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Gene Silencing

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it covers all the responsible factors & conditions for gene silent very nicely....

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Gene Silencing

  1. 1. Gene Silencing Presented by Vishnu kumar Dhakad AB & MB Department Dr. RPCAU,pusa
  2. 2.  1. Types:  1.1 Transcriptional level A . Genomic Imprinting  B . Paramutation  C . Transposon silencing (or Histone Modifications)  D . Transgene silencing  E . Position effect  F . RNA-directed DNA methylation  1.2 Post transcriptional level A . RNAi  B . SiRNA  C . miRNA  D . Dicer  E . RISC 1.3 Meiotic  2. Research methods:  2.1 Antisense oligonucleotides  2.2 Ribozymes  2.3 SiRNA  2.4 microRNA  3. Application :  4. Advantages & disadvantages  5. Conclusions  6. Reference
  3. 3.  1.Epigenetics and aging .  2. Is epigenetics a cause or effect of aging ?  3How could natural selection prefer a genome that destroys itself and cuts off its own reproduction?.
  4. 4.  Interruption or suppression of the expression of a gene at transcriptional or translational levels.  It is used as regulation of gene expression & prevent the expression of a certain gene.  1980's - Antisense oligodeoxynucleotides (ODNs)  1990's - Ribozymes  2000's - RNA interference (RNAi)  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.
  5. 5. Agents Mechanism Result Most drugs Bind to target protein Protein inhibition RNase H-independent ODNs Hybridize to target mRNA Inhibition of translation of the target protein RNase H-dependent ODNs Hybridize to target mRNA Degradation of the mRNA by RNase H Ribozymes and DNA enzymes Catalyze cleavage of target mRNA Degradation of the mRNA siRNA Hybridize to target mRNA by its antisense strand and guide it into endoribonuclease enzyme complex (RISC) Degradation of the mRNA
  6. 6.  1 . Genomic imprinting : It is an epigenetic process that involves DNA methylation and histone methylation without altering the genetic sequence. These epigenetic marks are established ("imprinted") in the germline (sperm or egg cells) of the parents and are maintained through mitotic cell divisions in the somatic cells of an organism.  2 . Paramutation : In epigenetics, 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
  7. 7. • 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.
  8. 8. • 4. Transgene silencing: • Unfortunate insertion of transgene in to a transcriptionally inactive part of genome. When an insertion of any transgene it does not show activity as per desire and this is because of it’s instability. • The lose of transgene stability is because of gene silencing. • E.g. slow fruit softening tomato, by reducing expression of polygalactouronase enzyme.
  9. 9. • 5. 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).
  10. 10. Discovery of RNA interference (1998) silencing of gene expression with dsRNA Cenorhabditis elegans
  11. 11.  1. RNA interference (RNAi) is a natural process used by cells to regulate gene expression.  2. 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.  3.The double-stranded molecule is then cut into small double- stranded fragments by an enzyme called Dicer.  4. These small fragments, which include small interfering RNAs (siRNA) and microRNA (miRNA), are approximately 21–23 nucleotides in length.  5. 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.
  12. 12.  6. One strand of the molecule, called the "guide" strand, binds to RISC, while the other strand, known as the "passenger" strand is degraded.  7. The guide or antisense strand of the fragment that remains bound to RISC directs the sequence-specific silencing of the target mRNA molecule.  8. 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.  9. 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.
  13. 13.  Small (or short) interfering RNA (siRNA) is the most commonly used RNA interference (RNAi) tool for inducing short-term silencing of protein coding genes. siRNA is a synthetic RNA duplex designed to specifically target a particular mRNA for degradation.
  14. 14.  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.
  15. 15.  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:  Dicer is a ribonuclease (RNAse III family) with 4 distinct domains:  1) Amino-terminal helicase domain  2) Dual RNAse III motifs in the carboxyl terminal segment  3) dsRNA binding domain  4) PAZ domain (110-130 amino-acid domain present in protein like Argo, Piwi..);it is thought to be important for protein-protein interaction
  16. 16.  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.
  17. 17.  Antisense RNA (asRNA) is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell.  Discovered in 1978 by Paul Zamecnik and Mary Stephenson.  The antisense oligonucleotides can affect gene expression in two ways: by using an RNase H-dependent mechanism or by using a steric blocking mechanism.  Some authors have used the term micRNA (mRNA-interfering complementary RNA) to refer to these RNAs but it is not widely used.  Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it. Antisense RNA may be created by expression of antisense RNA & may be created by other mechanisms including secondary RNA structures.  Antisense RNA is expressed in some GMOs plants such as flvar savr, and two cultivar of ringspot resistant papaya.  Example; hok/sok system of the E. coli R1 plasmid.
  18. 18. • 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- sepecific cleavage ribozymes.
  19. 19.  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.
  20. 20.  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 analysing 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
  21. 21.  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.  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
  22. 22.  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.  Now recently CIRSPR- Cas 9 system is discovered , which targets specific nucleotides sequences to cleavage and modified DNA and RNA.
  23. 23. Thank you
  24. 24.  1. "Gene Silencing". National Center for Biotechnology Information. Retrieved 11 November 2013.  2. Molecular biology of the cell 5th edition by whatson, baker , bell, gann, levinn, losick.]  3. Hood E (March 2004). "RNAi: What's all the noise about gene silencing?". Environmental Health Perspectives. 112 (4): A224–9.  4. Kole R, Krainer AR, Altman S (February 2012). "RNA therapeutics: beyond RNA interference and antisense oligonucleotides". Nature Reviews. Drug Discovery.  5. Shampo MA, Kyle RA, Steensma DP (October 2012). "Sidney Altman--Nobel laureate for work with RNA". Mayo Clinic Proceedings.  6. Phylactou, L. (1 September 1998). "Ribozymes as therapeutic tools for genetic disease". Human Molecular Genetics.  7. Dias N, Stein CA (March 2002). "Antisense oligonucleotides: basic concepts and mechanisms". Molecular Cancer Therapeutics.

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