Gene silencing last

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

Transcript of Gene silencing last

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GENE SILENCING

AND ITS APPLICATION

By-

Mahendra Patidar

MSc. Biotechnology

Centre of Biotechnology,

University of Allahabad

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Contents

• Introduction

• Process of gene silencing

• Gene silencing methods used in research

• RNA interference

• Application of RNAi

• Conclusion

• References

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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.

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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.).

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• 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

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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

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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.

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Cenorhabditis

elegans

Discovery of RNA interference

(1998)- silencing of gene expression with dsRNA

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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

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Different classes of small RNA

molecules

During dsRNA cleavage, different RNA classes are produced:

– siRNA

– miRNA

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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

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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).

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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)

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Source : Cheng JC,. Moore TB ,Sakamoto KM. RNA interference and human disease.

Molecular Genetics and Metabolism 80 (2003) 121–128.

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siRNA miRNA

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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

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• 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

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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.

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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

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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.

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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

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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).