Regulation of Plant Gene Expression by Antisense Rna - PPT

REGULATION OF PLANT
GENE EXPRESSION BY
ANTISENSE RNA
SUBMITTED TO-
DR. BAPI GHOSH
ASSISTANT PROFESSOR
SUBMITTED BY-
SHILPA MONDAL
M.SC. 4TH SEMESTER
CYTOGENETICS & PLANT BREEDING
❖ Introduction
CONTENTS
❖ History
❖ Mechanism
❖ Levels of gene silencing
❖ Transcriptional gene silencing
❖ Post transcriptional gene silencing
❖ Salient features of RNAi
❖ Mechanism of RNAi
❖ Applications
❖ Advantages and disadvantages
❖ Future perspectives
❖ Case study
❖ Conclusion
INTRODUCTION
ANTISENSE RNA
• Antisense RNA is a Single Stranded RNA that is complementary to

messenger RNA (mRNA) strand transcribed within a cell.
• Antisense RNA introduced into a cell to inhibit translation of a
complementary mRNA by base pairing to it and creating barrier to the
translation machinery.
• E.g.
hok/sok system of the E. Coli R1 Plasmid.
• Powerful technology that permits controlled silencing of a specific gene.
• Formulated by Dr. Hal Weintranb and colleagues at the basic science
division in early 1980s.
• Tool used for inhibition of gene expression.
 Antisense RNA can be produced, for example, by inverting the coding region of
a gene with respect to its promoter. The antisense RNA can hybridize with its
corresponding mRNA, making it double-stranded. The double-stranded mRNA
no longer can be recognized by the protein synthesizing machinery (the
ribosomes), and thus expression of this mRNA is suppressed. Also, in many
systems double-stranded mRNA is very unstable and is broken down quickly.
Thus, one can inactivate specific genes while not interfering with others.
Theories on how inhibition works
 Exact mechanism by which translation is blocked is unknown.
 Several theories include-
 That the ds RNA prevents ribosomes from binding to the sense RNA and
translating (kimball, November 2002)
 The ds RNA cannot be transported from within the nucleus to the cytosol
which is were the translation occurs(tritton,1998)
 ds RNA is susceptible to endoribonucleases that would otherwise not effect ss
RNA but degrade ds RNA ( Kimball, November 2002)
ANTISENSE TECHNOLOGY
HISTORY
❖ 1990- Richard Jorgenson gave the phenomenon of ‘cosupression of gene

expression’.
❖ 1992- Romano and Macino gave the phenomenon of as Virus Induced

Gene Silencing , set of such phenomenon was termed as post
transcriptional gene silencing.
❖ 1995-RNA silencing was first documented in animals by Guo and

Kemphues, and for this phenomenon they coined the term antisense
mediated silencing.
❖ 1998-Andrew Fire and Craig C.Mello coined the term RNA interference
(RNAi).
❖ 2001-Thomas Tuschl, discovered with his colleagues that RNAi could
be prompted through the use of shorter pieces of RNA known as small
interfering RNAs (siRNAs).
❖ 2001-Gregory Hannon identified, described, and named the "Dicer"

enzyme, which chops dsRNA into siRNAs, as well as the RNA induced
silencing complex (RISC), which mediates the silencing
process by degrading the homologous mRNA7.
❖ 2002-Effect named "short hairpin-activated gene silencing" or

SHAGging was introduced.
❖ 2004 Morris et al. Observed that siRNA silences genes at the

transcriptional level.
MECHANISM
• It has recently been shown that ds RNA in the cytoplasm triggers as

yet poorly understood cascade of events leading to the suppression
of the transcription of the gene producing the specific mRNA
involved in the cytoplasmic RNA duplex.
Several possible sequence specific sites of antisense inhibition. Antisense oligodeoxynucleotides are
represented by black bars. Antisense oligodeoxynucleotides can interfere with (I) splicing, (II) transport of
the nascent mRNA from the nucleus to the cytoplasm, (III) binding of initiation factors, (IV) assembly of
ribosomal subunits at the start codon or (V) elongation of translation. Inhibition of capping
and polyadenylation is also possible. (VI) Antisense oligodeoxynucleotides that activate RNase H
(e.g., oligonucleotides with phosphodiester and phosphorothioate backbones) can also inhibit gene
expression by binding to their target mRNA, catalyzing the RNase H cleavage of the mRNA into
segments that are rapidly degraded by exonucleases
• The inhibition of gene expression by antisense molecules is believed to occur by a combination

of two mechanisms:
(a) ribonuclease H (RNase H) degradation of the RNA and
(b) steric hindrance of the processing of the RNA(which physically prevent or inhibit the progression
of splicing or the translational machinery)
(c) RNase H is a ubiquitous enzyme that hydrolyzes the RNA strand of an RNA/DNA duplex.
Oligonucleotide-assisted RNase H-dependent reduction of targeted RNA expression can be quite
efficient, reaching 80–95% down-regulation of protein and mRNA expression. Furthermore, in
contrast to the steric-blocker oligonucleotides, RNase H-dependent oligonucleotides can inhibit
protein expression when targeted to virtually any region of the mRNA. Thus, whereas most
steric-blocker oligonucleotides are efficient only when targeted to the 5′or AUG initiation codon
region, phosphorothioate oligonucleotides, e.g., can inhibit protein expression when targeted to
widely separated areas in the coding region
• In order for an antisense oligonucleotide to down-regulate gene expression, it
must penetrate into the targeted cells. To date, the precise mechanisms
involved in oligonucleotide penetration are not clear.
• Uptake occurs through active transport, which in turn depends on
temperature , the structure and the concentration of the oligonucleotide , and
the cell line.
• At the present time, it is believed that adsorptive endocytosis and fluid phase
pinocytosis are the major mechanisms of oligonucleotide internalization, with
the relative proportions of internalized material depending on oligonucleotide
concentration.
• At relatively low oligonucleotide concentration, it is likely that internalization
occurs via interaction with a membrane-bound receptor . De Diesbach et al.
have recently purified and partially characterized one of these receptors. At
relatively high oligonucleotide concentration, these receptors are saturated,
and the pinocytotic process assumes larger importance.
LEVELS OF GENE SILENCING
1.Transcriptional gene silencing (TGS)

It Causes gene silencing by:
• DNA methylation.
• Heterochromatin formation.
• Programmed DNA elimination.
2. Post transcriptional gene silencing (PTGS)

It is known commonly as RNA interference
(RNAi). It causes silencing by destruction of
the mRNA of the gene to which the siRNA
shows perfect complementarity.
Transcriptional gene silencing (TGS)
Transcriptional gene silencing - Result of modifications

of either the histones or DNA.
Gene silencing by modification of Histones and DNA
❖ Modification of nucleosomes alter the accessibility of the gene to the

transcriptional machinery and regulatory proteins
❖ Heterochromatin is commonly involved in gene silencing, and
affects large sections of DNA.
❖ Methylation of particular DNA sequences can also silence
transcription in many eukaryotes.
Post Transcriptional gene silencing (PTGS)
RNA interference (RNAi) is a molecular mechanism in

which fragments of double stranded nucleic acid (dsRNA)
interfere with the expression of a particular gene.
The dsRNA can be either, MicroRNA (miRNA) or Small

interference RNA (siRNA)
SALIENT FEATURES OF MICRO RNA
❖ Double stranded RNA rather than single–stranded

antisense RNA is the interfering agent.
❖ High degree of specific gene silencing with less effort.
❖ Highly potent and effective, only a few double stranded
RNA molecules per cell are required for effective
interference.
❖ Silencing can be introduced in different developmental
stages
❖ Systemic silencing
❖ Avoids problems with abnormalities caused by a knocked
out gene in early stages which could mask desired
observations.
❖ Silencing effects passed through generations
MECHANISM OF RNAi
Initiation step:
1.Double stranded RNA(dsRNA)
molecule
is cleaved to form 21-23 bp double
stranded
fragments called short interfering
RNAs(siRNAs).
Effector step:
1.siRNA is unbound by helicase
activity
associated with a multiprotein
complex
known as RNA-induced silencing
complex(RISC).
2.The antisense RNA complexed with
RISC binds to its corresponding mRNA
which is cleaved by the enzyme Slicer
rendering it inactive.
SIGNIFICANCE & APPLICATIONS
❖In biological functions

• immunity
• downregulation of genes
• upregulation of genes
• Evolution
❖In technology
• gene knockdown
• functional genomics
• medicines
• biotechnology
ADVANTAGES & DISADVANTAGES
ADVANTAGES DISADVANTAGES
1. This process is able to affect 1. Delivery, i.e., getting those
only selected genes which the exquisitely specific siRNAs to
RNAi is complementary to. the appropriate sites in the
2. RNAi will bind to most of the appropriate amounts to ensure
complementary genes it appropriate uptake and the
encounters, making it intended silencing remains a
highly efficient as well as considerable challenge
robust
3. Shown to work in the 2. Off-target effects i.e., when
laboratory and shows siRNA can affect unintended
potential in mammalian cell genes in the organism which
4. Able to be used on a large may be vital
scale
FUTURE PERSPECTIVE
❖ Since 1998 ,RNAi discovery has been touted as a technical
breakthrough in biological research.
❖ Even with RNAi's rapid development over the years, it is still

in its infancy stage. A better understanding of the
mechanisms that take place will help reduce problems such as
off-target effects.
❖ In 2001 RNAi was used to treat hepatitis in mice
❖ With further knowledge about the mechanisms of RNAi it

may be the gateway for other emerging technologies such as
transgenic studies, gene therapy and gene-wide screening..
❖ Whilst still in process, it opens the doors of what can be

achieved, and infact realises a small part of the hope - that
nothing is untreatable.
CASE STUDY
FLAVR SAVR TOMATO
❖ The first FDA approved genetically modified food

❖ Licensed in 1994
❖ will not soften while ripening on the vine.
❖ Increased shelf life , tomatoes can be shipped safely, keep
their colour, and have their natural flavours.
BEFORE THE FLAVR SAVR

❖ Picked before ripe and gassed with ethylene to give red
colour – keeps them from becoming spoiled
❖ Tomatoes lose their taste and taste more like “cardboard.”
MAKING OF FLAVR SAVR
❖ Enzyme Polygalacturonase
breaks down structural
polysaccharide pectin in wall
of a plant.
❖ This is part of the natural
decay process in a plant
❖ Flavr savr tomatoes have been
constructed that express an
antisense mRNA complementary
to mRNA for an enzyme involved in
ethylene production
❖ These tomatoes make only 10%
of normal amount of enzyme thus
delaying ethylene production.
G.M VS TRADITIONAL TOMATO
OUTCOMES
❖ Basically, the gene in the tomato stops the tomato from

softening during ripening so that it is easier to ship but keeps
its natural flavours too.
❖ The tomato also has a much longer shelf life and keeps from
spoiling quickly.
PROBLEMS WITH FLAVR SAVR:
❖ Safety- health risks, some environmental risks

❖ Possible monopolies for businesses
❖ Ethical concerns
❖ Only rich countries can afford it
CONCLUSION
The Antisense RNA technology shows the

potential for diverse applications to basic
research and therapy. Antisense technology
offers almost unlimited scope for the
development of new methods of drug design
and one of the most approved approaches
among several others, for inactivating a
single chosen gene.
However, the full commitment of this

promise is yet to be established.
THANK YOU

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REGULATION OF PLANT

• Antisense RNA is a Single Stranded RNA that is complementary to

❖ 1990- Richard Jorgenson gave the phenomenon of ‘cosupression of gene

❖ 1992- Romano and Macino gave the phenomenon of as Virus Induced

❖ 1995-RNA silencing was first documented in animals by Guo and

❖ 2001-Gregory Hannon identified, described, and named the "Dicer"

❖ 2002-Effect named "short hairpin-activated gene silencing" or

❖ 2004 Morris et al. Observed that siRNA silences genes at the

• It has recently been shown that ds RNA in the cytoplasm triggers as

• The inhibition of gene expression by antisense molecules is believed to occur by a combination

1.Transcriptional gene silencing (TGS)

2. Post transcriptional gene silencing (PTGS)

Transcriptional gene silencing - Result of modifications

Gene silencing by modification of Histones and DNA

❖ Modification of nucleosomes alter the accessibility of the gene to the

RNA interference (RNAi) is a molecular mechanism in

The dsRNA can be either, MicroRNA (miRNA) or Small

❖ Double stranded RNA rather than single–stranded

❖In biological functions

❖ Even with RNAi's rapid development over the years, it is still

❖ In 2001 RNAi was used to treat hepatitis in mice

❖ With further knowledge about the mechanisms of RNAi it

❖ Whilst still in process, it opens the doors of what can be

❖ The first FDA approved genetically modified food

BEFORE THE FLAVR SAVR

❖ Basically, the gene in the tomato stops the tomato from

PROBLEMS WITH FLAVR SAVR:

❖ Safety- health risks, some environmental risks

The Antisense RNA technology shows the

However, the full commitment of this

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