian cells
this facilitates the study of essential genes whose
due to the interferon response, the use of ds RNAs
inactivation would lead to lethality or extremely
shorter than ~30 nts are proving to be successful. The
severe pleiotropic phenotypes.
very ﬁrst application of RNAi were mainly in the
treatment of macular degeneration and respiratory
·RNAi can be controlled in a tissue–speciﬁc or
syncytial virus knockdown of host receptors and
time–dependent manner RNAi can be quickly
coreceptors for HIV,the silencing of hepatitis A and
and easily used in a wide range of genotypes or
hepatitis B genes, silencing of inﬂuenza gene
even species, whereas insertion mutant
expression, and inhibition of measles viral replication.
collections are limited to just a few due to the
Potential treatments for neurodegenerative diseases
have also been proposed, with particular attention
·RNAi can be used to reduce the expression of
being paid to the polyglutamine diseases such as
several related genes in parallel by targeting
Huntington's disease. Despite the proliferation of
conserved regions of the genes, facilitating the
promising cell culture studies for RNAi–based drugs,
study of redundant gene functions.
some concern has been raised regarding the safety of
RNA interference, especially the potential for "off–
target" effects in which a gene with a coincidentally
There is derepression of the centromeric outer–
similar sequence to the targeted gene is also repressed.
transposon repeats in RNAi mutants deﬁcient for RNAi
A computational genomics study estimated that the
components. This led to the proposal that small RNAs
error rate of off–target interactions is about 10%.
function as guides to target the chromatin modiﬁcations
that are typical of heterochromatin (57). There is reduced
amounts of H3K9 methylation and repeat–associated
This RNAi technology has proven to be an eco–friendly
Swi6 in the RNAi mutants. Second evidence was that a
technique for crop improvement. Through this
transgene that is located in the centromeric repeats,
technique the genes which are responsible for inducing
which would usually be silenced, was activated in RNAi
various stresses in plants are silenced and novel traits
mutants. Apart from centromeric heterochromatin
like disease resistance are incorporated into plants. It
formation, RNAi pathway is also implicated in the
has emerged as a method of choice for gene targeting in
targeting of non–centromeric, interstitial sites in
fungi,(61) viruses,(62,63) bacteria(64) and plants (65).
euchromatin for silencing. In D. melanogaster, dispersed
It has been used for applications in biotechnology,
transgenes that are inserted at several sites in
particularly in the engineering of food plants that
euchromatin are silenced at the transcriptional level
produce lower levels of natural plant toxins as shown in
through association with the POLYCOMB
Table 2. Other plant traits that have been engineered in
COMPLEX(58)through histone modiﬁcations which is
the laboratory include the production of non–narcotic
dependent on components of RNAi pathway. The
natural products by the opium poppy, resistance to
Polycomb–dependent silencing involves histone
common plant viruses, and fortiﬁcation of plants such
modiﬁcations and is known to keep the chromatin in the
as tomatoes with dietary antioxidants.
closed or compact conformation.Heterochromatin
formation requires that histone H3 of the chromatin is
ﬁrst deacetylated and then methylated at lysine 9. This
DISADVANTAGES OF RNA INTERFERENCE
methylated lysine is subsequently bound by a
heterochromatin binding protein, HP1 in highly speciﬁc
In large–scale screens in animals it was observed that
manner and with a very high afﬁnity (59).
the silencing effects were also seen in genes that were
46 ERA’S JOURNAL OF MEDICAL RESEARCHJan.–June.2015VOL.2 NO.1
silencing vectors that are able to operate in a temporally
not the predicted targets of RNAi. The major difﬁculty is
and spatially controlled manner. In coming years better
the limited sequence speciﬁcity of siRNAs, as few as
and comprehensive understanding of RNAi would
seven nucleotides of sequence complementarity between
allow the researchers to work effectively and efﬁciently
a siRNA and an mRNA can lead to the inhibition of
in order to work more on improvement of crop plants
nutritionally and in managing various diseases of crop
A second speciﬁcity problem can occur via 'transitive
plants. Finally, the discovery of RNAi has not only
silencing', whereby RNAi against a gene–speciﬁc
provided us with a powerful new experimental tool to
sequence 'spreads' into neighbouring sequences
study the function of genes but also raises expectations
conserved between the target mRNA and mRNAs from
about future applications of RNAi in medicine.
related genes, which become silenced in turn.
Inefﬁcacy and instability
CONFLICT OF INTEREST
RNAi inhibition can have widely varying effects
The authors declare that they have no competing
depending on the target gene, the region of the transcript
that is targeted and even between sibling plants carrying
identical RNAi constructs. The instability can result
from silencing of the transgene long hairpin transgenes
appear to be particularly sensitive to transcriptional
1. Agrawal N, Dasaradhi PVN, Mohmmed A,
silencing leading to a loss of RNAi phenotypes over
Malhotra P, Bhatnagar RK, Mukherjee SK.
RNA Interference: Biology, Mechanism, and
Applications. Microbiology and Molecular
Conclusion and Future Prospects
Biology Reviews. 2003; 67” 657–685.
RNA interference is an area of intense, upfront basic
research and holds the key to various technological
2. Bernstein E., Caudy AA., Hammond SM., and
applications in future due to their higher silencing
Hannon GJ. Role for a bidentate ribonuclease in
efﬁciency and shorter time requirements for screening
the initiation step of RNA interference. Nature.
and to analyses functions of wide variety of genes in
2001; 409” 363–366.
different organisms. The RNA silencing technology
apart from being highly sequence speciﬁc is also
3. Zamore PD. RNA Interference: big applause for
technologically facile and economical. Therefore, this
silencing in Stockholm. Cell. 2006: 127”
technique has great potential in agriculture speciﬁcally
for nutritional improvement of plants and the
management of various plant diseases. Future directions
4. Tan FL. and Yin JQ. Application of RNAi to
will focus on developing ﬁnely tuned RNAi–based gene
cancer research and therapy. Front. Biosci.
Table1. Application of RNAi in treating human disease (66)
Lymphoblastic leukemia Using siRNA’s speciﬁc for the BCR–ABL transcript to silence
Bladder cancer Treatment by miRNA’s as biomarkers
HIV Downregulation of cellular cofactors required for HIV infection
Viral hepatitis Inhibition of Fas expression by siRNA
Ocular diseases Shutting down production of VEGF by siRNA
Metabolic disease and
Treatment of these diseases by miRNA’s as potencial
neurodegenerative disorders targets.
Cardiovascular and cerebro Used to reduce damage to heart tissues and brain cells
Pancreatic and colon Use of retroviral vectors to introduce interfering RNAs speciﬁc
carcinomasfor an oncogenic variant of K–Ras
47 AN INSIGHT TO RNA INTERFERENCE BASED GENE SILENCING
Table 2. Application of RNAi in plants (67)
Application Case study
Increasing the level of lysine Reduction of lysine catabolism and improving seed germination
generating a dominant high–lysine maize variant by knocking out the expression
of the 22–kD maize zein storage protein
Barley and Rice Resistance of barley to BYDV and producing a rice variety called
LGC–1 (low glutenin content 1)
Banana Production of banana varieties resistant to the Banana Bract Mosaic Virus
Cotton Transgenic cotton plants expressing a RNAi construct of the d–cadinene
synthase gene of gossypol synthesis fused to a seed–speciﬁc promoter caused
seed–speciﬁc reduction of Gossypol
Wood and fruit quality –Down regulation of lignin biosynthesis pathways.
–Producing transgenic hypoallergenic apples and a possible solution for the
undesirable separation of juice into clear serum and particulate phase
Coffee RNAi technology has enabled the creation of varieties of Coffee that produces
natural coffee with low or very low caffeine content
Healthier oil Using RNAi to silence the gene in cotton which codes for the enzyme that
converts oleic acid into a different fatty acid
Tomato RNAi–mediated suppression of DET1 expression under fruit–speciﬁc promoters
has recently shown to improve carotenoid and ﬂavonoid levels in tomato fruits
with minimal effects on plant growth
Gentian Producing white–ﬂowered transgenic gentians by suppressing the chalcone
synthase (CHS) gene .
Blue Rose Producing blue transgenic rose by knock downing the cyanidin genes in rose and
Pest control Combining Bt technology with RNAi would both enhance product performance
and further guard against the development of resistance to Bt proteins
Fig1. Schematic representation of four–step gene silencing pathway (68)
1st step, generation
ADP + Pi, DICER
Argonaute family member
ADP + Pi
active RISC* formation
additional step in plants, fungi
and C. elegans including the generation
4th step, recognition
of secondary siRNAs by RdRp activity
and mRNA cleavage
48 ERA’S JOURNAL OF MEDICAL RESEARCHJan.–June.2015VOL.2 NO.1
2005: 10” 1946–1960.
16. Cogoni, C., and G. Macino. Gene silencing in
Neurospora crassarequires a protein
5. Lee YS, Nakahara K, Pham JW, Kim K, He Z,
homologous to RNA–dependent RNA
Sontheimer EJ, Carthew RW, Distinct roles for
polymerase. Nature. 1999: 399” 166–169.
Drosophila Dicer–1 and Dicer–2 in the
siRNA/miRNA silencing. Cell. 2004: 117(1)” 69–
17. Elbashir S. M., Lendeckel W., and Tuschl T.
RNA interference is mediated by 21– and 22–
nucleotide RNAs. Genes Dev. 2001: 15” 188–
6. Deleris A, et al. Hierarchical action and inhibition
of plant Dicer–like proteins in antiviral defense.
Science. 2006: 313” 68–71.
18. Yang D., Lu H., and Erichson J. W. Evidence
that processed small dsRNA may mediate
7. Blevins T, Rajeswaran R, Shivaprasad PV, et al.
sequence speciﬁc mRNA degradation during
Four plant Dicers mediate viral small RNA
RNAi in Drosophila embryos. Curr. Biol. 2000:
biogenesis and DNA virus induced silencing.
Nucleic Acids Res. 2006: 34(21)” 6233–46.
19. Talmor–Neiman M, Stay R, Klipcan L, Kobi B,
8. Xie Z, Allen E, Wilken A, Carrington JC. Dicer–
Baulcombe DC, Arazi T. Identiﬁcation of trans–
like 4 functions in trans–acting small interfering
acting siRNAs in moss and an RNA–dependent
RNA biogenesis and vegetative phase change in
RNA polymerase required for their biogenesis.
Arabidopsis thaliana. Proc Natl Acad Sci USA.
Plant J. 2006: 48(4)” 511–21.
2005: 102(36)” 12984 – 9.
20. Fahlgren N, Montogomery TA, Howell MD, et
9. Hammond SM, Boettcher S, Caudy AA,
al. Regulation of AUXIN RESPONSE
Kobayashi R, Hannon GJ. Argonaute2, a link
FACTOR3 by TAS3 ta–siRNA affects
between genetic and biochemical analyses of
developmental timing and patterning in
RNAi. Science. 2001: 293” 1146–1150.
Arabidopsis. Curr Biol. 2006: 16(9)” 939–44.
10. Caudy AA., Myers M., Hannon GJ. and
21. Montgomery TA, Howell MD, Cuperus JT.
Hammond SM. Fragile X–related protein and
Speciﬁcity of ARGONAUTE7–miR390
VIG associate with RNA interference machinery.
interaction and dual functionality in TAS3
Genes Dev. 2002: 16” 2491–2496.
trans–acting siRNA formation. Cell. 2008:
11. Cerutti L, Mian N, Bateman A. Domains in gene
silencing and cell differentiation proteins: the
22. Liu J, Carmell MA, Rivas FV, Marsden CG,
novel PAZ domain and redeﬁnition of the Piwi
Thomson JM, et al. Argonaute2 is the catalytic
domain. Trends Biochem. Sci.2000: 25” 481–82
engine of mammalian RNAi. Science. 2004:
12. Okamura K, Ischizuka A, Siomi H, Siomi MC.
Distinct roles for Argonaute proteins in small
RNA– directed RNA cleavage pathways. Genes
23. Grad Y, Aach J, Hayes GD, Reinhart GD,
Dev. 2004: 18(14)” 1655–66.
Church GM, Ruvkun G, et al. Computational
and experimental identiﬁcation of C. elegans
13. Zheng X, Zhu J, Kapoor A, Zhu JK. Role of
microRNAs. Mol Cell. 2003: 11(5)” 1253–63.
Arabidopsis AGO6 insiRNA accumulation, DNA
methylation and transcriptional gene silencing.
24. Bartel DP. MicroRNAs: genomics, biogenesis,
EMBO J. 2007: 26(6)” 1691–701.
mechanism, and function. Cell. 2004: 116(2)”
14. Katiyar–Agarwal S, Gao S, Vivian–Smith A, Jin
H. A novel class f bacteria–induced small RNAs in
25. Pillai, RS, Bhattacharyya SN, Artus CG, Zoller
Arabidopsis. Genes Dev. 2007: 21(23)” 3123–34.
T, Cougot N, Basyuk E, et al. Inhibition of
translational initiation by let–7 MicroRNA in
15. Cogoni C. and Macino G. Conservation of
human cells. Science 2005: 309” 1573–1576.
transgene–induced post–transcriptional gene
silencing in plants and fungi. Trends Plant Sci.
26. Aukerman, M.J. and Sakai, H. Regulation of
1997: 2” 438–443.
49 AN INSIGHT TO RNA INTERFERENCE BASED GENE SILENCING
ﬂowering time and ﬂoral organ identity by a
microRNA and its APETALA2–like target genes.
38. Sheth U, and Parker R. Decapping and decay of
Plant Cell. 2003: 15” 2730–2741
messenger RNA occur in cytoplasmic
processing bodies. Science. 2003: 300”
27. Okamura K, Hagen JW, Duan H, Tyler DM, Lai
EC. The mirtron pathway generates microRNA–
class regulatory RNAs in Drosophila. Cell. 2007:
39. Cougot N, Babajko S, Seraphin B. Cytoplasmic
foci are sites of mRNA decay in human cells. J
Cell Biol. 2004: 165(1)” 31–40.
28. Ruby JG, Jan CH, Bartel DP. Intronic microRNA
precursors that bypass Drosha processing.
40. Liu J, Valencia–Sanchez MA, Hannon GJ,
Nature. 2007: 448(7149)” 83–6.
Parker R. MicroRNA–dependent localization of
targeted mRNAs to mammalian P–bodies. Nat
29. Llave C, Xie Z, Kasschau KD and Carrington JC.
Cell Biol. 2005: 7(7)” 719–23.
Cleavage of Scarecrow–like mRNA targets
directed by a class of Arabidopsis miRNA.
41. Bhattacharyya SN, Habermacher R, Martine U,
Science. 2002: 297” 2053–2056.
Closs EI, Filipowicz W. Relief of microRNA
mediated translational repression in human
30. Rhoades MW, Reinhart BJ, Lim LP, Burge CB,
cells subjected to stress. Cell 2006: 125(6)”
Bartel B et al. Prediction of plant microRNA
targets. Cell. 2002: 110(4)” 513–20.
42. Song L, Han MH, Lesicka J, Fedoroff N.
31. Kasschau K, Xie Z, Allen E, Llave C, Chapman E,
Arabidopsis primary microRNA processing
Krizan K et al. P1/HC–Pro, a viral suppressor of
proteins HYL1 and DCL1 deﬁne a nuclear body
RNA silencing, interferes with Arabidopsis
distinct from the cajal body. Proc Natl Acad Sci
development and miRNA function. Dev. Cell.
USA. 2007: 104(13)” 5437–42.
2003: 4” 205–217.
43. Hamilton AJ, Baulcoumbe DC. A species of
32. Tang G, Galili G. Using RNAi to improve plant
small antisense RNA in posttranscriptional gene
nutritional value: from mechanism to application.
silencing in plants. Science. 1999: 286(5441)”
TRENDS in Biotechnology. 2003: 22(9)” 463–
44. Nykanen A, Haley B, Zamore PD. ATP
33. Lewis B, Burge C, Bartel D. Conserved seed
requirements and small interfering RNA
pairing, often ﬂanked by adenosines, indicates
structure in the RNA interference pathway. Cell
that thousands of human genes are microRNA
2001: 107” 309–21.
targets. Cell. 2005: 120(1)” 15–20.
45. Hutvagner G, Zamore PD. RNAi: nature abhors
34. Brennecke J, Stark A, Russell RB, Cohen SM.
a double–strand. Curr Opin Genet Dev. 2002:
Principles of microRNA– target recognition.
PLoS Biol. 2005: 3(3)” 85.
46. Anderson J. S. and Parker R. P. The 3´ to 5´
35. Reinhart BJ, Weinstein EG, Rhodes MW, Bartel
degradation of yeast mRNAs is a general
B and Bartel DP. MicroRNAs in plants.Genes
mechanism for mRNA turnover that requires
Dev. 2002: 16” 1616–1626.
the SKI2 DEVH box protein and 3´to 5´
exonucleases of the exosome complex. EMBO
36. Blow MJ, Grocock RJ, van Dongen S. RNA
J. 1998: 17” 1497–1506
editing of human microRNAs. Genome Biol.
2006: 7(4)” R27.
47. Manche L, Green SR, Schmedt C. and Mathews
MB. Interactions between double–stranded
37. Wakiyama M, Takimoto K, Ohara O, Yokoyama
RNA regulators and the protein kinase DAI.
S. Let–7 microRNA– mediated mRNA
Mol. Cell. Biol. 1992: 12” 5238–5248.
deadenylation and translational repression in a
mammalian cell–free system. Genes & Dev. 2007:
48. Kim DH, Behlke MA, Rose SD, Chang MS,
50 ERA’S JOURNAL OF MEDICAL RESEARCHJan.–June.2015VOL.2 NO.1
58. Fire A. RNA triggered gene silencing. Trends
Choi S. and Rossi JJ. Synthetic dsRNA Dicer
substrates enhance RNAi potency and efﬁcacy.
Genet. 1999: 15” 358–363.
Nat. Biotechnol. 2004: 23” 222–226.
59. Bannister AJ, Zegerman P, Partridge JF, Miska
49. Siolas D, Lerner C, Burchard J, Ge W, Linsley PS,
EA et al. Selective recognition of methylated
Paddison PJ. Synthetic shRNAs as potent RNAi
lysine 9 on histone H3 by the HP1 chromo
triggers. Nat. Biotechnol. 2004: 23” 227–231.
domain. Nature. 2001; 410” 120–124.
50. Smardon A, Spoerke JM, Stacey SC, Klein N,
60. Hannon GJ and Rose JJ. Unlocking the potential
Mackin ME, and, Maine EM. EGO–1 is related to
of the human genome with RNA interference.
RNA–directed RNA polymerase and functions in
Nature 2004; 431” 371–378.
germ–line development and RNA interference in
C. elegans. Curr. Biol. 2000: 10” 169–178.
61. Nakayashiki. RNA Silencing in Fungi:
Mechanisms and Applications. Federation of
51. Ketting RF, Fischer SE, Bernstein E, Sijen T,
European Biochemical Societies Letters. 2005;
Hannon GJ, and Plasterk RHA. Dicer functions
in RNA interference and in synthesis of small RNA
involved in developmental timing in C.
62. Baulcombe DC. RNA silencing in Plants.
elegans.Genes Dev. 2001: 15” 2654–2659
Nature. 2004. 431” 356–363.
52. Knight SW, Bass BL. A role for the RNase III
63. Wani SH. and Sanghera GS. Genetic
enzyme DCR–1 in RNA interference and germ
Engineering for Viral Disease Management in
line development in Caenorhabditis elegans.
Plants. Notulae Scientia Biologicae. 2010; 2”
Science 293” 2269–71.2001:
53. Bohmert K, Camus I, Bellini C, Bouchez D,
64. Escobar MA, Civerolo EL, Summerfelt KR. and
Caboche M, and Benning C. AGO 1 deﬁnes a
Dandekar AM. RNAi–Mediated Oncogene
novel class of Arabidopsis controlling leaf
Silencing Confers Ressitance to Crown Gall
development. EMBO J. 1998: 17” 170–180.
Tumorigenesis. Proceedings of the National
Academy of Sciences USA. 2001; 68(23)”
54. Jacobsen SE, Running MP and Meyerowitz EM.
Disruption of an RNA helicase/RNAseIII gene in
Arabidopsis causes unregulated cell division in
65. Brodersen P and Voinnet O. The Diversity of
ﬂoral meristems. Development 1999: 126” 5231–
RNA Silencing Pathways in Plants. Trends in
Genetics 2006; 22” 268–280.
55. Grishok A, Pasquinelli AE, Conte D et al. Genes
66. Angaji SA, Hedayati SS, Poor RH et al.
and mechanisms related to RNA interference
Application of RNA interference in treating
regulate expression of the small temporal RNAs
human diseases. J. Genet. 2010; 89” xx–xx
that control C. elegans developmental timing.
Cell. 2001: 106” 23–34.
67. Angaji SA, Hedayati SS, Poor RH. et al.
Application of RNA interference in plants. POJ
56. Dykxhoorn DM, Novina CD, and Sharp PA.
2010; 3(3)” 77–84.
Killing the messenger: short RNAs that silence
gene expression. Nat. Rev. Mol. Cell. Biol. 2003:
68. Zoﬁa Szweykowska–Kuliñska, Artur
Jarmolowski and Marek Figlerowicz. RNA
interference and its role in the regulation of
57. Volpe TA. Regulation of heterochromatic
eucaryotic gene expression. Acta Biochimica
silencing and histone H3 lysine–9 methylation by
Polonica. 2003; 50(1/2003)” 217–229
RNAi. Science 2002: 297” 1833–1837.