gene silencing is promising field for human welfare

1. GENE SILENCING
AND ITS APPLICATION
By-
Mahendra Patidar
MSc. Biotechnology
Centre of Biotechnology,
University of Allahabad

2. Contents
• Introduction
• Process of gene silencing
• Gene silencing methods used in research
• RNA interference
• Application of RNAi
• Conclusion
• References

3. INTRODUCTION
• Gene silencing is a general term describing epigenetic
processes of gene regulation.
• The term gene silencing is generally used to describe the
"switching off" of a gene by a mechanism other than genetic
modification.
• Gene silencing occurs when RNA is unable to make a protein
during translation.

4. PROCESS OF GENE SILENCING
• Genes are regulated at either the
transcriptional or post-transcriptional level.
• Transcriptional gene silencing is 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.).

5. • 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).
• The blocking of the gene occurs through the activity of
silencers, which bind to repressor regions. A common
mechanism of post-transcriptional gene silencing is RNAi

6. Gene silencing methods used in
research
• Antisense oligonucleotides were 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.
Antisense oligonucleotides

7. 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 catalytic RNA molecules.

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

9. RNA interference ( RNAi), is a technique in which exogenous,
double-stranded RNAs ( dsRNAs ) that are complimentary to
known mRNAs, are introduced into a cell to specifically
destroy that particular mRNA, thereby diminishing or
abolishing gene expression .
RNA interference was known by other names, including post
transcriptional gene silencing and quelling .
RNA Interference

10. Different classes of small RNA
molecules
During dsRNA cleavage, different RNA classes are produced:
– siRNA
– miRNA

11. Small interfering RNA ( siRNA ):
• Short interfering RNA or silencing RNA
• 21–23 nucleotide-long double-stranded RNA
•
• Can be exogenously (artificially) introduced into cells by transfection

12. Source-Hammond, S. M., Caudy, A. A. & Hannon, G. J. Post-transcriptional gene
silencing by double-stranded RNA. Nature Rev. Genet. 2, 110-119 (2001).

13. MicroRNAs ( miRNA ):
• Single-stranded RNA molecules, 22–25 nucleotides
• Encoded by genes but are not translated into protein (non-coding RNA)
• Primary transcript (a pri-miRNA ) is processed to a short structure called a pre-
miRNA and finally into a functional miRNA .
• First miRNA is lin-4 MicroRNA(miRNA)

14. Source : Cheng JC,. Moore TB ,Sakamoto KM. RNA interference and human disease.
Molecular Genetics and Metabolism 80 (2003) 121–128.

15. siRNA miRNA

16. Gene knockdown
• The RNA interference pathway is often exploited in experimental biology to
study the function of genes in cell culture and in vivo in model organisms.
• Repressing a gene from being expressed allows for testing of the protein and
its role in life of a cell or a larger organism.
APPLICATION

18. • Ras genes are frequently mutated in human cancers
• RNAi in the field of tumor therapy have targeted the commonly mutated Ras
oncogene
• Brummelkamp et al. report success inhibiting K-RAS expression in human
pancreatic carcinoma cells using a RNAi retroviral system
• which led to the loss of anchorage-independent growth and tumorgenicity.
• Furthermore,selective inhibition of mutant K-RAS (Kirsten-rat sarcoma 2 viral
oncogene) and not wild-type exemplifies the specificity of RNAi
• Researchers have also used siRNAs to selectively regulate the expression of
cancer-related genes.
• 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.
RNAi in cancer

19. RNAi as a treatment for HIV
• Viral genes and host genes that are required for viruses to replicate or enter the
cell,
• 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.
• co-receptors CCR5 have been successfully down regulated by RNAi, resulting
in the inhibition of HIV replication in numerous human cell lines and in
primary cells including T lymphocytes and haematopoietic stem-cell-derived
macrophages
• siRNA was used to silence the primary HIV receptor chemokine receptor 5
(CCR5).[22] This prevented the virus from entering the human peripheral blood
lymphocytes and the primary hematopoietic stem cells.

20. Respiratory diseases
Ribozymes, antisense oligonucleotides, and more recently RNAi have been
used to target mRNA molecules involved in asthma.
Neurodegenerative disorders
Huntington’s disease (HD) results from a mutation in the huntingtin gene that
causes an excess of CAG repeats.
The gene then forms a mutated huntingtin protein with polyglutamine repeats
near the amino terminus This disease is incurable and known to cause
motor,cognitive, and behavioral deficits.
Gene silencing can be used to treat HD by targeting the mutant huntingtin
protein

21. Application of RNAI in crop improvement
• Crop quality traits : Sunilkumar et al., 2006. reduced the
toxic terpenoid gossypol in cotton seeds and cotton oil by
engineering small RNAs for the cadinene synthase gene in the
gossypol biosynthesis pathway.

22. RNAi technology can be considered an eco-friendly, biosafe 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.
:
Conclusion

23. REFERENCES
• S.M. Elbashir, W. Lendeckel, T. Tuschl, RNA interference is mediated by 21- and 22-nucleotide
RNAs, Genes Dev. 15 (2) (2001) 188–200
• Hutvagner, G., McLachlan, J., Pasquinelli, A. E., Balint, E., Tuschl, T., and Zamore, P. D. (2001).
A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small
temporal RNA. Science 293(5531), 834–838.
• Chendrimada, T. P., Gregory, R. I., Kumaraswamy, E., Norman, J., Cooch, N., Nishikura, K., and
Shiekhattar, R. (2005). TRBP recruits the Dicer complex to Ago2 for microRNA processing and
gene silencing. Nature 436(7051), 740–744.
• Kole,R., Krainer,A.R. and Altman,S. (2012) RNA therapeutics: beyond RNA interference and
antisense oligonucleotides. Nat. Rev. Drug Discov., 11, 125–140.
• Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis
elegans. Nature 391, 806–811 (1998).