PROJECT REPORT

ON
RNAi AND ITS APPLICATIONS IN LIFE SCIENCES

SUBMITTED TO
HIMACHAL PRADESH UNIVERSITY,SUMMER HILL , SHIMLA (H.P)
FOR THE PARTIAL FULFILLMENT OF BACHELORS OF SCIENCE
(HONS) IN BIOTECHNOLOGY
PROJECT SUPERVISOR:

SUBMITTED BY:

Mr. Rajesh Kumar Gupta

Ishan Chauhan
Univ. Roll no. - M-5113

SHOOLINI INSTITUTE OF LIFE SCIENCES AND BUSINESS

MANAGEMENT ANAND CAMPUS, THE MALL, SOLAN-173212

CONTENTS
1. CERTIFICATE
2. ACKNOWLEDGEMENT
3. INTRODUCTION
4. REVIEW OF LITERATURE
5. SIGNIFICANCE OF THE DISCOVERY OF RNAi
6. CELLULAR MECHANISM
7. APPLICATIONS IN MEDICINE
8. BIOLOGICAL APPLICATIONS
9. APPLICATIONS IN AGRICULTURE
10. CONCLUSION
11. REFERENCES

ACKNOWLEDGEMENT

It is the great privilege for me to express my sincere gratitude and indebtness

to Mr. Rajesh Kumar Gupta for giving me opportunity to work under his
expert guidance.
I am indebt to my supervisor who has been model mentor offering constant
encouragement, support and expert advice throughout my project.

Last but not least Im thank full to my parents and room mates for their
inspiration and constant support consoled me whenever I was depressed, their
support and encouragement helped me to perform my task in efficient way
during the critical time.
Mere words would be less to express my respect and love for my parents,
mentor and friends who always stood by me at difficult moments and boosted
my morale.
I solely claim all responsibility for shortcoming and limitations in this project
work.

Ishan Chauhan

Place: - S.I.L.B
Date:-

INTRODUCTION

RNA interference is a natural cellular mechanism involved in regulation of

gene expression. It involves double stranded ribonucleic acid (dsRNA) that
can specifically silence the expression of 2 genes with sequences, which are
complementary to the dsRNA. The components of double stranded RNA
initiate the process of RNAi when an antisense RNA strand is activated,
which targets a complementary gene transcript such as a messenger RNA for
cleavage by a ribonuclease. RNAi has been shown to be an important
regulator of gene expression in many eukaryotes including plants, animals and
humans.

The enzyme dicer trims double stranded RNA, to form small interfering RNA
or microRNA. These processed RNAs are incorporated into the RNA-induced
silencing complex (RISC), which targets messenger RNA to prevent
translation. RNA interference (RNAi) is a system within living cells that helps
to control which genes are active and how active they are. Two types of small
RNA molecules microRNA (miRNA) and small interfering RNA (siRNA)
are central to RNA interference. RNAs are the direct products of genes, and
these small RNAs can bind to specific other RNAs and either increase or
decrease their activity, for example by preventing a messenger RNA from
producing a protein. RNA interference has an important role in defending
cells against parasitic genes viruses and transposons .
The RNAi pathway is found in many eukaryotes including animals and is
initiated by the enzyme Dicer, which cleaves long double-stranded RNA

(dsRNA) molecules into short fragments of ~20 nucleotides. One of the two
strands of each fragment, known as the guide strand, is then incorporated into
the RNA-induced silencing complex (RISC). The outcome is posttranscriptional gene silencing, which occurs when the guide strand base pairs
with a complementary sequence of a messenger RNA molecule and induces
cleavage by Argonaute, the catalytic component of the RISC complex. This
effect of RNAi on gene expression makes it a valuable research tool, both in
cell culture and in living organisms because synthetic dsRNA introduced into
cells can induce suppression of specific genes of interest. Exploitation of the
pathway is also a promising tool in biotechnology and medicine.
Double-stranded RNA synthesized within the cell can reduce or abolish gene
activity by RNAi. This control system for gene expression has proven to be
important for both the development of an organism and the physiological
functions of cells and tissues. Furthermore, RNAi protects against RNA virus
infections, especially in plants and invertebrate animals, and secures genome
stability by keeping mobile elements silent. Today, double-stranded RNA is
used as a powerful tool to experimentally determine the function of essentially
any gene in a cell.

REVIEW OF LITERATURE
Around 1990, Scientists working on petunia plants
reported some unexpected genetic expression while performing the

experiments to breed petunia plants to increase the colour intensity of the

flowers. For this, they introduced cloned gene encoding a key enzyme for red
pigmentation of flowers. Surprisingly, many of the petunia plants with
additional copies of this gene showed petals which appeared fully white or
partially white instead of the expected colour. Thus explained the
phenomenon as post transcriptional gene inhibition or co-suppression of gene
expression.

petunia plants in which genes for pigmentation are silenced by RNAi. Parent
plant contains transgenes that induce suppression of both transgene and
endogenous gene expression, giving rise to the unpigmented white areas of the
flower.
Plant virologists working on improving plant resistance to
viral diseases observed a similar phenomenon. While it was known that
plants expressing virus-specific proteins showed enhanced tolerance or
resistance to viral infection, it was not expected that plants carrying only
short, non-coding regions of viral RNA sequences would show similar levels
of protection. It was believed that viral RNA produced by transgenes could
also inhibit viral replication. Experiment, in which short sequences of plant
genes were introduced into viruses, showed that the targeted gene was
suppressed in an infected plant. This phenomenon was known as "virusinduced gene silencing" (VIGS), and the set of such phenomena were
collectively called post transcriptional gene silencing.
First discovered in plants the nematode Caenorhabditis
elegans, the production of small interfering RNAs (siRNAs) that bind to and
induce the degradation of specific endogenous mRNAs is now recognized as a
mechanism that is widely employed by eukaryotic cells to inhibit protein
production at a post-transcriptional level. The endogenous siRNAs are
typically 19- to 23-base double-stranded RNA oligonucleotides, produced

from much larger RNAs that upon binding to target mRNAs recruit RNases to
a protein complex that degrades the targeted mRNA. Methods for expressing
siRNAs in cells in culture and in vivo using viral vectors, and for transfecting
cells with synthetic siRNAs, have been developed and are being used to
establish the functions of specific proteins in various cell types and organisms.
RNA interference methods provide several major advantages over prior
methods (antisense DNA or antibody-based techniques) for suppressing gene
expression. Recent preclinical studies suggest that RNA interference
technology holds promise for the treatment of various diseases.
Pharmacologists have long dreamed of the ability to selectively antagonize or
eliminate the function of individual proteinsRNAi technology may
eventually make that dream a reality. Prior to the discovery of RNAi,
scientists applied various methods such as insertion of T-DNA elements, and
transposons, treatment with mutagens or irradiation and antisense.
RNA suppression to generate loss-of-function mutations.
These approaches allowed scientists to study the functions of a gene or gene
family of interest in an organism.
Transposons and T-DNA elements were found to occasionally insert
randomly in the genome resulting in highly variable gene expression.
Furthermore, in many instances the particular phenotype or a trait could not be
correlated with the function of a gene of interest eventually leading to the
discovery of RNAi. Antisense RNA suppression was an early form of RNA
silencing. This process involved the introduction of the antisense strand of
RNA into the cell that corresponded to the target
mRNA, the transcript intended to silence (Brantl 2002; Knee and Murphy
1997). After entry into the cell, the introduced antisense RNA and the native
target mRNA would bind via complementary base pairing preventing the
translation of mRNA. This is due to the
inability of ribosomes to bind to dsRNA (Arenz and Schepers 2003; Brantl
2002). This process, however, did not always result in a loss of function of a
targeted gene. This led concerned scientists to continue the search for other
methods of gene silencing.
Several forms of RNA as well as two highly conserved enzymes were studied
(a) Dicer in animals and

Dicer-like elements in plants; (b) RISC (RNA-induced Silencing Complex)

that are involved in RNAi. RNAi technology thus was used as a powerful tool
in the production of crop plants with specific traits Craig C. Mello and
Andrew Fire's reported a potent gene silencing effect after injecting double
stranded RNA into Caenorhabditis elegans. In investigating the regulation of
muscle protein production, they observed that neither mRNA nor antisense
RNA injections had an effect on protein production, but double-stranded RNA
successfully silenced the targeted gene. As a result of this work, they coined
the term RNAi. Fire and Mello's discovery represented the first identification
of the causative agent for the phenomenon. Fire and Mello were awarded the
Nobel Prize in Physiology or Medicine in 2006 for their work.
The main results presented by Fire and Mello were:
Silencing was triggered efficiently by injected dsRNA, but weakly or not at all
by sense or antisense single-stranded RNAs and silencing was specific for an
mRNA homologous to the dsRNA; other mRNAs were unaffected.
The dsRNA had to correspond to the mature mRNA sequence; both both
neither intron nor promoter sequences triggered a response nor the targeted
mRNA disappeared suggesting that it was degraded.
Only a few dsRNA molecules per cell were sufficient to accomplish full
silencing. This indicated that the dsRNA was amplified and/or acted
catalytically rather than stoichiometrically. The dsRNA effect could spread
between tissues and even to the progeny, suggesting a transmission of the
effect between cells.

Phenotypic effect after injection of single stranded or double stranded unc-22

RNA into the gonad of C.elegans. Injected double stranded RNA induced the
twitching phenotype in the progeny.

The effect on mex-3 mRNA content in embryos after injection of singlestranded or double-stranded mex-3 RNA into the gonad of C. elegans. mex-3
mRNA is abundant in the gonads and early embryos. The mRNA was lost
after injection of double-stranded RNA, while injection of antisense RNA
only reduced the content of mRNA to some extent. The extent of brown
colour reflects the amount of mRNA present.
Within a year, the presence of RNAi had been documented in many other
organisms, including fruit flies, trypanosomes, plants, planaria, hydra and
zebrafish.

SIGNIFICANCE OF THE DISCOVERY OF RNAi

1. RNAi protects against viral infections: RNAi constitute a defence
mechanism against viral attacks. Plant cells have an efficient defence against
viruses based on the PTGS phenomenon. When it became apparent that PTGS
is the plant equivalent to RNAi, this proposed that RNAi is involved in
protecting cells from viral attacks.

2. RNAi secures genome stability: RNAi/PTGS in C. elegans and plants

block the action of transposons (mobile elements in the genome).
Subsequently, when components of the RNAi machinery are mutated in C.
elegans, transposons are activated and the mobile elements cause disturbances
in the function of the genome. Transposons-containing regions of the genome
both DNA strands are transcribed, dsRNA is formed, and the RNAi process
eliminates these undesirable products
The RNAi machinery can recognize invading double-stranded viral RNA (or
the double-stranded replicative form of the viral RNA) and suppress the
infection by degradation of the RNA. The RNAi system thus shares important
features with the vertebrate immune system: it recognizes the invading
parasite (dsRNA) raises an initial response and subsequently amplifies the
response to eliminate the foreign element.
3. RNAi-like mechanisms repress protein synthesis and regulate the
development of organisms: There is a class of endogenous RNA molecules of
the same size in worms, flies, mice and humans; this small RNA was called
micro RNA (miRNA). Plants also contain this class of endogenous RNA. The
miRNAs can regulate gene expression by base-pairing to mRNA, which
results in either degradation of the mRNA or suppression of translation.
Today, it is estimated that there are about 500 miRNAs in mammalian cells,
and that about 30% of all genes are regulated by miRNAs. It is known that
miRNAs play an important role during development in plants, C. elegans and
mammals.

Cellular processes dependent on the RNAi machinery. The Dicer and RISC
complexes play a central role in the destruction of invading viral RNA
(a) The elimination of transcripts from mobile elements (transposons) and
repetitive DNA
(b) The block of protein synthesis brought about by small RNAs generated
within the cell
(c) RNAi mediated suppression of transcription
(d) The machinery is also utilized when siRNA is introduced into the cell to
inhibit activity of specific genes
(e) Dicer and RISC complexes can vary dependent on cellular process.

4. RNAi-like mechanisms keep chromatin condensed and suppress

transcription: It was known from work in plants that gene silencing could take
place at the transcriptional level (TGS). After RNAi discovery, it was shown
that TGS in plants operates via RNAi-like mechanisms. In the fission yeast
Schizosaccharomyces pombe, and later on in Drosophila and vertebrates, it
was found that similar processes keep heterochromatic regions condensed and
transcriptionally suppressed. In addition, the RNAi-like machinery regulates
the activity of genes in the immediate vicinity of the condensed blocks of
chromatin which helps in functioning of the genome and maintenance of
genome integrity.
5. RNAi offers a new experimental tool to repress genes specifically. The
action of RNAi phenomenon could be utilized as a general method to suppress
specific genes and look for the resulting phenotypic effect. It was also soon
evident that this could be accomplished in such an efficient manner that
essentially any gene in an organism could be studied functionally. The
targeted gene silencing by RNAi has already had a tremendous impact in

studies of the function of individual genes. It is now exploited not only in

cultured cells but also in transgenic organisms.

CELLULAR MECHANISM
The dicer protein from Giardia intestinalis, which catalyzes the cleavage of
dsRNA to siRNAs. The RNases domains are colored green, the PAZ domain
yellow, the platform domain red, and the connector helix blue.

RNAi is an RNA-dependent gene silencing process that is controlled by the

RNA-induced silencing complex (RISC) and is initiated by short doublestranded RNA molecules in a cell's cytoplasm, where they interact with the
catalytic RISC component argonaute. When the dsRNA is exogenous (coming
from infection by a virus with an RNA genome or laboratory manipulations),
the RNA is imported directly into the cytoplasm and cleaved to short
fragments by the enzyme dicer. The initiating dsRNA can also be endogenous
(originating in the cell).

dsRNA CLEAVAGE
Exogenous dsRNA initiates RNAi by activating the ribonuclease protein
Dicer, which binds and cleaves double-stranded RNAs (dsRNA) s to produce
double-stranded fragments of 2125 base pairs with a few unpaired overhang
bases on each end. Bioinformatics studies on the genomes of multiple
organisms suggest this length maximizes target-gene specificity and
minimizes non-specific effects. These short double-stranded fragments are
called small interfering RNAs (siRNAs). These siRNAs are then separated
into single strands and integrated into an active RISC complex. After
integration into the RISC, siRNAs base-pair to their target mRNA and induce
cleavage of the mRNA, thereby preventing it from being used as a translation
template. Exogenous dsRNA is detected and bound by an effectors protein,
known as RDE-4 in C. elegans and R2D2 in Drosophila, that stimulates dicer
activity.

MICRO RNA
The stem-loop secondary structure of a pre-microRNA from Brassica
oleracea.

MicroRNAs (miRNAs) are genomically encoded non-coding RNAs that help

regulate gene expression. The phenomenon of RNA interference includes the
endogenously induced gene silencing effects of miRNAs as well as silencing
triggered by foreign dsRNA. Mature miRNAs are structurally similar to
siRNAs produced from exogenous dsRNA. An miRNA is expressed from a
much longer RNA-coding gene as a primary transcript known as a primiRNA .The siRNAs derived from long dsRNA precursors differ from
miRNAs in that miRNAs, especially those in animals, typically have
incomplete base pairing to a target and inhibit the translation of many
different mRNAs with similar sequences. In contrast, siRNAs typically basepair perfectly and induce mRNA cleavage only in a single, specific target. In
Drosophila and C. elegans, miRNA and siRNA are processed by distinct
argonaute proteins and dicer enzymes.

A full-length argonaute protein from the archaea species Pyrococcus furious

and the PIWI domain of an argonaute protein in complex with doublestranded RNA.

RISC ACTIVATION AND CATALYSIS

The active components of an RNA-induced silencing complex (RISC) are
endonucleases called argonaute proteins, which cleave the target mRNA
strand complementary to their bound siRNA . As the fragments produced by
dicer are double-stranded, they could each in theory produce a functional
siRNA. However, only one of the two strands, which is known as the guide
strand, binds the argonaute protein and directs gene silencing. The other antiguide strand or passenger strand is degraded during RISC activation .
Although it was first believed that an ATP-dependent helicase separated these
two strands, the process is actually ATP-independent and performed directly
by the protein components of RISC. The structural basis for binding of RNA
to the argonaute protein was examined by X-ray crystallography

TRANSCRIPTIONAL SILENCING
Components of the RNA interference pathway are also used in many
eukaryotes in the maintenance of the organisation and structure of their
genomes. Modification of histones and associated induction of
heterochromatin
formation serves to downregulate genes pretranscriptionally; this process is referred to as RNA-induced transcriptional
silencing (RITS), and is carried out by a complex of proteins called the RITS
complex. In maintenance of existing heterochromatin regions, RITS forms a
complex with siRNAs complementary to the local genes and stably binds
local methylated histones, acting co-transcriptionally to degrade any nascent
pre-mRNA transcripts that are initiated by RNA polymerase.

VARIATION AMONG ORGANISMS

The effects of RNA interference can be both systemic and heritable in plants
and C. elegans, although not in Drosophila or mammals. In plants, RNAi is
thought to propagated by the transfer of siRNAs between cells through
plasmodesmata (channels in the cell walls that enable communication and
transport). The heritability comes from methylation of promoters targeted by
RNAi. A broad general distinction between plants and animals lies in the
targeting of endogenously produced miRNAs; in plants, miRNAs are usually
perfectly or nearly perfectly complementary to their target genes and induce
direct mRNA cleavage by RISC, while animals' miRNAs tend to be more

divergent in sequence and induce translational repression. This translational

effect may be produced by inhibiting the interactions of translation initiation
factors with the messenger RNA's polyadenine tail
Some eukaryotic protozoa such as Leishmania major and Trypanosoma cruzi
lack the RNAi pathway entirely. Most or all of the components are also
missing in some fungi, most notably the model organism Saccharomyces
cerevisiae .A recent study however reveals the presence of RNAi in other
budding yeast species such as Saccharomyces castellii and Candida albicans,
further demonstrating that inducing two RNAi-related proteins from S.
castellii facilitates RNAi in S. cerevisiae.

APPLICATIONS IN MEDICINE
It may be possible to exploit RNA interference in medical therapy. Interferon
responses in mammalian cells interfere with the introduction of long dsRNA
strands.
RNAi has been shown to cause the reversal of induced liver failure in mouse
system. The applications of RNAi holds good in antiviral therapies, including
the suppression of viral gene expression in cancerous cells, knockdown of
host receptors and co-receptors for HIV8, the silencing of hepatitis A9 and
hepatitis B genes, silencing of influenza gene expression, and inhibition of
measles viral replication. It is considered to be effective in treatment of
neurodegenerative diseases particularly focused on polyglutamine diseases
such as Huntingtons disease.
Other applications that are considered promising are to treat cancer by
silencing genes- differentially up regulated in tumor cells or genes involved in
cell division. A major area of concern, however, associated with RNAi
application is that it poses a risk because of its potent "offtarget"effects in
which a gene with a coincidentally similar sequence to the targeted gene is
also silenced.
RNAi has also become a powerful tool for drug target discovery.
Consequently, interest is rapidly growing for extension of its application to in
vivo systems, such as animal disease models and human therapeutics. Studies
on RNAi have resulted in two basic methods for its use for gene selective
inhibition: 1) cytoplasmic delivery of short dsRNA oligonucleotides (siRNA),
which mimics an active intermediate of an endogenous RNAi mechanism and
2) nuclear delivery of gene expression on-viral gene delivery systems are a
diverse collection of technologies that are applicable to both of these forms of
RNAi.

Therapeutic Applications of RNA interference

The most obvious clinical uses of RNAi are for diseases in which selective
depletion of one or a few specific proteins would be expected to slow or halt
the disease process in the affected cells. Ideally this would be accomplished
with no or tolerable side effects.

Cancer
There are two general abnormalities in cancer cells they exhibit
dysregulation of the cell cycle resulting in uncontrolled growth and they are
resistant to death as a result of abnormalities in one or more proteins that
mediate apoptosis. The goals for RNAi approaches for cancer therapy are
therefore to knock out the expression of a cell cycle gene and/or an antiapoptotic gene in the cancer cells thereby stopping tumor growth and killing
the cancer cells. To selectively eliminate cancer cells without damaging
normal cells, the RNAi would be targeted to a gene specifically involved in
the growth or survival of the cancer cell, or the siRNA would be selectively
delivered into the cancer cells.
siRNA was shown to be greater than an order of magnitude more potent than
antisense DNA in suppressing gene expression in human hepatoma and
pancreatic cancer cell lines. Viral vectors have also been used to express
siRNAs and inhibit cancer cell growth and tumorogenicity. For example, a
retroviral vector was used to specifically and stably inhibit expression of the
oncogenic K-RAS (V12) allele in human tumor cells. In addition to blocking
the expression of normal genes that are required for cancer cell growth and
survival, RNAi can be used to target specific cancer-causing mutations.

Infectious Diseases

Diseases caused by viruses and bacteria continue to be major causes of death

worldwide and are an increasing concern because of the emergence of
resistant strains and the potential use of infectious pathogens. Currently, HIV
infection has reached epidemic proportions in many African countries. Other
prominent infectious diseases include influenza, hepatitis, Lyme disease, and
West Nile virus. The ability of RNAi to inhibit the replication or cellular
uptake of viruses and other infectious agents has been clearly demonstrated in
cell culture studies and, therefore, holds promise for the treatment of human
patients. The ability of HIV-1 to infect cells and replicate can be severely
compromised by targeting of viral genes using siRNAs. Examples include the
suppression of HIV-1 replication in human cells transfected with siRNA
directed against tat and the rev gene.Transfection of human cells with siRNAs
directed against different genes in the poliovirus genome resulted in resistance
of the cells to infection with poliovirus.

Cardiovascular and Cerebrovascular Diseases

Cardiovascular disease is the leading cause of death in the United States and
many other industrialized countries. It most commonly results from the
progressive occlusion of arteries in a process called atherosclerosis,
Atherosclerosis involves damage to vascular endothelial cells, local
production of inflammatory cytokines, and the recruitment of macrophages to
the site forming foam cells; It may be possible to use RNAi technology to
intervene in the process of atherosclerosis or to reduce the damage to heart
tissue and brain cells that patients suffer following a myocardial infarction or
stroke. A key step in the process of atherosclerosis is the up-regulation of cell
adhesion molecules in vascular endothelial cells, which play an essential role
in the recruitment of macrophages to the site of endothelial damage. In
another study relevant to the pathogenesis of atherosclerosis, it was shown
that mevastatin, an inhibitor of cholesterol synthesis, suppresses cell
proliferation by inhibiting cyclin-dependent kinase-2.

Neurodegenerative Disorders
Alzheimer's diseases, Parkinsons disease, Huntingtons disease are examples
of relatively common age-related neurodegenerative disorders that are
increasing as average life expectancy increases. Each disorder is characterized

by the dysfunction and deaths of specific populations of neurons.There have

therefore been two different strategies for preventative and therapeutic
interventions in neurodegenerative disorders. One strategy is to block the
disease-specific events whereas the second strategy targets downstream events
in the neurodegenerative disorders. For example, an abnormality in the
proteolytic processing of the amyloid precursor protein is believed to be a key
event in Alzheimer's disease pathogenesis, and two enzymes called - and secretases that are responsible for cleaving of amyloid precursor protein to
generate the neurotoxic amyloid -peptide. RNAi has recently been used to
identify additional proteins, such as APH-1, that are critical for production of
amyloid -peptide (Lee et al., 2002b).
Remarkably, the RNAi machinery can handle double-stranded RNA entering
the cell as well as double-stranded RNA generated within the cell. The
development of an organism and proper function of its cells and tissues are
dependent on intact RNAi machinery. Infection by RNA viruses can be
blocked by RNAi, especially in plants and lower animals, and foreign
elements in the genome (viruses and transposes) can be kept silent. Finally,
the discovery of RNAi has not only provided us with a powerful new
experimental tool to study the function of genes but also raises expectations
about future applications of RNAi in medicine.
The discovery of RNAi has already had an immense impact on biomedical
research and will most likely lead to novel medical applications in the future.

Biological functions
Macromolecular Synthesis and Degradation
Regulated synthesis of nucleic acids, proteins, and lipids is essential for cell
survival and functions. RNAi makes possible understanding of the regulation
of macromolecular synthesis and degradation. For example, depletion of
galactosyltransferase II using siRNAs revealed a critical role for this enzyme
in the biosynthesis of the linkage region of glycosaminoglycans (Bai et al.,
2001). Transfection of cultured HeLa and HepG2 cells with siRNAs directed
against the mRNA encoding the acyl-CoA binding protein resulted in
cessation of cell proliferation, cell detachment, and death.

Cell Motility
The migration of cells within and among tissues, and extensions of cells such
as the axons and dendrites of neurons, is central to the structural and
functional organization of all tissues. RNAi methods elucidate roles for
specific proteins in regulating cell motility. Depletion of the cytoskeletal
linker protein trypanin using siRNAs resulted in a loss of directional cell
motility in African trypanosomes that is caused by impaired coordination of
the flagellar beating (Hutchings et al., 2002).

Cell Death
In many tissues throughout the body, cells have a finite life span and then
undergo apoptosis, a form of programmed cell death in which the cell dies in a
well controlled manner and is removed from the tissue without adversely
affecting adjacent healthy cells. Apoptosis also plays a key role in forming the
cellular structure of tissues during embryonic and postnatal development.Of
course, abnormal cell death is a major problem in a variety of diseases,
including neurodegenerative disorders such as Alzheimer's and Parkinson's

diseases.. RNAi establishes roles for specific genes in apoptotic and antiapoptotic pathways. For example, a critical role for p73 in p53-mediated
apoptosis was demonstrated by showing that depletion of p73 using siRNA
prevents cell death. RNAi was used to establish a role for the calcium-binding
protein calreticulin in necrotic cell death in C. elegans (Xu et al., 2001 .RNAi
was used to deplete cells of apoptosis-inducing factor (AIF), thereby
preventing apoptosis (Wang et al., 2002).

Viral Invasion/Replication
It is believed that endogenous RNAi mechanisms evolved, at least in part, to
protect cells against infectious pathogens such as viruses. For example, by
producing siRNAs directed against genes required for viral replication, the
infected cells prevented propagation of the virus (Lindenbach and Rice, 2002).
The development of RNAi technology has verified the importance of RNAi
mechanisms in viral invasion.

Immunity
RNA interference is a vital part of the immune response to viruses and other
foreign genetic material, especially in plants where it may also prevent selfpropagation by transposes. Plants such as Arabidopsis thaliana express
multiple dicer homologs that are specialized to react differently when the
plant is exposed to different types of viruses. Induced gene silencing in plants
can spread throughout the plant, and could be transferred from stock to scion
plants via grafting. RNAi in some animals has also been shown to produce an
antiviral response. In both juvenile and adult Drosophila, RNA interference is
important in antiviral innate immunity and is active against pathogens such as
Drosophila X virus.

Downregulation of genes
Endogenously expressed miRNAs, including both intronic and intergenic
miRNAs, are most important in translational repression and in the regulation
of development, especially on the timing of morphogenesis and the
maintenance of undifferentiated cell types such as stem cells. The role of
endogenously expressed miRNA in downregulating gene expression was first
described in C. elegans in 1993. In plants this function was discovered in
Arabidopsis .In many organisms, including humans, miRNAs have also been

linked to the formation of tumors and dysregulation of the cell cycle. Here,
miRNAs can function as both oncogenes and tumor suppressors.

Upregulation of genes
RNA sequences (siRNA and miRNA) that are complementary to parts of a
promoter can increase gene transcription, a phenomenon, RNA activation.
Part of the mechanism for how these RNA upregulate genes involves dicer
and argonaute and there is histone demethylation.

An adult C. elegans worm, grown under RNAi suppression of a nuclear

hormone receptor involved in desaturase regulation. These worms have
abnormal fatty acid metabolism but are viable and fertile.

Gene knockdown
The RNA interference pathway is often exploited in experimental biology to
study the function of genes in cell culture and in vivo in organisms. Doublestranded RNA is synthesized with a sequence complementary to a gene of
interest and introduced into a cell or organism, where it is recognized as
exogenous genetic material and activates the RNAi pathway. Using this
mechanism decrease in the expression of a targeted gene can be done. Since
RNAi may not totally abolish expression of the gene, this technique is
sometimes referred as a "knockdown", to distinguish it from "knockout"
procedures in which expression of a gene is entirely eliminated.

APPLICATIONS IN AGRICULTURE
RNA interference has been used particularly in the engineering of food plants
that produce lower levels of natural plant toxins. Such techniques take
advantage of the stable and heritable RNAi phenotype in plant stocks.
Cotton seeds are rich in dietary protein but naturally contain the toxic
terpenoid product gossypol, making them unsuitable for human consumption.
RNAi has been used to produce cotton stocks whose seeds contain reduced
levels of delta-cadinene synthase, a key enzyme in gossypol production,
without affecting the enzyme's production in other parts of the plant, where
gossypol is important in preventing damage from plant pests. Similar efforts
have been directed toward the reduction of the cyanogenic natural product
linamarin in cassava plants.
RNAi-based genetic engineering efforts have successfully reduced the levels
of allergens in tomato plants and decreased the precursors of likely
carcinogens in tobacco plants. Other plant traits that have been engineered in
the laboratory include the production of non-narcotic natural products by the
opium poppy, resistance to common plant viruses, and fortification of plants
such as tomatoes with dietary antioxidants .
Previous commercial products, including the Flavr Savr tomato and two
cultivars of ringspot-resistant papaya, were originally developed using
antisense technology .
Kusuba and his team (Kusuba et al. 2003) have recently made significant
contribution by applying RNAi to improve rice plants. They were able to
reduce the level of glutenin and produced a rice variety called LGC-1 (low
glutenin content 1). The low glutenin content was a relief to the kidney

patients unable to digest glutenin. The trait was stable and was transmitted for
a number of generations
In Ethiopia, Bangladesh and India, the people in the lower socioeconomic
class use a leafy vegetable known as Lathyrus sativus. It is a leguminous crop
and contains a
Neurotoxin called -oxalylaminoalanine-L-alanine (BOAA) (Spencer et al.
1986). People consuming this vegetable suffer from a paralytic disease called,
lathyrism. The disease paralyses people both temporarily and permanently,
RNAi technology can be used to silence the gene(s) responsible for
production of BOAA.
Another instance where RNAi may be fruitfully applied is in the production of
banana varieties resistant to the Banana Bract Mosaic Virus (BBrMV),
currently devastating the banana population in Southeast Asia and India
(Rodoni et al. 1999).
The BBrMV infects banana plants destroying the fruit producing bract region,
rendering them useless to farmers. The virus is spread by small plant eating
insects called aphids, as well as through infected plant materials.
A possible application of RNAi involves the down regulation of a key enzyme
in the biosynthetic pathway of lignin in the two economically important
Corchorus species, namely, C. capsularis and C. olitorius.
Thus RNAi technology may prove to be a powerful molecular tool by
generating jute varieties with low lignin content, allowing for easier,
environmentally friendly and cost effective processing of fiber for the
production of various economically important commodities such as high
quality paper and cloth.

DNA Directed RNAi in Plants

Applications of RNAi in plants have relied on nonAgrobacterium-mediated methods of introduction of dsRNA into the cells. For
example, particle bombardment was used to show that RNAi worked at the
single cell level in cereals (Schweitzer et al. 2000), while Klink & Wolniak
(2000) and Stout et al. (2003) demonstrated that there is a direct uptake of
dsRNA by fern spores during imbibitions.
DNA-directed RNAi makes use of dsRNA-expressing vectors introduced into
plants via

Agrobacterium-mediated transformation. This approach has been shown to be

effective in reducing the amount of a specific gene product. One of the first
studies testing DNA-directed RNAi in plants was done to compare the ability
of sense, antisense or dsRNA at generating RNA-mediated virus resistance via
PTGS in tobacco and silencing of an endogenous GUS reporter gene in rice
(Waterhouse et al. 1998
Current agricultural technology needs more and more molecular tools to
reduce current crop loss and feed extra mouths, which according to a recent
estimate by the FAO (Food and Agriculture Organization) will increase by
two billion over the next 30 years. The RNAi technology describes one such
powerful innovation. If judiciously used, this technology may go a long way
to narrow the gap through production of disease-, insect- and virus resistant,
nutritionally rich and toxic-free crops.
The cost effectiveness is always a big question, fortunately which is not a
problem with RNAi technology
RNAi technology may be helpful in tackling this gigantic problem of feeding
an additional 2 billion people over the next 30 years from an increasingly
fragile natural resource base.
Since RNAi offers a great potential in understanding gene functions and
utilize them to improve crop quality and production soon well see RNAi
research in the farmers fields around the world.