Sandipayan seminar gene silencing

M.S.Ramaiah College
Of
Arts, Science & Commerce
Presented by,
Sandipayan Dutta.
SeminaronGeneSilencing
MSc Biotechnology
2nd Semester
Year 2014.

From DNA to Protein
• Transcription
Process where information held in the
DNA is transferred to RNA.
• Translation
Conversion of RNA to amino acid
sequence of protein.
© Copyright. Sandipayan Dutta. 2014. All rights
reserved.

Glossary– Dicer –
• DICER is a cytoplasmic RNase III enzyme that not only cleaves precursor miRNAs to produce
mature miRNAs but also dissects naturally formed/synthetic double-stranded RNAs to generate
small interfering RNAs (siRNAs).
– Interferon –
• A small and highly potent molecule that functions in an autocrine and paracrine manner, and that
induces cells to resist viral replication. This term is related to RNAi because in mammals introduction
of dsRNA longer than 30 nt induces a sequence-nonspecific interferon response.
– Micro-RNA –
• Micro-RNAs (miRNA) are single-stranded RNAs of 22-nt that are processed from ~70-nt hairpin RNA
precursors by Rnase III nuclease Dicer. Similar to siRNAs, miRNAs can silence gene activity via
destruction of homologous mRNA in plants or blocking its translation in plants and animals.
– Post-Transcriptional Gene Silencing –
• Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation system
designed to act as an anti-viral defense mechanism. A form of PTGS triggered by transgenic DNA,
called co-suppression, was initially described in plants and a related phenomenon, termed quelling,
was later observed in the filamentous fungus Neurospora crassa
– RNA Interference –
• RNA Interference (RNAi), a term coined by Fire et al in 1998, is a phenomenon that small double-
stranded RNA (referred as small interference RNA or siRNA) can induce efficient sequence-specific
silence of gene expression.
– RNA-Directed DNA Methylation –
• RNA-directed DNA methylation (RdDM) is an RNA directed silencing mechanism found in plants.
Similar to RNA interference (RNAi), RdDM requires a double-strand RNA that is cut into short 21-26-
nt fragments. DNA sequences homologous to these short RNAs are then methylated and silenced.
– RNA-Induced Silencing Complex –
• RNA-induced silencing complex (RISC) is an siRNA-directed endonuclease, catalyzing cleavage of
a single phosphodiester bond on the RNA target.
– RNAi Trigger –
• RNAi triggers are double-stranded RNAs containing 21-23 nt sense and antisens strands hybridized
to have 2 nt overhangs at both 3' ends.
– Small Interfering RNA –
• Small Interfering RNA (siRNA) is 21-23-nt double-strand RNA. It guides the cleavage and
degradation of its cognate RNA.
– Helicase –
• Enzyme responsible for unwinding double stranded molecule

DROSHA
• DROSHA is a nuclear RNase III enzyme responsible for cleaving primary
microRNAs (miRNAs) into precursor miRNAs and thus is essential for the
biogenesis of canonical miRNAs.

Gene Silencing
 Gene silencing – “Switching off” of a gene ,by a
machinery in the cell.
 Epigenetic process of gene regulation.
 Silencing is a position effect .
 Genes are silenced at either the transcriptional or post-
transcriptional level.
 Transcriptional gene silencing - Result of
modifications of either the histones or DNA.
e.g.:- Silencing at the yeast telomere.
 Post-transcriptional gene silencing -Result of the
mRNA of a particular gene being destroyed or blocked.
A common mechanism of PTGS is RNAi.

Causes of gene silencing
• Methylation of transgenes
• Degradation of transgenic
mRNA in cytoplasm
• Inactivation of homologous
gene by transcriptional and
post-transcriptional regulation
reserved.

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.
E.g.:- the telomere, silent mating -type locus and
rDNA gene in the budding yeast S.cerevisae
• Methylation of particular DNA sequences can also
silence transcription in many eukaryotes.
E.g. :-the human H19 gene
reserved.

Histone Tail Modification Status
Correlates with Transcriptional
Activity

Acetylation
• These reactions are catalyzed by enzymes with "histone
acetyltransferase" (HAT) or "histone deacetylase" (HDAC)
activity.
• It also reduces affinity of tail for adjacent nucleosomes, thus
affecting ability of nucleosome arrays to form more repressive
higher-ordered chromatin structures.

Methylation
• These reactions are catalyzed by enzymes "histone
methyltransferase”
• Methylation recruit silencing or regulatory proteins that bind
methylated histones.
• Chromodomain containing proteins interact with methylated
histone tails.

Histone Acetylation and Methylation

Silencing at the yeast telomere.
SIR proteins (Silent Information Regulation) form a
silencing complex. This complex is recruited by Rap1.

DNA methylation can recruit Histone
Deacetylases and Methylases
DNA
methyltransferase
methylate Cytosine
within promoter. This
modification binds
proteins (MeCP2),
which in turn recruit
complexes modifying
nucleosome and
switch off gene
expression. [In
Mammals]

Comparison of different gene
silencing strategies.
Agent Mechanism Result
Most drugs Bind to target protein Protein inhibition
RNase H-independent
ODNs
Hybridize to target
mRNA
Inhibition of translation
of the target protein
RNase H-dependent
ODNs
Hybridize to target
mRNA
Degradation of the
mRNA by RNase H
Ribozymes and DNA
enzymes
Catalyze cleavage of
target mRNA
Degradation of the
mRNA
siRNA
Hybridize to target
mRNA by its antisense
strand and guide it into
endoribonuclease
enzyme complex
(RISC)
Degradation of the
mRNA
reserved.

Transcriptional Gene silencing

Genomic imprinting
• Genomic imprinting is an epigenetic phenomenon by
which certain genes can be expressed in a parent-of-
origin-specific manner. In Homo sapiens, imprinted
alleles are silenced such that the genes are either
expressed only from the non-imprinted allele inherited
from the mother (e.g. H19 or CDKN1C), or in other
instances from the non-imprinted allele inherited from
the father (e.g. IGF-2). However, in plants parental
genomic imprinting can refer to gene expression both
solely or primarily from either parent's allele.
• Genomic imprinting is an epigenetic process that can
involve DNA methylation and histone modulation in
order to achieve monoallelic gene expression without
altering the genetic sequence. These epigenetic marks
are established in the germline and can be maintained
through mitotic divisions.

Paramutation
• A paramutation is an interaction between two
alleles at a single locus, whereby one allele induces
a heritable change in the other allele.
• Paramutation can result in a single allele of a gene
controlling a spectrum of phenotypes. At r1 in
maize, for example, the weaker expression state
adopted by a paramutant allele can range from
completely colorless to nearly fully colored kernels.
• paramutation is RNA-directed. Stability of the
chromatin states associated with paramutation and
transposon silencing requires the mop1 gene,
which encodes an RNA-dependent RNA
polymerase.
reserved.

Position effect
• Position effect is the effect on the expression of a
gene when its location in a chromosome is
changed, often by translocation. This has been well
described in Drosophila with respect to eye color
and is known as position effect variegation (PEV).
• Position effect is also used to describe the variation
of expression exhibited by identical transgenes that
insert into different regions of a genome. In this
case the difference in expression is often due to
enhancers that regulate neighbouring genes. These
local enhancers can also affect the expression
pattern of the transgene. Since each transgenic
organism has the transgene in a different location
each transgenic organism has the potential for a
unique expression pattern.

RNA-directed DNA methylation
• RNA-directed DNA methylation (RdDM) is an
epigenetic process first discovered in plants
(Wassenegger et al, 1994, Cell, Vol 76, 567-576).
During RdDM, double-stranded RNAs (dsRNAs)
are processed to 21-24 nucleotide small
interfering RNAs (siRNAs) and guide methylation
of homologous DNA loci. In plants dsRNAs may
be generated from three sources:
 Viral replication intermediates
 Products of the endogenous RNA-directed RNA
polymerase
 Transcribed inverted repeats

Post-Transcriptional Gene
Silencing (PTGS)
• Definition: the ability of exogenous double-
stranded RNA (dsRNA) to suppress the expression
of the gene which corresponds to the dsRNA
sequence.
• Process results in down-regulation of a gene at the
RNA level (i.e., after transcription)
• There is also gene silencing at the
transcriptional level (TGS)
– Examples: transposons, retroviral genes,
heterochromatin.
• PTGS is heritable, although it can be modified in
subsequent cell divisions or generations
– Ergo, it is an epigenetic phenomenon

• Epigenetics - refers to heritable changes in
phenotype or gene expression caused by
mechanisms other than changes in the
underlying DNA sequence.
reserved.

Difference
 Promoters active
 Gene
hypermethylated in
coding region
Transcriptional gene
silencing (TGS)
Posttranscriptional gene
silencing (PTGS)
Promoters
silenced
Genes
hypermethylated
in promoter
region
reserved.

Discovery of PTGS
• First discovered in plants
– (R. Jorgensen, 1990)
• When Jorgensen introduced a re-engineered gene
into petunia that had a lot of homology with an
endogenous petunia gene, both genes became
suppressed!
– Also called Co-suppression
– Suppression was mostly due to increased
degradation of the mRNAs (from the
endogenous and introduced genes)

• Involved attempts to manipulate pigment synthesis
genes in petunia
• Genes were enzymes of the flavonoid/ anthocyanin
pathway:
– CHS: chalcone synthase
– DFR: dihydroflavonol reductase
• When these genes were introduced into petunia using a
strong viral promoter, mRNA levels dropped and so did
pigment levels in many transgenics.

Jorgensen 1990
van der Krol 1990
Gene injection (pigmentation
Enzyme-petunias)
Expectation: more red color
Co-suppression of transgene
and endogenous gene.
Bill Douherty and Lindbo 1993
Gene injection with a complete tobacco
etch virus particle.
Expectation: virus expression
Co-suppression of transgene
and virus particles via RNA.
Hamilton and Baulcombe 1998
Identification of short antisense RNA
sequences
dsRNA?
How?Fire and Mello 1998
Injection of dsRNA into
C. elegans
RNA interference (RNAi)
or silencing
Ambros 1993 (2000)
Identification of small RNA in
C. elegans (micro RNA)

Post-transcriptional gene silencing
reserved.

“siRNA” molecule:
“Dicer” processes
long dsRNA into
short siRNA:
“Guide” (antisense)
strand
incorporated into RISC
complex:
guides RISC to
complementary
sequences in target
mRNAs
(RNA-induced silencing
complex)
Post-transcriptional gene silencing (PTGS)

Nonsense-mediated decay
• Nonsense-mediated mRNA decay (NMD) is a
surveillance pathway that exists in all eukaryotes.
Its main function is to reduce errors in gene
expression by eliminating mRNA transcripts that
contain premature stop codons.
reserved.

A tale of two pathways
• RNA interference (RNAi) pathway: produces
small interfering RNAs (siRNAs) that silence
complementary target genes
• MicroRNA pathway: produces microRNAs
(miRNAs) that silence complementary target
genes
Mechanisms involve transcriptional gene silencing
(TGS) and/or post-transcriptional gene silencing
(PTGS)
Pathways are conserved among most all
eukaryotic organisms (fungi, protozoans, plants,
nematodes, invertebrates, mammals)

RNAi pathway
• Double-stranded RNA (dsRNA)
is processed by Dicer, an
RNase III family member, to
produce 21-23nt small
interfering RNAs (siRNAs)
• siRNAs are manipulated by a
multi-component nuclease
called the RNA-induced
silencing complex (RISC).
• RISC specifically cleaves
mRNAs that have perfect
complementarity to the siRNA
strand

RNA Interference (RNAi)
• RNA interference (RNAi) is a biological process
in which RNA molecules inhibit gene expression,
typically by causing the destruction of specific
mRNA molecules.
• Historically, it was known by other names,
including co-suppression, post transcriptional
gene silencing (PTGS), and quelling.
reserved.

• RNAi discovered in C. elegans (first animal)
while attempting to use antisense RNA in
vivo
Craig Mello Andrew Fire (2006 Nobel Prize in
Physiology & Medicine)
– Control “sense” RNAs also produced suppression of
target gene!
– sense RNAs were contaminated with dsRNA.
– dsRNA was the suppressing agent.

Antisense RNA (c) or
dsRNA (d) for the mex-3
(mRNA) was injected
into C. elegans ovaries,
and then mex-3 mRNA
was detected in
embryos by in situ
hybridization with a mex-
3 probe.
(a) control embryo
(b) control embryo hyb.
with mex-3 probe
Conclusions: (1) dsRNA reduced mex-3 mRNA better than antisense
mRNA. (2) the suppressing signal moved from cell to cell.
Double-stranded RNA (dsRNA) induced
interference of the Mex-3 mRNA in the
nematode C. elegans.

Core components of the RNAi
pathway
• Dicer
Dicer family proteins contain an N-terminal helicase
domain, a C-terminal segment containing dual RNase III
domains, and one or more dsRNA-binding motifs. Family
members also contain a PAZ domain.
•Member of RNAseIII family of enzymes.
• Recognize and process dsRNA into siRNA.
• Dicer family proteins are ATP-dependent nucleases.
• Dicer homologs exist in many organisms
includingC.elegans, Drosophila, yeast and humans.

• Argonaute (RISC complex)
•RNA-Induced Silencing Complex
(RISC)
Argonaute family members are highly basic, ~100 kD
proteins that contain PAZ and PIWI domains.
• Large (~500-kDa) RNA-multiprotein complex, which
triggers mRNA degradation in response to siRNA.
• The active component of RISC are endonucleases
called argonaute proteins .
• The strand binding to the argonaute protein - ‘guide
strand’.
• The other ‘anti-guide strand or passenger strand is
degraded during RISC activation.
• The strand chosen is the one whose 5’ end is least
paired to its complement.
• The process is ATP independent.

Mechanism of RNAi: Role of Dicer
1. Cells (plants and animals) undergoing RNAi
contained small fragments (~25 nt) of the RNA
being suppressed.
2. A nuclease (Dicer) was purified from Drosophila
embryos that still had small RNA fragments
associated with it, both sense and antisense.
3. The Dicer gene is found in all organisms that
exhibit RNAi, and mutating it inhibits the RNAi
effect.
Conclusion: Dicer is the endonuclease that
degrades dsRNA into 21-24 nt fragments, and in
higher eukaryotes also pulls the strands apart
via intrinsic helicase activity.

Model for RNAi
21-23 nt RNAs
ATP-dependent
Helicase or Dicer
Very efficient
process
because many
small
interfering
RNAs
(siRNAs)
generated from
a larger
dsRNA.

Biological roles of RNAi
Cellular immune response to viruses
(some organisms)
• In certain organisms (especially plants),
RNAi serves as a first line of defense
against viral infection, as virus may
contain or viral replication can produce
dsRNA
• • To this point, a number of plant
viruses encode proteins that specifically
bind and sequester siRNAs as a means of
countering the cellular immune response
of RNAi

Genetic stability
• RNAi represses transposable genetic elements in C.
elegans and S. pombe
• Disruption of Dicer or Argonaute increases the
relative abundance of transposon RNA and
increases transposon mobility
• RNAi is required to establish and maintain
heterochromatin formation and gene silencing at
mating type loci and centromeres in S. pombe
• Disruption of Dicer or Argonaute eliminates
silencing, decreases histone and DNA methylation,
and causes aberrant chromosome segregation
• Highly repetitive DNA is often associated with
heterochromatin which is transcriptionally silent.

PTGS (RNAi) occurs in wide
variety of Eukaryotes:
– Angiosperms
– C. elegans (nematode)
– Drosophila
– Mammalian cells
– Chlamydomonas (unicellular
– Neurospora, but not in Yeast!
reserved.

Recent applications of RNAi
Modulation of HIV-1 replication by RNA interference.
Hannon(2002).
Potent and specific inhibition of human immunodeficiency
virus type 1 replication by RNA interference.
An et al.(1999)
Selective silencing of viral gene expression in HPV-positive
human cervical carcinoma cells treated with siRNA, a primer
of RNA interference.
Jung et al. 2002.
RNA interference in adult mice.
Mccaffrey et al.2002
Successful inactivation of endogenous Oct-3/4 and c-mos
genes in mouse pre implantation embryos and oocytes using
short interfering RNAs.
Le Bon et al.2002

Significance of RNAi
• Protects against viral infection.
• Secures the genome stability by keeping
mobile elements silent.
• Repress protein synthesis and regulate
development of organism.
• Keep chromatin condensed and suppress
transcription.
• Experimental tool to elucidate the function of
any gene.
• Biotechnology – engineering of food plants.
• Useful approach in future gene therapy.

Schematic illustration of systemic viral spread as well as
RNAi and subsequent viral recovery in plants.

Why RNAi silencing?
• Most widely held view is that RNAi evolved
to protect the genome from viruses (and
perhaps transposons or mobile DNAs).
• Some viruses have proteins that suppress
silencing:
1. HCPro - first one identified, found in plant
potyviruses (V. Vance)
2. P19 - tomato bushy stunt virus, binds to siRNAs
and prevents RISC formation (D. Baulcombe).
3. Tat - RNA-binding protein from HIV

RNAi is a conserved mechanism
– RNAi is a universal, omnipresent
conserved mechanism in eukaryotic
cells.
– The cellular mechanism of RNAi
Predates evolutionary divergence of
plants and worms.
– key proteins involved in RNAi in
disparate organisms are highly
conserved
reserved.

THE SILENCING MECHANISM
• Two-step model to explain RNAi.
– I. dsRNA is diced by an ATP-
dependent ribonuclease (Dicer)
into short interfering RNAs
(siRNAs).
• duplexes of 21 23 nucleotides
bearing two-nucleotide 3'
overhanging ends.
– II. siRNAs are transferred to a
second enzyme complex,
designated RISC for RNAi-
induced silencing complex.
The siRNA guides RISC to the
target mRNA, leading to its
destruction.
• the antisense strand of the siRNA
is perfectly complementary

Two-step model for the mechanism of gene silencing induced by double-stranded RNA. In
step I, dsRNA is cleaved by the Dicer enzyme to produce siRNAs.

The classical RNA interference (RNAi)
pathway in Drosophila
– Long double-stranded RNAs
(dsRNAs) are processed by the
R2D2/Dicer heterodimer into
small interfering RNAs
(siRNAs).
– The duplexed siRNA is
unwound in an ATP-dependent
manner*.
• *starting at the 5' terminus that
has the lowest relative free
energy of base pairing.
– This strand of the siRNA, the
guide strand, is also
preferentially taken up by the
RNA-induced silencing complex
(RISC).
– The single-stranded siRNA
guides the endonuclease activity
of the activated RISC
("holoRISC") to the homologous
site on the mRNA, cleaving the
mRNA.

Discovery of miRNAs
 siRNAs
1990: Transgenic introduction of a gene silenced endogenous
gene expression in plant(Petunia).
Mechanism: Introduced dsRNA is processed by Dicer into a 21-
23 nt small interfering
RNA (siRNA). Dicer (RNase III-like RNase) plays a role in post-
transcriptional gene silencing (PTGS) or transgene quelling .
 microRNAs regulate developmental timing (heterochronic
gene pathway) in C. elegans.
C. elegans lin-4 (identified in 1993) controls developmental
timing.
Andrew Fire and Craig Mello (2006 Nobel Prize in Physiology or
Medicine) reported in 1998 that small regulatory RNAs
(microRNAs) interfere with target gene expression. [Potent and
specific genetic interference by double-stranded RNA in
C.elegans. Nature (1998) 391:806-11]. Also found that double-
stranded RNA mixtures caused potent and specific interference
in animals.

microRNA classification
siRNA
Small interfering RNA (20-24 nt)
• from invasive nucleic acids (viruses or
foreign genes introduced for experiemntal
and clinical purposes, or from other
environmental sources)
• perfect complementarity to their mRNA
targets
• targeted sites may disperse throughout the
entire transcripts
• primarily cause mRNA degradation

miRNA
microRNA (20-24 nt)
• most mammalian miRNAs contain
mismatches
• usually target untranslated regions of
mRNAs
• primarily cause translational suppression
or can also facilitate RNA
• degradation if not perfectly match to the
target

rasiRNA
Repeat-associated small interfering RNA (24-
29 nt)
• derived from repetitive elements within
the genome (heterochromatin regions
including centromeres and telomeres, and
rertotransposons).
• arise mainly from the antisense strand
• cause transcriptional silencing via
chromatin remodeling

piRNA
Piwi-interacting RNA (26-31 nt)
• in the germ line
• associated with Piwi
• predicted to have function in gametogenesis

tasiRNA
trans-acting RNA (24-29 nt) in plant
• endogenous siRNAs derived from noncoding
transcripts that are cleaved by a microRNA (miRNA)
• facilitate protein-coding mRNA degradation
• function in plant stress responses
• similar siRNAs (tiny noncoding RNA) found in
C. elegans

mirtrons
• short hairpin introns provide an alternative source
for animal microRNA biogenesis and use the
splicing machinery to bypass Drosha cleavage in
initial maturation.

LINE (L1) retrotransposons can generate dsRNAs
from both sense and
• antisense promoters in germ cells. These
dsRNAs can be processed into
• siRNAs. L1-specific siRNAs target to the 5’UTR of
L1 transcripts and
• cause its degradation. Thus, siRNAs suppress L1
retrotransposition in
• germ cells.

microRNA (abbreviated miRNA)
A microRNA is a small non-coding RNA molecule
(containing about 22 nucleotides) found in plants,
animals, and some viruses, which functions in
transcriptional and post-transcriptional regulation of
gene expression. Encoded by eukaryotic nuclear
DNA in plants and animals and by viral DNA in
certain viruses whose genome is based on DNA,
miRNAs function via base-pairing with
complementary sequences within mRNA molecules.
As a result, these mRNA strands are silenced
because they can no longer be translated into
proteins by ribosomes, and such complexes are
often actively disassembled by the cell.

• miRNA Biogenesis
– Transcribed from endogenous gene as pri-miRNA
• Primary miRNA: long with multiple hairpins
• Imperfect internal sequence complementarity
– It is processed into 70-nt hairpins by the RNase III family
member Drosha to become the pre-miRNA.
• Note: How does it identify pri-miRNA?
– Hairpin terminal loop size
– Stem structure
– Hairpin flanking sequences
– The pre-miRNA is exported to the cytoplasm by Exportin 5.
– It is cleaved by the R2D2/Dicer heterodimer into the mature
miRNA.
• Symmetric 2nt 3’ overhangs, 5’ phosphate groups

DCL1 mutant
Comparison of Mechanisms of MiRNA Biogenesis and Action
Better complementarity of MiRNAs and targets in plants.

Summary of differences between plant and animal MiRNA
systems
Plants Animals
miRNA genes: 100-200 100-500
Location in genome: intergenic regions Intergenic regions, introns
Clusters of miRNAs: Uncommon Common
MiRNA biosynthesis: Dicer-like Drosha, Dicer
Mechanism of repression mRNA cleavage Translational repression
Location of miRNA
target in a gene: Predominantly Predominantly the 3′-UTR
the open-reading frame
miRNA binding
sites in a target gene: Generally one Generally multiple
Functions of known
target genes: Regulatory genes Regulatory genes—crucial
crucial for development, for development, structural
enzymes proteins, enzymes

miRNAs and Cancer – A Summary
• miRNAs control cell cycle, cell differentiation and
apoptosis by regulating oncogenes and tumor supressor
genes
• miRNAs are misexpressed in cancer and are therefore
excellent diagnostic/prognostic markers in cancer
• Some miRNAs e.g. mir-155, can cause cancer and
oncogenic miRNAs may be therapeutic targets in cancer
• Other miRNAs like let-7, may prevent cancer and may
be therapeutic molecules themselves.
• MicroRNAs could augment current cancer therapies.

MicroRNAs Commonly Associated
with Human Cancer

microRNAs
1. Derived from an endogenous,
structured transcript (pre-miRNA)
2. One miRNA accumulates
3. Evolutionary conserved
4. Usually located away from
genes
5. Imperfect pairing blocks
translation
6. Incorporated into miRNP
7. Regulate expression of genes
encoded at another locus
8. miRNAs bind to the target 3'
UTRs through imperfect
complementarity at multiple sites
siRNAs
1. Derived from extended
dsRNA
2. Each dsRNA gives multiple
siRNAs
3. Less conservation
4. Nearly complementary to
target RNA (self-targeting)
5. Perfect pairing induces
target RNA cleavage
6. Incorporated into RISC
7. Regulate the locus from
which their sequence derives
8. siRNAs often form a perfect
duplex with their targets at only
one site.
miRNAs and siRNAs — what's the
difference?

Gene Knockout: Introduce dsRNA as
hairpin RNA (hpRNA) to silence
some specific gene.
Trick the plant in to shutting down or
re-routing specific molecular
pathways to alter biological
activities.
e.g. - flowering time
- leaf shape
- yield index
- oil quality

Small Interfering RNA (siRNA)
• 21-25 nucleotide dsRNA with 2-nt 3’
overhangs on either end.
• Produced in vivo by cleavage of dsRNA
or exogenously introduced in the cell.
• Amplification by an RNA-dependent RNA
polymerase (RdRP) may occur.
• Incorporated into the RISC guiding it to
mRNA
• A single base pair difference between the
siRNA template and the target mRNA is
enough to block the process.

siRNAs have a defined structure
19 nt duplex
2 nt 3’
overhangs
reserved.

siRNAs
 Small interfering RNAs that have an integral role in
the phenomenon of RNA interference(RNAi), a form
of post-transcriptional gene silencing
 RNAi: 21-25 nt fragments, which bind to the
complementary portion of the target mRNA and tag it
for degradation
 A single base pair difference between the siRNA
template and the target mRNA is enough to block
the process.

siRNA design
• Target Sequence- 21-nucleotides long , 50-
100 bp downstream from start codon (AUG)
• Search for seq. motif AA(N19).
• Avoid sequences with > 50% G+C content.
• Avoid targeting introns.
• Avoid stretches of 4 or more nucleotide
repeats.
• Avoid sequences that share a certain degree
of homology with other related or unrelated
genes.
reserved.

from Mittal, Nature Rev.Genet. 5, 355 (2004)
The Design of Optimal siRNAs
21 nt RNA that contains 2 nt
3’-
overhangs and
phosphorylated 5’-ends
Lower stability at the 5’-end
of the antisense terminus
Low stability in the RISC
cleavage site
Low secondary structure in
the
targeted region of the mRNA

How can RNAi be used ?
C. elegans
D. melanogaster
Planaria
Trypanosomes
Hydra
Xenopus
Mammalian tissue culture cells
Mice
• biological research
– Define gene function (gene knockdown)
– Define biochemical pathways
– Identify and validate targets
– Generate knockdown models without
developmental complications
• therapeutic treatment
– viral infection
– parasitic infection
– cancer
– neurodegeneration

RNAi as a powerful therapeutic
drug
• Exquisitely selective like the fabled “magic
bullet”.
• May work synergistically with other drug and
treatment regimes.
• Endogenous pathway so allows the
development of safe and efficacious drugs.
• Potent- very low concentrations of siRNA
are required.
• The clinical applications appear endless.
reserved.

from Dykxhoorn and Lieberman, Cell 126, 231 (2006)
Delivery of siRNA for Therapy
siRNA is not taken up by
most mammalian cells
Cholesterol-conjugated siRNA is
taken up by the LDL receptor
siRNA bound to targeted antibody
linked to protamine can achieve
cell-specific siRNA delivery

Therapeutic siRNAs
siRNA target gene Disease
p53 mutant
K- Ras
BCR-ABL
MDR1
C-RAF
Bcl-2
VEGF
PKC-
Β- Catenin
Cancer
reserved.

Therapeutic siRNAs
siRNA target
gene
Disease
HIV-Tat
HIV-Rev
HIV- Vif, - Hef
HPV-E6 and –E7
HBV-S1, -S2, -S, -
X
CCR5, CXCR4
CD4
Viral Infection
Fas receptor
Caspase-8
Acute Liver Failure
TNF- Sepsis

Conclusion
A different variety was selected in nature during evolution by
using gene silencing
Gene Silencing balances and satisfy the biosafety concern
as non transgenic variety has no implications
Down regulation Of allergins
• Gene silencing can down regulate allergin or potentially
toxic substances eg – allergic proteins of rice is
downregulated by antisense method
Sexual seggregation of transgene constructs
• 1 transgene construct may inactivate other.but they can
be seperated by sexual reproduction
• Thus poorly expressed gene become highly
expressed.but it impose concerns of biosafety

Cancer treatments
– knock-out of genes required for cell proliferation
– knock-out of genes encoding key structural proteins.
Powerful for analyzing unknown genes in
sequence genomes.
efforts are being undertaken to target every human
gene via miRNAs
Assesment of biosafety for transgeneses
Biosafety concerns with transgenecity
An experiment for this assessment concluded that new
ecological environment created by trangenesis can
promote new type of recombinant virus

© Copyright. Sandipayan Dutta. 2014. All rights reserved.
BOOKS READ:
 Molecular Biology_
David P.clark
ELSEVIER
 Molecular biology of the cell
5th edition
Whatson,Baker,Bell,Gann, Levinn,Losick
WEBSITES VISITED:
 Search on www.google.com
 Search on http://www.ncbi.nlm.nih.gov
References

I, Sandipayan Dutta would like to express my heartfelt
gratitude towards our respected principal Dr. A .
Nagarathna and our Head of the Department Mrs.
Asha.K.K .
I would like to give special thanks to Dr.Manjula Dutt for
assigning me the topic for this presentation.
My family and friends have always been by my side and
it falls as my duty to mention them in this event.
reserved.
Acknowledgement

reserved.
Thank You !

reserved.

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