Gene silencing

1. Gene Silencing
Md. Dilshad Karim
ID: 19BMBJD07M
Dept. of Biochemistry and Molecular Biology
BAU, Mymensingh

2. Outlines
• Introduction
• History of gene silencing
• How does it works
• Types of Gene silencing
• Antisense Oligonucleotides
• Ribozymes
• RNA Interference (RNAi)
• Disadvantages of gene silencing
• Advantages of gene silencing
• Biological role of gene silencing
• Application of gene silencing
• Conclusion

3. Introduction
• 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 removed from organism’s
genome and thus have no expression.

4. History of gene silencing
1990 Jorgensen:
• To develop the pigmentation in petunias introduction of transgenes
homologous to endogenous genes often resulted in plants with both
gene suppressed called co-suppression. Resulted in degradation of the
endogenous and transgene mRNA.
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.

5. 1998 Mello and Fire:
• Extension of above experiments, combination of sense and antisense
RNA(=dsRNA) was 10 times more effective than single strand RNA. The
discovery of the mechanism of RNA interference by ds RNA by prof.
Andrew Fire and prof. Craig Mello in 1998, gave them the Nobel prize in
2006.
Cont.

6. How does it works
• This is accomplished by binding a specific strand of RNA to an
existing m-RNA strand.
• The m-RNA creates a copy of DNA strand.
• By binding the RNA to the m-RNA, m-RNA is prevented from
replicating that portion of the DNA.
• Specific genes can be targeted and prevented from replicating in to
new DNA strands.

7. Types of gene silencing
There are mainly two types of gene silencing
• Transcriptional gene silencing
• Post transcriptional gene silencing
• Transcriptional gene silencing-is the process of silence the gene by
modify the Gene.
• Post transcriptional gene silencing-means double stranded RNA
(dsRNA) suppress the expression of the gene.
It have three type of mechanisms
• Antisense Oligonucleotides
• Ribozymes
• RNA Interference (RNAi)

8. Antisense oligonucleotides
• Antisense therapy was first demonstrated in 1970s Zamecnik and
Stephenson in rous sarcoma virus.
• It is slightly differed from the RNAi process. Instead of gene
cutting mechanism, the Oligonucleotide was introduced to silence
the gene.
• Antisense therapy helps in the study of Gene function.
• It uses to prevent the synthesis of specific protein.

9. Mechanism
• This process was done with the help of antisense DNA Oligo
nucleoside (AS-ON). These all are the short segments of single strand
DNA.
• The AS-ON usually consists of 15-20 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
RNaseH. It cleave the mRNA by the hydrolytic mechanism.
• Because of that cleavage the translation process should be blocked.

10. Figure: Antisense Oligonucleotides process

11. 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.
• 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- specific cleavage ribozymes.

12. Mechanism
• 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.
• These motifs can self-splice by cleaving and joining together
phosphodiester bonds.
• 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.
• The general catalytic mechanism used by ribozymes is similar to the
mechanism used by protein ribonucleases.

13. • 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.
• This catalytic mechanism has been increasingly used by scientists to
perform sequence-specific cleavage of target mRNA molecules.
• In addition, attempts are being made to use ribozymes to produce
gene silencing therapeutics, which would silence genes that are
responsible for causing diseases.
Cont.

14. Figure: Ribozymes process
Ribozyme mediated
cut introduced into
RNA message
Cut RNA message

15. RNA Interference (RNAi)
• Andrew Fire and C. Mello pooled the 2006 Nobel Prize in Physiology
or Medicine for their work on RNA interference in the nematode worm
Caenorhabditis elegans, which they published in 1998.
• RNA interference (RNAi) is a biological process in which RNA
molecules inhibit gene expression or translation, by neutralizing
targeted mRNA molecules.
• RNAi was known by other names, including co-suppression, Post-
transcriptional gene silencing (PTGS), and quelling.

16. Mechanism
• During RNAi, long dsRNA is cut or "diced" into small fragments ~21
nucleotides long by an enzyme called "Dicer".
• These small fragments, referred to as small interfering RNAs (siRNA),
then it bind to the mRNA sequence which derived from the transcription
process.
• It bind to messenger RNA with the help of the multi protein complex
named RISC (ribonucleoprotein).
• RISC protein helps that siRNA to crop the mRNA into small DNA
fragments.
• These small DNA fragments can’t undergoes to the translation process.
So the gene express was inhibited.

17. Figure: RNA interference (RNAi) process

18. 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
utilization in a clinical set-up.
• Liposomes/cationic encapsulated siRNA may also be toxic to the host and may
cause severe host immune responses.
• Other emerging strategies includes chemical modification of siRNA molecules
and encapsulated with different molecules are still in their infancy and need to
be thoroughly investigated before used in therapeutic applications.

19. 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 analyzing 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.

20. Biological role of gene silencing
• Protection against pathogens including viruses.
• Production of resistant plants.
• Protection against endogenous transposable elements.
• Protection against foreign or duplicated genes.

21. Application of gene silencing
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.

22. 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.
Cont.

23. Conclusion
RNAi technology can be considered an eco-friendly, biosafety and ever
green technology as it eliminates even certain risks associated with
development of transgenic. RNAi triggers the formation of dsRNA
molecules that target and facilitate the degradation of the gene of
interest as well as the transgene itself to avoid problems arising from the
synthesis of gene sequences as well as non coding regions of gene, thus
limiting undesirable recombination events.

24. Thank you