2. Gene silencing
Gene silencing refers to reduction of a certain gene’s
expression.
• 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.
• 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 removed from organism’s genome and thus have
no expression.
3. Types of Gene Silencing
There are mainly two types of gene silencing
1- Transcriptional gene silencing
2-Post transcriptional gene silencing
6. DNA methylation
DNA methylation is a biochemical process involving the addition of a
methyl group to the cytosine or adenine DNA nucleotides.
It is the addition of methyl group to C-5 position of pyrimidine ring to
form 5-methylcytosine.
Methyl groups are transferred from S-adenocyl methionine in a
reaction catalysed by DNA methyltransferases or methylases.
SAM is then converted to SAH (S-adenosyl homocysteine).
7. In DNA where methylations are happening?
CpG island
8.
9. Histone modification:
It is the introduction of an Methyl functional group to Lysine or
Arginine of the histone tail.
These reactions are catalyzed by enzymes with "histone
methyltransferase”
‘Arg’ can be methylated once or twice, and ‘Lys’ once, twice of
trice.
10. Methylation generally associated with transcription repression,
but specific methylations result in activation.
They can loosen the tail allowing transcription factors to
access DNA or encompass the tails around DNA restricting
access.
12. 1 . Genomic imprinting:
Genomic imprinting is the inheritance out of Mendelian borders. It is an
epigenetic process that involves DNA methylation and histone methylation
without altering the genetic sequence.
Humans inherit two alleles from mother and father, both are functional for the
majority of the genes, but sometimes one is turned off or “stamped” and doesn’t
show in offspring, that gene is imprinted. Imprinting means that that gene is
silenced, and gene from other parent is expressed.
13. Genomic imprinting is a process of silencing
genes through DNA methylation. The repressed
allele is methylated, while the active allele is
unmethylated.
example:
PraderWilli syndrome, and Angelman syndrome. Both
of these syndromes can be caused by imprinting or
other errors involving genes on the long arm of
chromosome 15.
Prader–Willi syndrome (PWS) is a genetic
disorder due to loss of function of specific
genes. In newborns, symptoms include weak
muscles, poor feeding, and slow development.
About 74% of cases occur when part of the
father's chromosome 15 is deleted. In another 25%
of cases, the person has two copies of chromosome
15 from their mother and none from their father. As
parts of the chromosome from the mother
are turned off, they end up with no working copies
of certain genes. PWS is not generally inherited,
but instead the genetic changes happen during the
formation of the egg or sperm.
14. Angelman syndrome (AS) is a genetic disorder that mainly
affects the nervous system. Symptoms include a small head and a
specific facial appearance, severe intellectual disability, developmental
disability, speaking problems, balance and movement problems,
seizures, and sleep problems. Children usually have a happy
personality and have a particular interest in water.
Angelman syndrome is due to a lack of function of part
of chromosome 15 inherited from a person's mother.
15. 2- paramutation
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.
The allele inducing the change is said to be
paramutagenic, while the allele that has been
epigenetically altered is termed
paramutable. A paramutable allele may have
altered levels of gene expression. Alleles
unaffected by exposure to a paramutagenic
allele are called neutral alleles.
16. Crossing a plant carrying a paramutable allele with a plant carrying a
paramutagenic allele results in reduced gene expression of the
susceptible allele. When these hybrids are out crossed to plants
carrying paramutable alleles, most of the progeny exhibit the low-
expression phenotype in this and the following generations,
demonstrating a heritable change in expression state for the
paramutable allele.
In some cases, the formerly paramutable allele becomes
paramutagenic and can now silence other paramutable alleles; this is
called secondary paramutation.
17. For example:
(A)In maize, the paramutable B-I allele is silenced by the paramutagenic B9 allele in
the first generation. In the next generation, the newly silenced B-I allele is
paramutagenic and silences a naive B-I allele.
(B) In maize, the paramutable R-r:standard allele is expressed in the first generation
when heterozygous with the paramutagenic R-stippled. When the heterozygote is
crossed to a plant homozygous for a neutral r allele, R-r:standard is silenced in
the second generation progeny
(c) In Arabidopsis autotetraploids, cross of hygromycin-resistant plants (RRRR)
with hygromycin- sensitive plants (SSSS) carrying a silent transgene produces first
generation progeny that are hygromycin resistant. Self-fertilization of the hybrids
produces mainly hygromycin-sensitive (silenced) progeny.
18. 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.
19. 4. 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)
20. B- Post transcriptional gene silencing:(PTGS)
Post-transcriptional gene silencing is the result of mRNA of a
particular gene being destroyed or blocked.
• The destruction of the mRNA prevents translation to form an
active gene product (in most cases, a protein).
common mechanism of post-transcriptional gene
silencing
1-Ribozymes
2-RNA Interference (RNAi)
3-Antisense Oligonucleotides
The Collective use of these techniques is called Antisense
Technology
21.
22. The silencing of a gene could be achieved by:
i) Drugs: These bind to target protein and cause protein
inhibition
ii) RNase H–independent ODNs: These oligodeoxy
nucleotides hybridize to target mRNA and cause inhibition of
translation of target protein.
iii) RNase H–dependent ODNs: These hybridize to target
mRNA and mediate its degradation by RNase H.
iv) Ribozymes: These catalyze cleavage of mRNA and hence
cause its degradation.
v) SiRNA and miRNA: These hybridize to target mRNA by
antisense strand and guide it into endo ribonuclease enzyme
complex, thereby causing its degradation or inhibition of
translation.
23. 1-Ribozymes
Ribozymes are catalytic RNA molecules used to inhibit gene
expression. These molecules work by cleaving mRNA molecules,
essentially silencing the genes that produced them.
idney Altman and Thomas Cech first discovered in 1989 catalytic
RNA molecules
Several types of ribozyme motifs exist,
including hammerhead, hairpin, hepatitis delta virus, group
I, group II, and RNase P ribozymes.
Hammerhead, hairpin, and hepatitis delta virus (HDV) ribozyme
motifs are generally found in viruses or viroid RNAs.
These motifs are able to self-cleave a specific phosphodiester
bond on an mRNA molecule.
Lower eukaryotes and a few bacteria contain group I and group II
ribozymes.
The last ribozyme motif, the RNase P ribozyme, is found
in Escherichia coli and is known for its ability to cleave the
phosphodiester bonds of several tRNA precursors when joined to
a protein cofactor.
.
24. Mechanism
The general catalytic mechanism used by ribozymes is
similar to the mechanism used by
protein ribonucleases. These catalytic RNA molecules bind
to a specific site and attack the neighboring phosphate in
the RNA backbone with their 2' oxygen, which acts as
a nucleophile, resulting in the formation of cleaved products
with a 2'3'-cyclic phosphate and a 5' hydroxyl terminal
end. .
29. What is RNA interference (RNAi)?
RNA interference (RNAi) is a mechanism that inhibits gene expression at the
stage of translation or by hindering the transcription of specific genes.
30. Mechanism of RNAi
In Interference
Dicer : produces 20-25 nt cleavages that initiate RNAi
Drosha : cleaves base hairpin in to form pre miRNA; which is later
processed by Dicer
RISC: RNA induced Silencing Complex, that cleaves mRNA
Enzymes
RNA
siRNA: dsRNA 21-25 nt.
miRNA: ssRNA 19-25nt. Encoded by non protein coding genome
31. Dicer
Dicer is an endoribonuclease in the RNase III family that cleaves
double-stranded RNA (dsRNA) and pre-microRNA (miRNA)
into short double-stranded RNA fragments called small
interfering RNAs (siRNAs) about 20-25 nucleotides long,
usually with a two-base overhang on the 3' end.
32. Dicer’s domains
Dicer is a ribonuclease (Rnase III family) with 4 distinct domains
Dicer contains:
1. Amino-terminal helicase domain
2. two RNase III domains
3. dsRNA binding domain
4. PAZ domain is important for protein-protein interaction (110-
130 amino-acid domain present in protein).
Loss of dicer: loss of silencing
33. Drosha : cleaves base hairpin in to form pre miRNA; which is
later processed by Dicer
34. RNA Induced Silencing
Complex (RISC
The RNA-induced silencing complex, or
RISC, is a multiprotein complex,
specifically a ribonucleoprotein, which
incorporates one strand of a single-
stranded RNA (ssRNA) fragment,
such as microRNA (miRNA), or
double-stranded small interfering RNA
(siRNA).
The single strand acts as a template for
RISC to recognize complementary
messenger RNA (mRNA) transcript.
The active components of an RISC are
endonucleases called argonaute proteins
which cleave the target mRNA strand.
35. siRNAs
The siRNA called small interfering or short interfering RNA
The smaller double-stranded piece of RNA having a
dinucleotide overhang at the 3’ end which functionally,
degrade the mRNA and prevent the protein synthesis are
siRNAs.
RNAi: 21-25 nt fragments,
The source of siRNA is exogeneous
Functionally, it blocks gene expression
The siRNA is a double-stranded structure in which one strand
is known as the guide strand and another strand is called the
passenger strand.
Each strand of siRNA has:
a. 5’-phosphate termini
b. 3’-hydroxyl termini
c. 2/3-nucleotide 3’ overhangs
36. miRNA:
• the miRNA is known as microRNA.
• the miRNA is made up to 19-25.
•the miRNA are endogenous single-stranded, non-coding RNA
molecule, by forming a hairpin structure, it becomes duplex.
37. Mechanism of RNAi
1-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.
2.The double-stranded molecule is then cut into small double stranded
fragments by an enzyme called Dicer
3. These small fragments, which include small interfering RNAs (siRNA)
and micro RNA (miRNA), are approximately 19–25 nucleotides in
length.
4. 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.
5. One strand of the molecule, called the "guide" strand, binds to RISC,
while the other strand, known as the "passenger" strand is degraded.
38. 6. The guide or antisense strand of the fragment that remains
bound to RISC directs the sequence-specific silencing of the
target mRNA molecule.
7. 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.
8. 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.
39.
40. 3- Antisense oligonucleotides
Discovered in 1978 by Paul Zamecnik and Mary Stephenson.
Oligonucleotides, which are short nucleic acid fragments, bind to
complementary target mRNA molecules when added to the cell.
The antisense oligonucleotides can affect gene expression in two
ways:
1- by using an RNase H-dependent mechanism
2-by using a steric blocking mechanism
RNase H-dependent oligonucleotides cause the
target mRNA molecules to be degraded, While steric-blocker
oligonucleotides prevent translation of the mRNA molecule because
The ribosome cannot gain access to the nucleotides in the mRNA.
41. Mechanism
• This process was done with the help of antisense DNA
Oligonucleoside (AS-ON).
These all are the short segments of single strand DNA.
• The AS-ON usually consists of 13-25 nucleotides, which are
complementary to their mRNA.
• It will bind to the target RNA.
When it bind to the mRNA, a DNA RNA hybrid will be formed.
• After the hybrid formation it will be degraded by the enzyme
Rnase H.
It cleave the mRNA by the hydrolytic mechanism.
• Because of that cleavage the translation process should be
blocked.
42.
43. Antisense Therapy
• Antisense therapy is a mode of treatment for genetic
disorder or infections.
• A complementary mRNA strand is synthesized on the
basis of the known pathogenic sequence, and which
switch ‘off’ the pathogenic gene by activating the
degrading enzyme RNase H.
• Antisense drugs are being researched to treat cancers,
HIV, CMV etc.
• Formivirsen is the first antisense antiviral drug
developed to treat CMV. It was licensed by FDA in Aug,
1998.
• It is a synthetic 21 member oligonucleotide with
phosphorothioate linkages (which are resistant to
degradation by nucleases) and has the sequence: 5'-
GCG TTT GCT CTT CTT CTT GCG-3‘
44.
45. Challenges to antisense technology
1. One major challenge to antisense technology (and RNAi) is the
difficulty of getting it into the body. Delivery of the treatment to the
brain, for use in diseases like HD, is especially challenging
because it must cross the blood brain barrier.
2.The second major challenge to antisense technology is its
inevitable toxic effects. Although antisense technology is
engineered to be very specific, it can still cause unintended
damage because it would regulate both the mutant and normal
Huntington alleles.
46. CRISPR-cas System
An array of short repeated sequences separated by
spacers in a region of DNA called Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR).
The spacers derived from nucleic acid of viruses and
plasmids and act as recognition elements to find matching
virus genomes
• Antiviral defense system
• Targets DNA or RNA protecting against viruses and other
mobile genetic elements.
The CRISPR locus, first observed in Escherichia coli
• Found on both chromosomal and plasmid DNA
• CRISPR activity requires the presence of a set of
CRISPRassociated (cas) genes
• Code for proteins essential to the immune response
• Offspring inherit the protection due to genome
modification by spacer acquisition
47. Stages of CRISPR-cas mediated defense
The key steps of CRISPR-cas immunity.
• 1) Adaptation: insertion of new spacers into the
CRISPR locus.
• 2) Expression: transcription of the CRISPR
locus into long precursor CRISPR RNA and
processing of pre-CRISPR RNA into mature
crRNA by cas proteins and accessory factors
• 3) Interference: detection and degradation of
mobile genetic elements by CRISPR RNA and
cas protein
48. CRISPER cas 9
CRISPR - Clustered Regularly Interspaced Short Palindromic
Repeats
CRISPR-Cas9 is a unique technology that enables geneticists
and medical researchers to edit parts of the genome by cutting
out, replacing or adding parts to the DNA sequence.
The CRISPR-Cas9 system consists of two key molecules
that introduce a change (mutation) into the DNA. These are:
1- cas9
2- gRNA
49. an enzyme called Cas9. This acts as a pair of ‘molecular
scissors’ that can cut the two strands of DNA at a specific
location in the genome so that bits of DNA can then be
added or removed
sgRNA:
Single guide RNA is a combination of tracr RNA and cr RNA
crRNA:
Contains the guide RNA that locates the correct section of
host DNA along with a region that binds to tracrRNA forming
an active complex.
tracrRNA:
Trans activating crisper RNA (tracer RNA) is another
important molecule that plays a critical role in the processing
of pre-crRNA. It is a short RNA sequence and is
complementary to the CRISPR repeat. It binds to crRNA and
and forms an active complex.
Single guide RNA is a combination of tracr RNA and cr RNA
50. Applications of Gene silencing
• In Agriculture:
1- production of virus resistant plants and engineering of
food plants that produce lower levels of natural plant
toxins.
2-Such techniques take advantage of the stable and
heritable RNAi phenotype in plant stocks.
For example, 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
51. 3- in Rice reduced the level of glutein by Produced
variety LGC-1, was a relief of the kidney patients
unable to digest glutein.
4- in Tomato improve carotenoid and flavonoid levels
in tomato fruits with minimal effects on plant growth
and fruit quality parameters (Davuluri et al., 2005).
5- -in Barley Production of resistant plants to BYDV
(Barley yellow dwarf virus) (Wang et al., 2000).
6- in Potato Production of Resistance plant to late
blight .
7- Caffeine producing gene in Coffee produce Low or
very low caffeine content (Van Uyen, 2006 ).
8- In scientific research, knock out genes with known
sequence to study their functions under functional
genomics
52. Medicine
1.RNA interference is seen as a promising way to treat
cancer by silencing genes differentially upregulated in
tumor cells or genes involved in cell division.
2-AIDS: It has been shown that siRNAs can inhibit HIV
replication effectively in culture. HIV infection can also be
blocked by targeting either viral genes (for example, gag,
rev, tat and env) or human genes (for example, CD4, the
principal receptor for HIV) that are involved in the HIV life
cycle. Thus promising antiviral therapies that can attack
multiple viral and cellular targets circumvent genetic
resistance of HIV
3-Hepatitis: This has provided the first tangible evidence
for RNAi as a therapy for diseases in live animals. Early
RNAi studies noted that RNA silencing was prominent in
the liver, which made this organ an attractive target for
53. REFERENCES
Parveen, P., Deepti Brundavani K., Mahathi K., Bhavani M.S and
Shaheda Sultana SK (2019). Gene Silencing and DNA Methylation .
American Journal of Phytomedicine and Clinical Therapeutics
Kumar, G. et al. Gene Silencing, Mechanism and Applications. DHR
International Journal Of Biomedical and Life Sciences (DHR-IJBLS),
Vol. 3(1), 2012 .
Kole,R., Krainer,A.R. and Altman,S. (2012) RNA therapeutics: beyond
RNA interference and antisense oligonucleotides. Nat. Rev. Drug
Discov., 11, 125–140
"Gene Silencing". National Center for Biotechnology Information.
Retrieved 11 November 2013
https://www.sciencedirect.com/.../gene-silencing
https://www.ncbi.nlm.nih.gov
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