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Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
Sandipayan seminar gene silencing
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Sandipayan seminar gene silencing

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gene silencing

gene silencing

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  • 1. M.S.Ramaiah College Of Arts, Science & Commerce Presented by, Sandipayan Dutta. SeminaronGeneSilencing MSc Biotechnology 2nd Semester Year 2014.
  • 2. From DNA to Protein • Transcription Process where information held in the DNA is transferred to RNA. • Translation Conversion of RNA to amino acid sequence of protein. © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 3. © Copyright. Sandipayan Dutta. 2014. All rights
  • 4. Glossary– Dicer – • DICER is a cytoplasmic RNase III enzyme that not only cleaves precursor miRNAs to produce mature miRNAs but also dissects naturally formed/synthetic double-stranded RNAs to generate small interfering RNAs (siRNAs). – Interferon – • A small and highly potent molecule that functions in an autocrine and paracrine manner, and that induces cells to resist viral replication. This term is related to RNAi because in mammals introduction of dsRNA longer than 30 nt induces a sequence-nonspecific interferon response. – Micro-RNA – • Micro-RNAs (miRNA) are single-stranded RNAs of 22-nt that are processed from ~70-nt hairpin RNA precursors by Rnase III nuclease Dicer. Similar to siRNAs, miRNAs can silence gene activity via destruction of homologous mRNA in plants or blocking its translation in plants and animals. – Post-Transcriptional Gene Silencing – • Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation system designed to act as an anti-viral defense mechanism. A form of PTGS triggered by transgenic DNA, called co-suppression, was initially described in plants and a related phenomenon, termed quelling, was later observed in the filamentous fungus Neurospora crassa – RNA Interference – • RNA Interference (RNAi), a term coined by Fire et al in 1998, is a phenomenon that small double- stranded RNA (referred as small interference RNA or siRNA) can induce efficient sequence-specific silence of gene expression. – RNA-Directed DNA Methylation – • RNA-directed DNA methylation (RdDM) is an RNA directed silencing mechanism found in plants. Similar to RNA interference (RNAi), RdDM requires a double-strand RNA that is cut into short 21-26- nt fragments. DNA sequences homologous to these short RNAs are then methylated and silenced. – RNA-Induced Silencing Complex – • RNA-induced silencing complex (RISC) is an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. – RNAi Trigger – • RNAi triggers are double-stranded RNAs containing 21-23 nt sense and antisens strands hybridized to have 2 nt overhangs at both 3' ends. – Small Interfering RNA – • Small Interfering RNA (siRNA) is 21-23-nt double-strand RNA. It guides the cleavage and degradation of its cognate RNA. – Helicase – • Enzyme responsible for unwinding double stranded molecule © Copyright. Sandipayan Dutta. 2014. All rights
  • 5. DROSHA • DROSHA is a nuclear RNase III enzyme responsible for cleaving primary microRNAs (miRNAs) into precursor miRNAs and thus is essential for the biogenesis of canonical miRNAs. © Copyright. Sandipayan Dutta. 2014. All rights
  • 6. Gene Silencing  Gene silencing – “Switching off” of a gene ,by a machinery in the cell.  Epigenetic process of gene regulation.  Silencing is a position effect .  Genes are silenced at either the transcriptional or post- transcriptional level.  Transcriptional gene silencing - Result of modifications of either the histones or DNA. e.g.:- Silencing at the yeast telomere.  Post-transcriptional gene silencing -Result of the mRNA of a particular gene being destroyed or blocked. A common mechanism of PTGS is RNAi. © Copyright. Sandipayan Dutta. 2014. All rights
  • 7. Causes of gene silencing • Methylation of transgenes • Degradation of transgenic mRNA in cytoplasm • Inactivation of homologous gene by transcriptional and post-transcriptional regulation © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 8. Gene silencing by modification of Histones and DNA • Modification of nucleosomes alter the accessibility of the gene to the transcriptional machinery and regulatory proteins. • Heterochromatin is commonly involved in gene silencing, and affects large sections of DNA. E.g.:- the telomere, silent mating -type locus and rDNA gene in the budding yeast S.cerevisae • Methylation of particular DNA sequences can also silence transcription in many eukaryotes. E.g. :-the human H19 gene © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 9. Histone Tail Modification Status Correlates with Transcriptional Activity © Copyright. Sandipayan Dutta. 2014. All rights
  • 10. Acetylation • These reactions are catalyzed by enzymes with "histone acetyltransferase" (HAT) or "histone deacetylase" (HDAC) activity. • It also reduces affinity of tail for adjacent nucleosomes, thus affecting ability of nucleosome arrays to form more repressive higher-ordered chromatin structures. © Copyright. Sandipayan Dutta. 2014. All rights
  • 11. Methylation • These reactions are catalyzed by enzymes "histone methyltransferase” • Methylation recruit silencing or regulatory proteins that bind methylated histones. • Chromodomain containing proteins interact with methylated histone tails. © Copyright. Sandipayan Dutta. 2014. All rights
  • 12. Histone Acetylation and Methylation © Copyright. Sandipayan Dutta. 2014. All rights
  • 13. Silencing at the yeast telomere. SIR proteins (Silent Information Regulation) form a silencing complex. This complex is recruited by Rap1. © Copyright. Sandipayan Dutta. 2014. All rights
  • 14. DNA methylation can recruit Histone Deacetylases and Methylases DNA methyltransferase methylate Cytosine within promoter. This modification binds proteins (MeCP2), which in turn recruit complexes modifying nucleosome and switch off gene expression. [In Mammals] © Copyright. Sandipayan Dutta. 2014. All rights
  • 15. Comparison of different gene silencing strategies. Agent 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 © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 16. Transcriptional Gene silencing © Copyright. Sandipayan Dutta. 2014. All rights
  • 17. Genomic imprinting • Genomic imprinting is an epigenetic phenomenon by which certain genes can be expressed in a parent-of- origin-specific manner. In Homo sapiens, imprinted alleles are silenced such that the genes are either expressed only from the non-imprinted allele inherited from the mother (e.g. H19 or CDKN1C), or in other instances from the non-imprinted allele inherited from the father (e.g. IGF-2). However, in plants parental genomic imprinting can refer to gene expression both solely or primarily from either parent's allele. • Genomic imprinting is an epigenetic process that can involve DNA methylation and histone modulation in order to achieve monoallelic gene expression without altering the genetic sequence. These epigenetic marks are established in the germline and can be maintained through mitotic divisions. © Copyright. Sandipayan Dutta. 2014. All rights
  • 18. Paramutation • A paramutation is an interaction between two alleles at a single locus, whereby one allele induces a heritable change in the other allele. • Paramutation can result in a single allele of a gene controlling a spectrum of phenotypes. At r1 in maize, for example, the weaker expression state adopted by a paramutant allele can range from completely colorless to nearly fully colored kernels. • paramutation is RNA-directed. Stability of the chromatin states associated with paramutation and transposon silencing requires the mop1 gene, which encodes an RNA-dependent RNA polymerase. © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 19. 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 color and is known as position effect variegation (PEV). • Position effect is also used to describe the variation of expression exhibited by identical transgenes that insert into different regions of a genome. In this case the difference in expression is often due to enhancers that regulate neighbouring genes. These local enhancers can also affect the expression pattern of the transgene. Since each transgenic organism has the transgene in a different location each transgenic organism has the potential for a unique expression pattern. © Copyright. Sandipayan Dutta. 2014. All rights
  • 20. RNA-directed DNA methylation • RNA-directed DNA methylation (RdDM) is an epigenetic process first discovered in plants (Wassenegger et al, 1994, Cell, Vol 76, 567-576). During RdDM, double-stranded RNAs (dsRNAs) are processed to 21-24 nucleotide small interfering RNAs (siRNAs) and guide methylation of homologous DNA loci. In plants dsRNAs may be generated from three sources:  Viral replication intermediates  Products of the endogenous RNA-directed RNA polymerase  Transcribed inverted repeats © Copyright. Sandipayan Dutta. 2014. All rights
  • 21. Post-Transcriptional Gene Silencing (PTGS) • Definition: the ability of exogenous double- stranded RNA (dsRNA) to suppress the expression of the gene which corresponds to the dsRNA sequence. • Process results in down-regulation of a gene at the RNA level (i.e., after transcription) • There is also gene silencing at the transcriptional level (TGS) – Examples: transposons, retroviral genes, heterochromatin. • PTGS is heritable, although it can be modified in subsequent cell divisions or generations – Ergo, it is an epigenetic phenomenon © Copyright. Sandipayan Dutta. 2014. All rights
  • 22. • Epigenetics - refers to heritable changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence. © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 23. Difference  Promoters active  Gene hypermethylated in coding region Transcriptional gene silencing (TGS) Posttranscriptional gene silencing (PTGS) Promoters silenced Genes hypermethylated in promoter region © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 24. Discovery of PTGS • First discovered in plants – (R. Jorgensen, 1990) • When Jorgensen introduced a re-engineered gene into petunia that had a lot of homology with an endogenous petunia gene, both genes became suppressed! – Also called Co-suppression – Suppression was mostly due to increased degradation of the mRNAs (from the endogenous and introduced genes) © Copyright. Sandipayan Dutta. 2014. All rights
  • 25. • Involved attempts to manipulate pigment synthesis genes in petunia • Genes were enzymes of the flavonoid/ anthocyanin pathway: – CHS: chalcone synthase – DFR: dihydroflavonol reductase • When these genes were introduced into petunia using a strong viral promoter, mRNA levels dropped and so did pigment levels in many transgenics. © Copyright. Sandipayan Dutta. 2014. All rights
  • 26. Jorgensen 1990 van der Krol 1990 Gene injection (pigmentation Enzyme-petunias) Expectation: more red color Co-suppression of transgene and endogenous gene. Bill Douherty and Lindbo 1993 Gene injection with a complete tobacco etch virus particle. Expectation: virus expression Co-suppression of transgene and virus particles via RNA. Hamilton and Baulcombe 1998 Identification of short antisense RNA sequences dsRNA? How?Fire and Mello 1998 Injection of dsRNA into C. elegans RNA interference (RNAi) or silencing Ambros 1993 (2000) Identification of small RNA in C. elegans (micro RNA) © Copyright. Sandipayan Dutta. 2014. All rights
  • 27. Post-transcriptional gene silencing © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 28. “siRNA” molecule: “Dicer” processes long dsRNA into short siRNA: “Guide” (antisense) strand incorporated into RISC complex: guides RISC to complementary sequences in target mRNAs (RNA-induced silencing complex) Post-transcriptional gene silencing (PTGS) © Copyright. Sandipayan Dutta. 2014. All rights
  • 29. Nonsense-mediated decay • Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that exists in all eukaryotes. Its main function is to reduce errors in gene expression by eliminating mRNA transcripts that contain premature stop codons. © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 30. A tale of two pathways • RNA interference (RNAi) pathway: produces small interfering RNAs (siRNAs) that silence complementary target genes • MicroRNA pathway: produces microRNAs (miRNAs) that silence complementary target genes Mechanisms involve transcriptional gene silencing (TGS) and/or post-transcriptional gene silencing (PTGS) Pathways are conserved among most all eukaryotic organisms (fungi, protozoans, plants, nematodes, invertebrates, mammals) © Copyright. Sandipayan Dutta. 2014. All rights
  • 31. RNAi pathway • Double-stranded RNA (dsRNA) is processed by Dicer, an RNase III family member, to produce 21-23nt small interfering RNAs (siRNAs) • siRNAs are manipulated by a multi-component nuclease called the RNA-induced silencing complex (RISC). • RISC specifically cleaves mRNAs that have perfect complementarity to the siRNA strand © Copyright. Sandipayan Dutta. 2014. All rights
  • 32. RNA Interference (RNAi) • RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. • Historically, it was known by other names, including co-suppression, post transcriptional gene silencing (PTGS), and quelling. © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 33. • RNAi discovered in C. elegans (first animal) while attempting to use antisense RNA in vivo Craig Mello Andrew Fire (2006 Nobel Prize in Physiology & Medicine) – Control “sense” RNAs also produced suppression of target gene! – sense RNAs were contaminated with dsRNA. – dsRNA was the suppressing agent. © Copyright. Sandipayan Dutta. 2014. All rights
  • 34. Antisense RNA (c) or dsRNA (d) for the mex-3 (mRNA) was injected into C. elegans ovaries, and then mex-3 mRNA was detected in embryos by in situ hybridization with a mex- 3 probe. (a) control embryo (b) control embryo hyb. with mex-3 probe Conclusions: (1) dsRNA reduced mex-3 mRNA better than antisense mRNA. (2) the suppressing signal moved from cell to cell. Double-stranded RNA (dsRNA) induced interference of the Mex-3 mRNA in the nematode C. elegans. © Copyright. Sandipayan Dutta. 2014. All rights
  • 35. Core components of the RNAi pathway • Dicer Dicer family proteins contain an N-terminal helicase domain, a C-terminal segment containing dual RNase III domains, and one or more dsRNA-binding motifs. Family members also contain a PAZ domain. •Member of RNAseIII family of enzymes. • Recognize and process dsRNA into siRNA. • Dicer family proteins are ATP-dependent nucleases. • Dicer homologs exist in many organisms includingC.elegans, Drosophila, yeast and humans.
  • 36. • Argonaute (RISC complex) •RNA-Induced Silencing Complex (RISC) Argonaute family members are highly basic, ~100 kD proteins that contain PAZ and PIWI domains. • Large (~500-kDa) RNA-multiprotein complex, which triggers mRNA degradation in response to siRNA. • The active component of RISC are endonucleases called argonaute proteins . • The strand binding to the argonaute protein - ‘guide strand’. • The other ‘anti-guide strand or passenger strand is degraded during RISC activation. • The strand chosen is the one whose 5’ end is least paired to its complement. • The process is ATP independent. © Copyright. Sandipayan Dutta. 2014. All rights
  • 37. Mechanism of RNAi: Role of Dicer 1. Cells (plants and animals) undergoing RNAi contained small fragments (~25 nt) of the RNA being suppressed. 2. A nuclease (Dicer) was purified from Drosophila embryos that still had small RNA fragments associated with it, both sense and antisense. 3. The Dicer gene is found in all organisms that exhibit RNAi, and mutating it inhibits the RNAi effect. Conclusion: Dicer is the endonuclease that degrades dsRNA into 21-24 nt fragments, and in higher eukaryotes also pulls the strands apart via intrinsic helicase activity. © Copyright. Sandipayan Dutta. 2014. All rights
  • 38. Model for RNAi 21-23 nt RNAs ATP-dependent Helicase or Dicer Very efficient process because many small interfering RNAs (siRNAs) generated from a larger dsRNA. © Copyright. Sandipayan Dutta. 2014. All rights
  • 39. Biological roles of RNAi Cellular immune response to viruses (some organisms) • In certain organisms (especially plants), RNAi serves as a first line of defense against viral infection, as virus may contain or viral replication can produce dsRNA • • To this point, a number of plant viruses encode proteins that specifically bind and sequester siRNAs as a means of countering the cellular immune response of RNAi © Copyright. Sandipayan Dutta. 2014. All rights
  • 40. Genetic stability • RNAi represses transposable genetic elements in C. elegans and S. pombe • Disruption of Dicer or Argonaute increases the relative abundance of transposon RNA and increases transposon mobility • RNAi is required to establish and maintain heterochromatin formation and gene silencing at mating type loci and centromeres in S. pombe • Disruption of Dicer or Argonaute eliminates silencing, decreases histone and DNA methylation, and causes aberrant chromosome segregation • Highly repetitive DNA is often associated with heterochromatin which is transcriptionally silent. © Copyright. Sandipayan Dutta. 2014. All rights
  • 41. PTGS (RNAi) occurs in wide variety of Eukaryotes: – Angiosperms – C. elegans (nematode) – Drosophila – Mammalian cells – Chlamydomonas (unicellular – Neurospora, but not in Yeast! © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 42. Recent applications of RNAi Modulation of HIV-1 replication by RNA interference. Hannon(2002). Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. An et al.(1999) Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Jung et al. 2002. RNA interference in adult mice. Mccaffrey et al.2002 Successful inactivation of endogenous Oct-3/4 and c-mos genes in mouse pre implantation embryos and oocytes using short interfering RNAs. Le Bon et al.2002 © Copyright. Sandipayan Dutta. 2014. All rights
  • 43. Significance of RNAi • Protects against viral infection. • Secures the genome stability by keeping mobile elements silent. • Repress protein synthesis and regulate development of organism. • Keep chromatin condensed and suppress transcription. • Experimental tool to elucidate the function of any gene. • Biotechnology – engineering of food plants. • Useful approach in future gene therapy. © Copyright. Sandipayan Dutta. 2014. All rights
  • 44. Schematic illustration of systemic viral spread as well as RNAi and subsequent viral recovery in plants.
  • 45. Why RNAi silencing? • Most widely held view is that RNAi evolved to protect the genome from viruses (and perhaps transposons or mobile DNAs). • Some viruses have proteins that suppress silencing: 1. HCPro - first one identified, found in plant potyviruses (V. Vance) 2. P19 - tomato bushy stunt virus, binds to siRNAs and prevents RISC formation (D. Baulcombe). 3. Tat - RNA-binding protein from HIV © Copyright. Sandipayan Dutta. 2014. All rights
  • 46. RNAi is a conserved mechanism – RNAi is a universal, omnipresent conserved mechanism in eukaryotic cells. – The cellular mechanism of RNAi Predates evolutionary divergence of plants and worms. – key proteins involved in RNAi in disparate organisms are highly conserved © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 47. THE SILENCING MECHANISM • Two-step model to explain RNAi. – I. dsRNA is diced by an ATP- dependent ribonuclease (Dicer) into short interfering RNAs (siRNAs). • duplexes of 21 23 nucleotides bearing two-nucleotide 3' overhanging ends. – II. siRNAs are transferred to a second enzyme complex, designated RISC for RNAi- induced silencing complex. The siRNA guides RISC to the target mRNA, leading to its destruction. • the antisense strand of the siRNA is perfectly complementary © Copyright. Sandipayan Dutta. 2014. All rights
  • 48. Two-step model for the mechanism of gene silencing induced by double-stranded RNA. In step I, dsRNA is cleaved by the Dicer enzyme to produce siRNAs.
  • 49. Two-step model for the mechanism of gene silencing induced by double-stranded RNA. In step I, dsRNA is cleaved by the Dicer enzyme to produce siRNAs.
  • 50. Two-step model for the mechanism of gene silencing induced by double-stranded RNA. In step I, dsRNA is cleaved by the Dicer enzyme to produce siRNAs.
  • 51. The classical RNA interference (RNAi) pathway in Drosophila – Long double-stranded RNAs (dsRNAs) are processed by the R2D2/Dicer heterodimer into small interfering RNAs (siRNAs). – The duplexed siRNA is unwound in an ATP-dependent manner*. • *starting at the 5' terminus that has the lowest relative free energy of base pairing. – This strand of the siRNA, the guide strand, is also preferentially taken up by the RNA-induced silencing complex (RISC). – The single-stranded siRNA guides the endonuclease activity of the activated RISC ("holoRISC") to the homologous site on the mRNA, cleaving the mRNA. © Copyright. Sandipayan Dutta. 2014. All rights
  • 52. Discovery of miRNAs  siRNAs 1990: Transgenic introduction of a gene silenced endogenous gene expression in plant(Petunia). Mechanism: Introduced dsRNA is processed by Dicer into a 21- 23 nt small interfering RNA (siRNA). Dicer (RNase III-like RNase) plays a role in post- transcriptional gene silencing (PTGS) or transgene quelling .  microRNAs regulate developmental timing (heterochronic gene pathway) in C. elegans. C. elegans lin-4 (identified in 1993) controls developmental timing. Andrew Fire and Craig Mello (2006 Nobel Prize in Physiology or Medicine) reported in 1998 that small regulatory RNAs (microRNAs) interfere with target gene expression. [Potent and specific genetic interference by double-stranded RNA in C.elegans. Nature (1998) 391:806-11]. Also found that double- stranded RNA mixtures caused potent and specific interference in animals. © Copyright. Sandipayan Dutta. 2014. All rights
  • 53. microRNA classification siRNA Small interfering RNA (20-24 nt) • from invasive nucleic acids (viruses or foreign genes introduced for experiemntal and clinical purposes, or from other environmental sources) • perfect complementarity to their mRNA targets • targeted sites may disperse throughout the entire transcripts • primarily cause mRNA degradation © Copyright. Sandipayan Dutta. 2014. All rights
  • 54. miRNA microRNA (20-24 nt) • most mammalian miRNAs contain mismatches • usually target untranslated regions of mRNAs • primarily cause translational suppression or can also facilitate RNA • degradation if not perfectly match to the target © Copyright. Sandipayan Dutta. 2014. All rights
  • 55. rasiRNA Repeat-associated small interfering RNA (24- 29 nt) • derived from repetitive elements within the genome (heterochromatin regions including centromeres and telomeres, and rertotransposons). • arise mainly from the antisense strand • cause transcriptional silencing via chromatin remodeling © Copyright. Sandipayan Dutta. 2014. All rights
  • 56. piRNA Piwi-interacting RNA (26-31 nt) • in the germ line • associated with Piwi • predicted to have function in gametogenesis © Copyright. Sandipayan Dutta. 2014. All rights
  • 57. tasiRNA trans-acting RNA (24-29 nt) in plant • endogenous siRNAs derived from noncoding transcripts that are cleaved by a microRNA (miRNA) • facilitate protein-coding mRNA degradation • function in plant stress responses • similar siRNAs (tiny noncoding RNA) found in C. elegans © Copyright. Sandipayan Dutta. 2014. All rights
  • 58. mirtrons • short hairpin introns provide an alternative source for animal microRNA biogenesis and use the splicing machinery to bypass Drosha cleavage in initial maturation. © Copyright. Sandipayan Dutta. 2014. All rights
  • 59. LINE (L1) retrotransposons can generate dsRNAs from both sense and • antisense promoters in germ cells. These dsRNAs can be processed into • siRNAs. L1-specific siRNAs target to the 5’UTR of L1 transcripts and • cause its degradation. Thus, siRNAs suppress L1 retrotransposition in • germ cells. © Copyright. Sandipayan Dutta. 2014. All rights
  • 60. © Copyright. Sandipayan Dutta. 2014. All rights
  • 61. microRNA (abbreviated miRNA) A microRNA is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals, and some viruses, which functions in transcriptional and post-transcriptional regulation of gene expression. Encoded by eukaryotic nuclear DNA in plants and animals and by viral DNA in certain viruses whose genome is based on DNA, miRNAs function via base-pairing with complementary sequences within mRNA molecules. As a result, these mRNA strands are silenced because they can no longer be translated into proteins by ribosomes, and such complexes are often actively disassembled by the cell. © Copyright. Sandipayan Dutta. 2014. All rights
  • 62. • miRNA Biogenesis – Transcribed from endogenous gene as pri-miRNA • Primary miRNA: long with multiple hairpins • Imperfect internal sequence complementarity – It is processed into 70-nt hairpins by the RNase III family member Drosha to become the pre-miRNA. • Note: How does it identify pri-miRNA? – Hairpin terminal loop size – Stem structure – Hairpin flanking sequences – The pre-miRNA is exported to the cytoplasm by Exportin 5. – It is cleaved by the R2D2/Dicer heterodimer into the mature miRNA. • Symmetric 2nt 3’ overhangs, 5’ phosphate groups
  • 63. DCL1 mutant Comparison of Mechanisms of MiRNA Biogenesis and Action Better complementarity of MiRNAs and targets in plants.
  • 64. Summary of differences between plant and animal MiRNA systems Plants Animals miRNA genes: 100-200 100-500 Location in genome: intergenic regions Intergenic regions, introns Clusters of miRNAs: Uncommon Common MiRNA biosynthesis: Dicer-like Drosha, Dicer Mechanism of repression mRNA cleavage Translational repression Location of miRNA target in a gene: Predominantly Predominantly the 3′-UTR the open-reading frame miRNA binding sites in a target gene: Generally one Generally multiple Functions of known target genes: Regulatory genes Regulatory genes—crucial crucial for development, for development, structural enzymes proteins, enzymes
  • 65. miRNAs and Cancer – A Summary • miRNAs control cell cycle, cell differentiation and apoptosis by regulating oncogenes and tumor supressor genes • miRNAs are misexpressed in cancer and are therefore excellent diagnostic/prognostic markers in cancer • Some miRNAs e.g. mir-155, can cause cancer and oncogenic miRNAs may be therapeutic targets in cancer • Other miRNAs like let-7, may prevent cancer and may be therapeutic molecules themselves. • MicroRNAs could augment current cancer therapies. © Copyright. Sandipayan Dutta. 2014. All rights
  • 66. MicroRNAs Commonly Associated with Human Cancer
  • 67. microRNAs 1. Derived from an endogenous, structured transcript (pre-miRNA) 2. One miRNA accumulates 3. Evolutionary conserved 4. Usually located away from genes 5. Imperfect pairing blocks translation 6. Incorporated into miRNP 7. Regulate expression of genes encoded at another locus 8. miRNAs bind to the target 3' UTRs through imperfect complementarity at multiple sites siRNAs 1. Derived from extended dsRNA 2. Each dsRNA gives multiple siRNAs 3. Less conservation 4. Nearly complementary to target RNA (self-targeting) 5. Perfect pairing induces target RNA cleavage 6. Incorporated into RISC 7. Regulate the locus from which their sequence derives 8. siRNAs often form a perfect duplex with their targets at only one site. miRNAs and siRNAs — what's the difference? © Copyright. Sandipayan Dutta. 2014. All rights
  • 68. © Copyright. Sandipayan Dutta. 2014. All rights Gene Knockout: Introduce dsRNA as hairpin RNA (hpRNA) to silence some specific gene. Trick the plant in to shutting down or re-routing specific molecular pathways to alter biological activities. e.g. - flowering time - leaf shape - yield index - oil quality
  • 69. Small Interfering RNA (siRNA) • 21-25 nucleotide dsRNA with 2-nt 3’ overhangs on either end. • Produced in vivo by cleavage of dsRNA or exogenously introduced in the cell. • Amplification by an RNA-dependent RNA polymerase (RdRP) may occur. • Incorporated into the RISC guiding it to mRNA • A single base pair difference between the siRNA template and the target mRNA is enough to block the process. © Copyright. Sandipayan Dutta. 2014. All rights
  • 70. siRNAs have a defined structure 19 nt duplex 2 nt 3’ overhangs © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 71. siRNAs  Small interfering RNAs that have an integral role in the phenomenon of RNA interference(RNAi), a form of post-transcriptional gene silencing  RNAi: 21-25 nt fragments, which bind to the complementary portion of the target mRNA and tag it for degradation  A single base pair difference between the siRNA template and the target mRNA is enough to block the process. © Copyright. Sandipayan Dutta. 2014. All rights
  • 72. siRNA design • Target Sequence- 21-nucleotides long , 50- 100 bp downstream from start codon (AUG) • Search for seq. motif AA(N19). • Avoid sequences with > 50% G+C content. • Avoid targeting introns. • Avoid stretches of 4 or more nucleotide repeats. • Avoid sequences that share a certain degree of homology with other related or unrelated genes. © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 73. from Mittal, Nature Rev.Genet. 5, 355 (2004) The Design of Optimal siRNAs 21 nt RNA that contains 2 nt 3’- overhangs and phosphorylated 5’-ends Lower stability at the 5’-end of the antisense terminus Low stability in the RISC cleavage site Low secondary structure in the targeted region of the mRNA © Copyright. Sandipayan Dutta. 2014. All rights
  • 74. How can RNAi be used ? C. elegans D. melanogaster Planaria Trypanosomes Hydra Xenopus Mammalian tissue culture cells Mice • biological research – Define gene function (gene knockdown) – Define biochemical pathways – Identify and validate targets – Generate knockdown models without developmental complications • therapeutic treatment – viral infection – parasitic infection – cancer – neurodegeneration © Copyright. Sandipayan Dutta. 2014. All rights
  • 75. RNAi as a powerful therapeutic drug • Exquisitely selective like the fabled “magic bullet”. • May work synergistically with other drug and treatment regimes. • Endogenous pathway so allows the development of safe and efficacious drugs. • Potent- very low concentrations of siRNA are required. • The clinical applications appear endless. © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 76. from Dykxhoorn and Lieberman, Cell 126, 231 (2006) Delivery of siRNA for Therapy siRNA is not taken up by most mammalian cells Cholesterol-conjugated siRNA is taken up by the LDL receptor siRNA bound to targeted antibody linked to protamine can achieve cell-specific siRNA delivery © Copyright. Sandipayan Dutta. 2014. All rights
  • 77. Therapeutic siRNAs siRNA target gene Disease p53 mutant K- Ras BCR-ABL MDR1 C-RAF Bcl-2 VEGF PKC- Β- Catenin Cancer © Copyright. Sandipayan Dutta. 2014. All rights reserved.
  • 78. Therapeutic siRNAs siRNA target gene Disease HIV-Tat HIV-Rev HIV- Vif, - Hef HPV-E6 and –E7 HBV-S1, -S2, -S, - X CCR5, CXCR4 CD4 Viral Infection Fas receptor Caspase-8 Acute Liver Failure TNF- Sepsis © Copyright. Sandipayan Dutta. 2014. All rights
  • 79. Conclusion A different variety was selected in nature during evolution by using gene silencing Gene Silencing balances and satisfy the biosafety concern as non transgenic variety has no implications Down regulation Of allergins • Gene silencing can down regulate allergin or potentially toxic substances eg – allergic proteins of rice is downregulated by antisense method Sexual seggregation of transgene constructs • 1 transgene construct may inactivate other.but they can be seperated by sexual reproduction • Thus poorly expressed gene become highly expressed.but it impose concerns of biosafety © Copyright. Sandipayan Dutta. 2014. All rights
  • 80. Cancer treatments – knock-out of genes required for cell proliferation – knock-out of genes encoding key structural proteins. Powerful for analyzing unknown genes in sequence genomes. efforts are being undertaken to target every human gene via miRNAs © Copyright. Sandipayan Dutta. 2014. All rights Assesment of biosafety for transgeneses Biosafety concerns with transgenecity An experiment for this assessment concluded that new ecological environment created by trangenesis can promote new type of recombinant virus
  • 81. © Copyright. Sandipayan Dutta. 2014. All rights reserved. BOOKS READ:  Molecular Biology_ David P.clark ELSEVIER  Molecular biology of the cell 5th edition Whatson,Baker,Bell,Gann, Levinn,Losick WEBSITES VISITED:  Search on www.google.com  Search on http://www.ncbi.nlm.nih.gov References
  • 82. I, Sandipayan Dutta would like to express my heartfelt gratitude towards our respected principal Dr. A . Nagarathna and our Head of the Department Mrs. Asha.K.K . I would like to give special thanks to Dr.Manjula Dutt for assigning me the topic for this presentation. My family and friends have always been by my side and it falls as my duty to mention them in this event. © Copyright. Sandipayan Dutta. 2014. All rights reserved. Acknowledgement
  • 83. © Copyright. Sandipayan Dutta. 2014. All rights reserved. Thank You !
  • 84. © Copyright. Sandipayan Dutta. 2014. All rights reserved.

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