Gene silencing is a technique that aims to reduce or eliminate the production of a protein from its corresponding gene. There are two main types of gene silencing: transcriptional gene silencing which occurs at the transcriptional level through histone modifications or DNA methylation, and post-transcriptional gene silencing which occurs after transcription through RNA interference (RNAi) or microRNA (miRNA) pathways. Gene silencing has many applications including cancer treatment, biotechnology, and studying gene function.

1. GENE SILENCING
BY,
PAVITHRA B (16BIOB12)
II M.SC.BIOTECH

2. Contents
• Introduction
• Types of gene silencing
-Transcriptional gene silencing
-Post transcriptional gene silencing
• Application
• Conclusion

3. • Gene silencing is a technique that aims to reduce or eliminate the production of a protein from it’s
corresponding gene.
• 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.
Introduction

4. • 1990 Jorgensen:
• To deepen the pigmentation in petunias, Introduction of transgenes homologous to endogenous genes often
resulted in plants with both gene suppressed called co suppression.
• 1995 Guo and Kemphues:
• Injection of either antisense or sense RNAs in the germline of C.elegans was equally effective at silencing at
homologous target genes.
• 1998 Mello and Fire:
• Extension of above experiments, combination of sense and antisense RNA(=dsRNA) was 10 times more
effective than single strand RNA.
Short History of gene silencing

5. • Genes are regulated at either the transcriptional level or post-transcriptional
level, therefore silencing can be induced either at transcriptional level or post-
transcriptional level.
• There are mainly two types of gene silencing
1. Transcriptional gene silencing
2. Post transcriptional gene silencing
Types of Gene silencing
Transcriptional gene silencing Post transcriptional gene
silencing
Promoter silenced Promoter active
Genes hypermethylated in
promoter region
Genes hyper methylated in
coding region

6. • The result of histone modifications, creating an environment of
heterochromatin around a gene that makes it inaccessible to transcriptional
machinery (RNA polymerase, transcription factors, etc.).
Genomic imprinting:
• genomic imprinting is a genetic phenomenon by which certain genes are
expressed in a parent-of-origin-specific manner. It is an inheritance process
independent of the classical Mendelian inheritance.
• genomic imprinting have been demonstrated in insects, mammals and
flowering plants.
Transcriptional gene silencing

7. Paramutation:
• Paramutation is an interaction
between two alleles of a single
locus, resulting in a heritable
change of one allele that is
induced by the other allele.
• Paramutation was first observed
by the effect it had on the
colour of corn kernels in maize
plants.

8. 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).

9. RNA Directed DNA Methylation:
• RNA-directed DNA methylation is
an epigenetic process first
elucidated in plants whereby small
double-stranded RNAs (dsRNA's)
are processed to guide methylation
to complementary DNA loci. In the
model plant organism Arabidopsis
thaliana

10. 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.

11. Transgene silencing:
• Unfortunate insertion of transgene in to a
transcriptionally inactive part of genome.
• The lose of transgene stability is because of gene
silencing.
• E.g. slow fruit softening tomato, by reducing
expression of polygalactouronase enzyme.

12. • The ability of exogenous or sometimes endogenous RNA to supress the expression of the gene which
corresponds to the m-RNA sequence.
RNA i (RNA interference):
• it is a post transcriptional process triggered by the introduction of double stranded RNA (ds RNA) which
leads to the gene silencing in a sequence specific manner.
• First evidence came from studies on nematode caenorhabditis elegans. further analysis in fruit fly drosophila
melanogaster.
• It is also known as post transcriptional gene silencing / co suppression and quelling.
Post transcriptional gene silencing

13. Over view of RNA i:
• During RNA i Double-stranded RNAs cut into short double-stranded RNAs, s(small)
i(interfering) RNAs, by an enzyme called Dicer. These then base pair to an mRNA
through a dsRNA-enzyme complex. This will lead to degradation of the mRNA strand
• Highly specific process
• So far only been seen in eukaryotes
• Evidence 30% of genome is regulated by RNA I
• RNA i pathway guided by, Si RNA (small interfering RNA):
• Small interfering RNAs that have an integral role in the phenomenon of RNA
interference (RNA i), a form of post- transcriptional gene silencing
• RNA i: 21-25 nt fragments, which bind to the complementary portion of the target
mRNA and tag it for degradation

14. 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:
• 1) Amino-terminal helicase domain
• 2) Dual RNAse III motifs in the carboxyl terminal segment
• 3) dsRNA binding domain
• 4) PAZ domain

15. • DICER’ s domain:

16. • A single base pair difference
between the Si RNA
template and the target
mRNA is enough to block
the process.
• Each strand of Si RNA has:
• a. 5’-phosphate termini
• b. 3’-hydroxyl termini
• c. 2/3-nucleotide 3’
overhangs

17. 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.

18. Steps of the RNA i pathway :
• I. ds RNA is sliced by an ATP-dependent
ribonuclease (Dicer) into short interfering RNAs
(si RNAs).
• duplexes of 21 23 nucleotides bearing two-
nucleotide 3' overhanging ends.
• II. Si RNAs are transferred to a second enzyme
complex, designated RISC for RNA i -induced
silencing complex.
The si RNA guides RISC to the target mRNA,
leading to its destruction.
• the antisense strand of the Si RNA is perfectly
complementary

19. 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.

20. Non sense Mediated Decay:
• Nonsense mediated decay (NMD) is a cellular
mechanism of mRNA surveillance that functions
to detect nonsense mutations and prevent the
expression of truncated or erroneous proteins.
• NMD is triggered by exon junction complexes
(EJCs) (components of the assembled RNP) that
are deposited during pre-mRNA processing.

21. Anti sense RNA technology:
• It blocks the activity of the m RNA in a
stoichiometric manner.
• Antisense RNA has the opposite sense to m RNA.
• The presence of complimentary sense and
antisense RNA in the in the same cell can lead to
the formation of a stable duplex which interferes
with gene expression at the level of RNA
processing or possible translation.
• This technology widely used in plants for gene
inhibition.

22. Advantages of gene silencing:
• Downregulation 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.
• Powerful tool for analysing unknown genes in sequenced genomes.
• Useful approach in future gene therapy.

23. 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.

24. Application of Gene silencing
• Cancer treatments
• RNA interference has been used for applications in biotechnology, particularly in the
engineering of food plants that produce lower levels of natural plant toxins.
• Such techniques take advantage of the stable and heritable RNA i phenotype in plant
stocks. For example, cotton seeds.
• Modulation of HIV-I replication by RNA i.
• Developing technologies for epigenomic analysis and clinical application of molecular
diagnosis.

25. • 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.
Conclusion

26. GENE REGULATION

27. Flow of genetic information

28. • Total no: genes: prokaryotes - 4000
eukaryotes - 35000
• Gene expression
Multistep process that results in the production of functional
gene product
• Importance
=>Cellular differentiation
=>Morphogenesis
=>Adaptability of organism

29. Types of genes
• Constitutive genes (House keeping genes):
Expressed at a reasonably constant rate
Not subjected to regulation
eg: enzymes of glycolysis
• Regulated genes
expressed only under certain conditions
expressed in all cells / subset of cells
eg: expression of insulin gene in pancreas

30. Types of genes
• Inducible gene:
expression increases in response to inducer or activator
eg: bile acid induces ALP
• Gratuitous inducers
Compounds structurally similar to substrates may act as
inducers
eg: IPTG (isopropylthiogalactoside) – lactose analog

31. • Repression
inhibition of gene expression by repressor
eg: inhibition of ALA synthase by heme
Catabolite repression:
Catabolite of a molecule inhibit gene expression

32. Types of gene expression regulation
• Positive regulation
increased gene expression mediated by positive
regulator / enhancer / activator
• Negative regulation
reduced gene expression mediated by negative
regulator / silencer / repressor

33. Regulation of Gene Expression

34. Regulation of Gene Expression
• Gene expression can be modulated by
• Control of transcription
• Post transcriptional modifications
• Gene amplification
• Gene rearrangements
• Control of translation
• Protein modification / stabilization
• Gene regulation is influenced by hormones, heavy metals and chemicals

35. Regulation of Gene Expression in prokaryotes
• Regulation occurs at transcription
• Operon
Genes involved in a metabolic pathway when present in a linear array
• Cistron
Smallest unit of gene expression
• Polycistronic mRNA: A single mRNA that encodes more than one separately
translated protein.
eg - lac operon mRNA

36. Operon concept of gene regulation
• Francois Jacob and Jacques Monod, 1961
• Lactose metabolism in E.Coli (intestinal bacteria)
• Lac Operon
structural gene
inhibitor gene
promoter / operator areas
• Repression – derepression mechanism

38. • Structural gene
lacZ : β- galactosidase (lactase)
lacY: galactoside permease
lacA: thiogalactoside transacetylase
• Inhibitor gene
lacI: LacI (lac operon repressor protein)
• Promoter site
transcription of structural genes
• Operator site
binding of lac repressor

39. Regulation lac operon
• Absence of lactose – reduced synthesis of lactose
metabolizing enzymes
• presence of lactose – increased enzyme synthesis
• Presence of lactose and glucose – reduced synthesis of
enzymes

40. Repression of Lac operon (absence of lactose)

41. Derepression of Lac operon (presence of lactose)

42. Catabolite repression (both glucose and lactose
present)

43. Catabolite repression
• Repression of lac operon by catabolite of glucose
(catabolite activator protein)
CAP + c AMP
facilitate binding of RNAP to the promoter
transcription of structural gene

44. Discovered in 1953 by Jacques Monod and colleagues, the trp operon in E. coli was the first
repressible operon to be discovered.
This operon contains five structural genes:
trp E,
trp D,
trp C,
trp B, and
trp A, which encodes tryptophan synthetase.
It also contains a promoter which binds to RNA polymerase and an operator which blocks
transcription when bound to the protein synthesized by the repressor gene (trp R) that binds to the
operator

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46. Amount of trp operon affect transcription

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48. Eukaryotic gene expression
• Eukaryote genome is complex
DNA is extensively folded and packed into chromatin (protein – DNA
complex)
Differs from prokaryotic gene regulation
• Genes are not organized into operon
• Separation of transcription and translation
• RNA processing : capping at 5’ ends,
addition of poly A tail at 3’ end
splicing

49. Regulation of eukaryotic gene expression
 Regulation at transcription level
Chromatin remodeling
Enhancers
Trans acting molecules – DNA binding proteins function as transcriptional factors
Cis-acting regulatory elements
eg: Hormone response elements
 Regulation by post transcriptional process
alternative mRNA splicing
mRNA editing
 Gene amplification
 Gene rearrangement

50. Eukaryotic DNA:
DNA + Histones
HIGHLY CONDENSED
LESS CONDENCED
EUCHROMATINS
HETEROCHROMATINS
NOT TRANSCRIPTED
TRANSCRIPTED
CHROMATINS

51. Mechanism
Histone acetylation
recruitement of remodelling complex
conformational changes in DNA
exposure of binding sites
RNAP binds to the site
transcription

52. • Chromatin remodeling
Heterochromatin / Euchromatin.
eg, Β-globin gene in reticulocytes and muscle
Importance:
1) increases the accesibility of promoters and regulatory regions
2) increases binding of RNA polymerase

53. Chromatin remodeling

54. Enhancers
• DNA elements which facilitate / enhance the initiation of
transcription at the promoter
Features
• Is active only when it exists within the same DNA molecule (cis to the
promoter)
• Work when located long distances from the promoter
• Work when upstream or downstream from the promoter
• Work when oriented in either direction

55. Mechanism of action
Enhancer work by :
• binding one or more proteins
• facilitating binding of the basal transcription complex to the promoter
• recruiting chromatin-modifying co regulatory complexes
• Insulators: prevents the enhancer function

56. DNA binding motifs
• Mediate DNA-protein interactions
Features
• Binds with high affinity to the specific site
• Maintained by hydrogen bonds, ionic interactions and van der Waals
forces
• Involved in providing:
trans -activation domains / ligand binding sites
surfaces for interaction with co-activators/repressors

57. Types of motifS
Helix turn Helix Leucine zipper Zinc finger

58. Types of motiFs
Binding motif Organism Regulatory protein
Helix-turn-Helix E.Coli
mammals
Lac repressor CAP
Pit 1, Oct 1
Zinc finger E.Coli
mammals
Gene 32 protein
Steroid receptor family
Leucine Zipper Yeast
mammals
GCN4
Fos, jun, c-myc

59. Gene amplification
• Increases the number of genes available for transcription
• Eg: resistance to methotrexate by cancer cells
cancer cells
increase the number of genes for DHFR
drug resistance

60. Gene rearrangement
• Occurs during immunoglobulin synthesis
• IgG light chain mRNAs are coded by different segments/domains
variable (VL) =300 coding sequences
joining (JL) = 5
constant (CL) = 10
• Difficult for immune cells to achieve immunological specificity

61. Immunoglobulin gene rearrangement

62. Alternative RNA processing
• Primary transcript is processed differently in different tissues
Achieved by
• Alternative transcription start sites
• Alternative polyadenylation site
• Alternative splicing and processing

63. Regulation of mRNA stability
• Ends of mRNA
5’ cap structure: prevents attack by 5’ exonuclease
3’ poly (A) tail: prevents attack by 3’ exonuclease
• Ribonucleoprotein particles

64. Different forms of tropomyosin by alternative gene
splicing.

65. GTT
CAA
CAA UAA
Apo protein B100
(4563 aa)
Apo protein B48
(2153 aa)
mRNA
transcript
Apo B gene
mRNA
C
U
Translation
Liver Intestine
Example of RNA Editing
mRNA

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