1.
Gene Silencing
Presented by
Vishnu kumar Dhakad
AB & MB Department
Dr. RPCAU,pusa

2.
 1. Types:
 1.1 Transcriptional level A . Genomic Imprinting
 B . Paramutation
 C . Transposon silencing (or Histone Modifications)
 D . Transgene silencing
 E . Position effect
 F . RNA-directed DNA methylation
 1.2 Post transcriptional level A . RNAi
 B . SiRNA
 C . miRNA
 D . Dicer
 E . RISC
1.3 Meiotic
 2. Research methods:
 2.1 Antisense oligonucleotides
 2.2 Ribozymes
 2.3 SiRNA
 2.4 microRNA
 3. Application :
 4. Advantages & disadvantages
 5. Conclusions
 6. Reference

3.
 1.Epigenetics and aging .
 2. Is epigenetics a cause or effect of aging ?
 3How could natural selection prefer a
genome that destroys itself and cuts off its
own reproduction?.

4.
 Interruption or suppression of the expression of a gene at
transcriptional or translational levels.
 It is used as regulation of gene expression & prevent the
expression of a certain gene.
 1980's - Antisense oligodeoxynucleotides (ODNs)
 1990's - Ribozymes
 2000's - RNA interference (RNAi)
 It generally describe the “switching off” of a gene by a mechanism other
than genetic modification.
 That is, a gene which would be expressed (“turned on”) under normal
circumstances is switched off by machinery in the cell.
 It occurs when RNA is unable to make a protein during translation.
 Gene silencing is same as gene knock down but is totally different from
gene knock out.
 When genes are knock down ,there expression is reduced , where in
contrast when genes are knocked out, they are completely erased from
organism’s genome and thus have no expression.

5.
Agents 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

6.
 1 . Genomic imprinting : It is an
epigenetic process that involves DNA
methylation and histone
methylation without altering the
genetic sequence. These epigenetic
marks are established ("imprinted") in
the germline (sperm or egg cells) of the
parents and are maintained
through mitotic cell divisions in
the somatic cells of an organism.
 2 . Paramutation : In epigenetics,
a paramutation is an interaction
between two alleles at a single locus,
whereby one allele induces a heritable
change in the other allele. The change
may be in the pattern of DNA
methylation or histone modifications

7.
• 3. Transposon silencing:
• Transposon silencing is a form of
transcriptional gene silencing
targeting transposons.
Transcriptional gene silencing is a
product of histone modification
that prevent the transcription of
that area of DNA.
• The “jumping” of transposon
generates the genomic instability
and cause the extremely
deleterious mutations.
• Transposable element insertion
have been linked to many disease
including haemophilia ,SCID and
predisposition to cancer.

8.
• 4. Transgene silencing:
• Unfortunate insertion of
transgene in to a
transcriptionally inactive part
of genome. When an insertion
of any transgene it does not
show activity as per desire and
this is because of it’s
instability.
• The lose of transgene stability
is because of gene silencing.
• E.g. slow fruit softening
tomato, by reducing
expression of
polygalactouronase enzyme.

9.
• 5. 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
colour and is known as position effect variegation (PEV).

10.
Discovery of RNA interference (1998)
silencing of gene expression with dsRNA
Cenorhabditis elegans

11.
 1. RNA interference (RNAi) is a natural process used by cells
to regulate gene expression.
 2. The process to silence genes first begins with the entrance
of a double-stranded RNA (dsRNA) molecule into the cell,
which triggers the RNAi pathway.
 3.The double-stranded molecule is then cut into small double-
stranded fragments by an enzyme called Dicer.
 4. These small fragments, which include small
interfering RNAs (siRNA) and microRNA (miRNA), are
approximately 21–23 nucleotides in length.
 5. The fragments integrate into a multi-subunit protein
called the RNA-induced silencing complex, which
contains Argonaute proteins that are essential
components of the RNAi pathway.

12.
 6. One strand of the molecule, called the "guide" strand,
binds to RISC, while the other strand, known as the
"passenger" strand is degraded.
 7. The guide or antisense strand of the fragment that
remains bound to RISC directs the sequence-specific
silencing of the target mRNA molecule.
 8. The genes can be silenced by siRNA molecules that
cause the endonucleatic cleavage of the target mRNA
molecules or by miRNA molecules that suppress
translation of the mRNA molecule.
 9. RNAi is thought to have evolved as a cellular defense
mechanism against invaders, such as RNA viruses, or to
combat the proliferation of transposons within a cell's
DNA. Both RNA viruses and transposons can exist as
double-stranded RNA and lead to the activation of RNAi.

13.
 Small (or short) interfering RNA (siRNA) is the most
commonly used RNA interference (RNAi) tool for inducing
short-term silencing of protein coding genes. siRNA is a
synthetic RNA duplex designed to specifically target a
particular mRNA for degradation.

14.
 Mi RNA (micro RNA):
 mi RNA Originate from capped &
polyadenylated full length precursors (pri-
miRNA)
 Hairpin precursor ~70 nt (pre-mi RNA)
Mature mi RNA ~22 nt (mi RNA)
 mi RNA originates with SS RNA that
forms a hairpin secondary structure.
 Mi RNA regulates post-transcriptional gene
expression and is often not 100%
complementary to the target.
 And also mi RNA help to regulate gene
expression, particularly during induction of
heterochromatin formation serves to down
regulate genes pre- transcriptionally (RNA
induced transcriptional silencing or RITS)
RITS.

15.
 Dicer:
 RNAse III-like dsRNA-specific ribonuclease
• Enzyme involved in the initiation of RNA i.
• It is able to digest dsRNA into uniformly sized small RNAs (si RNA)
 Dicer family proteins are ATP- dependent nucleases.
 RNAse III enzyme acts as a dimer
 Loss of dicer→loss of silencing processing in vitro
 Dicer homologs exist in many organisms including C.elegans,
Drosophila, yeast and humans (Dicer is a conserved protein)
 DICER’s domain:
 Dicer is a ribonuclease (RNAse III family) with 4 distinct domains:
 1) Amino-terminal helicase domain
 2) Dual RNAse III motifs in the carboxyl terminal segment
 3) dsRNA binding domain
 4) PAZ domain (110-130 amino-acid domain present in protein like
Argo, Piwi..);it is thought to be important for protein-protein
interaction

16.
 RISC (RNA Inducing Silencing Complex):
 RISC is a large (~500-kDa) RNA-multi protein complex, which
triggers mRNA degradation in response to Si RNA
 Unwinding of double- stranded Si RNA by ATP independent
helicase.
 The active components of an RISC are endonucleases called
argonaute proteins which cleave the target mRNA strand.

17.
 Antisense RNA (asRNA) is a single-stranded RNA that
is complementary to a messenger RNA (mRNA) strand transcribed
within a cell.
 Discovered in 1978 by Paul Zamecnik and Mary Stephenson.
 The antisense oligonucleotides can affect gene expression in two
ways: by using an RNase H-dependent mechanism or by using a
steric blocking mechanism.
 Some authors have used the term micRNA (mRNA-interfering
complementary RNA) to refer to these RNAs but it is not widely
used.
 Antisense RNA may be introduced into a cell to
inhibit translation of a complementary mRNA by base pairing to it.
Antisense RNA may be created by expression of antisense RNA &
may be created by other mechanisms including secondary RNA
structures.
 Antisense RNA is expressed in some GMOs plants such as flvar
savr, and two cultivar of ringspot resistant papaya.
 Example; hok/sok system of the E. coli R1 plasmid.

18.
• Ribozymes are catalytic RNA molecules used to inhibit gene
expression.
• These molecules work by cleaving mRNA molecules, essentially
silencing the genes that produced them.
• Sidney Altman and Thomas Cech first discovered in 1989 catalytic
RNA molecules.
• Several ribozymes motif such as hammerhead, haipin, hepatitis delta
virus, Rnase P (e coil ,degrade tRNA). Most of the ribozymes motif
are found in viruses and viroids.Mechanisms same as Ribonuclease .
Research to finding sequence- sepecific cleavage ribozymes.

19.
 Disadvantages of gene silencing:
 ”High pressure injection” and electroporation can cause significant
injection damage to the integrity of the normal tissues and organs
and thus preclude the utilisation in a clinical set-up.
 Liposomes/cationic encapsulated Si RNA may also be toxic to the
host and may cause severe host immune responses.
 Other emerging strategies includes chemical modification of Si
RNA molecules and encapsulated with different molecules are still
in their infancy and need to be thoroughly investigated before used
in therapeutic applications.

20.
 Advantages of gene silencing:
 Down regulation of gene expression simplifies "knockout"
analysis.
 Easier than use of antisense oligonucleotides. Si RNA more
effective and sensitive at lower concentration.
 Cost effective
 High Specificity middle region 9-14 are most sensitive With Si
RNA, the researcher can simultaneously perform experiments in
any cell type of interest Can be labelled Ease of transfection by use
of vector
 blocking expression of unwanted genes and undesirable substances.
 Inducing viral resistance
 Powerful tool for analysing unknown genes in sequenced genomes.
 Useful approach in future gene therapy.
 Oligonucleotides can be manufactured quickly, some within one
week; the sequence of the mRNA is all that is needed

21.
 RNAi as a treatment for HIV :
 siRNA was used to silence the primary HIV receptor chemokine receptor 5
(CCR5). This prevented the virus from entering the human peripheral blood
lymphocytes and the primary hematopoietic stem cells.
 siRNAs can inhibit HIV replication effectively in culture.
 RNAi in cancer :
 Ras genes are frequently mutated in human cancers .
 Antiapoptotic proteins, such as clusterin and survivin, are often expressed in cancer cells.
 Clusterin and survivin-targeting siRNAs were used to reduce the number of antiapoptotic
proteins and, thus, increase the sensitivity of the cancer cells to chemotherapy treatments.
 Respiratory diseases :
 Ribozymes, antisense oligonucleotides, and more recently RNAi have been used
to target mRNA molecules involved in asthma.
 Neurodegenerative disorders : Gene silencing can be used to treat HD by
targeting the mutant huntingtin protein.
 Crop quality traits : reduced the toxic terpenoid gossypol in cotton seeds and
cotton oil

22.
 It is the epigenetic regulation of gene
expression and widely used in agriculture and
in biotechnology.
 Besides the all types of gene silencing the
RNA i is the important post transcriptional
gene silencing.
 Now recently CIRSPR- Cas 9 system is
discovered , which targets specific
nucleotides sequences to cleavage and
modified DNA and RNA.

23.
Thank you

24.
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Retrieved 11 November 2013.
 2. Molecular biology of the cell 5th edition by whatson, baker , bell, gann,
levinn, losick.]
 3. Hood E (March 2004). "RNAi: What's all the noise about gene
silencing?". Environmental Health Perspectives. 112 (4): A224–9.
 4. Kole R, Krainer AR, Altman S (February 2012). "RNA therapeutics:
beyond RNA interference and antisense oligonucleotides". Nature
Reviews. Drug Discovery.
 5. Shampo MA, Kyle RA, Steensma DP (October 2012). "Sidney
Altman--Nobel laureate for work with RNA". Mayo Clinic Proceedings.
 6. Phylactou, L. (1 September 1998). "Ribozymes as therapeutic tools
for genetic disease". Human Molecular Genetics.
 7. Dias N, Stein CA (March 2002). "Antisense oligonucleotides: basic
concepts and mechanisms". Molecular Cancer Therapeutics.