Pavithra RDNA Technology
- 1. GENE SILENCING BY, PAVITHRA B (16BIOB12) II M.SC.BIOTECH
- 2. Contents • Introduction • Types of gene silencing -Transcriptional gene silencing -Post transcriptional gene silencing • Application • Conclusion
- 3. • 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 erased from organism’s genome and thus have no expression. Introduction
- 4. • 1990 Jorgensen: • To deepen the pigmentation in petunias, Introduction of transgenes homologous to endogenous genes often resulted in plants with both gene suppressed called co suppression. • 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. • 1998 Mello and Fire: • Extension of above experiments, combination of sense and antisense RNA(=dsRNA) was 10 times more effective than single strand RNA. Short History of gene silencing
- 5. • Genes are regulated at either the transcriptional level or post-transcriptional level, therefore silencing can be induced either at transcriptional level or post- transcriptional level. • There are mainly two types of gene silencing 1. Transcriptional gene silencing 2. Post transcriptional gene silencing Types of Gene silencing Transcriptional gene silencing Post transcriptional gene silencing Promoter silenced Promoter active Genes hypermethylated in promoter region Genes hyper methylated in coding region
- 6. • 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.). Genomic imprinting: • genomic imprinting is a genetic phenomenon by which certain genes are expressed in a parent-of-origin-specific manner. It is an inheritance process independent of the classical Mendelian inheritance. • genomic imprinting have been demonstrated in insects, mammals and flowering plants. Transcriptional gene silencing
- 7. Paramutation: • Paramutation is an interaction between two alleles of a single locus, resulting in a heritable change of one allele that is induced by the other allele. • Paramutation was first observed by the effect it had on the colour of corn kernels in maize plants.
- 8. 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).
- 9. RNA Directed DNA Methylation: • RNA-directed DNA methylation is an epigenetic process first elucidated in plants whereby small double-stranded RNAs (dsRNA's) are processed to guide methylation to complementary DNA loci. In the model plant organism Arabidopsis thaliana
- 10. 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.
- 11. Transgene silencing: • Unfortunate insertion of transgene in to a transcriptionally inactive part of genome. • The lose of transgene stability is because of gene silencing. • E.g. slow fruit softening tomato, by reducing expression of polygalactouronase enzyme.
- 12. • The ability of exogenous or sometimes endogenous RNA to supress the expression of the gene which corresponds to the m-RNA sequence. RNA i (RNA interference): • it is a post transcriptional process triggered by the introduction of double stranded RNA (ds RNA) which leads to the gene silencing in a sequence specific manner. • First evidence came from studies on nematode caenorhabditis elegans. further analysis in fruit fly drosophila melanogaster. • It is also known as post transcriptional gene silencing / co suppression and quelling. Post transcriptional gene silencing
- 13. Over view of RNA i: • During RNA i Double-stranded RNAs cut into short double-stranded RNAs, s(small) i(interfering) RNAs, by an enzyme called Dicer. These then base pair to an mRNA through a dsRNA-enzyme complex. This will lead to degradation of the mRNA strand • Highly specific process • So far only been seen in eukaryotes • Evidence 30% of genome is regulated by RNA I • RNA i pathway guided by, Si RNA (small interfering RNA): • Small interfering RNAs that have an integral role in the phenomenon of RNA interference (RNA i), a form of post- transcriptional gene silencing • RNA i: 21-25 nt fragments, which bind to the complementary portion of the target mRNA and tag it for degradation
- 14. 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: • 1) Amino-terminal helicase domain • 2) Dual RNAse III motifs in the carboxyl terminal segment • 3) dsRNA binding domain • 4) PAZ domain
- 15. • DICER’ s domain:
- 16. • A single base pair difference between the Si RNA template and the target mRNA is enough to block the process. • Each strand of Si RNA has: • a. 5’-phosphate termini • b. 3’-hydroxyl termini • c. 2/3-nucleotide 3’ overhangs
- 17. 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.
- 18. Steps of the RNA i pathway : • I. ds RNA is sliced by an ATP-dependent ribonuclease (Dicer) into short interfering RNAs (si RNAs). • duplexes of 21 23 nucleotides bearing two- nucleotide 3' overhanging ends. • II. Si RNAs are transferred to a second enzyme complex, designated RISC for RNA i -induced silencing complex. The si RNA guides RISC to the target mRNA, leading to its destruction. • the antisense strand of the Si RNA is perfectly complementary
- 19. 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.
- 20. Non sense Mediated Decay: • Nonsense mediated decay (NMD) is a cellular mechanism of mRNA surveillance that functions to detect nonsense mutations and prevent the expression of truncated or erroneous proteins. • NMD is triggered by exon junction complexes (EJCs) (components of the assembled RNP) that are deposited during pre-mRNA processing.
- 21. Anti sense RNA technology: • It blocks the activity of the m RNA in a stoichiometric manner. • Antisense RNA has the opposite sense to m RNA. • The presence of complimentary sense and antisense RNA in the in the same cell can lead to the formation of a stable duplex which interferes with gene expression at the level of RNA processing or possible translation. • This technology widely used in plants for gene inhibition.
- 22. Advantages of gene silencing: • Downregulation 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. • Powerful tool for analysing unknown genes in sequenced genomes. • Useful approach in future gene therapy.
- 23. 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.
- 24. Application of Gene silencing • Cancer treatments • RNA interference has been used for applications in biotechnology, particularly in the engineering of food plants that produce lower levels of natural plant toxins. • Such techniques take advantage of the stable and heritable RNA i phenotype in plant stocks. For example, cotton seeds. • Modulation of HIV-I replication by RNA i. • Developing technologies for epigenomic analysis and clinical application of molecular diagnosis.
- 25. • 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. Conclusion
- 26. GENE REGULATION
- 27. Flow of genetic information
- 28. • Total no: genes: prokaryotes - 4000 eukaryotes - 35000 • Gene expression Multistep process that results in the production of functional gene product • Importance =>Cellular differentiation =>Morphogenesis =>Adaptability of organism
- 29. Types of genes • Constitutive genes (House keeping genes): Expressed at a reasonably constant rate Not subjected to regulation eg: enzymes of glycolysis • Regulated genes expressed only under certain conditions expressed in all cells / subset of cells eg: expression of insulin gene in pancreas
- 30. Types of genes • Inducible gene: expression increases in response to inducer or activator eg: bile acid induces ALP • Gratuitous inducers Compounds structurally similar to substrates may act as inducers eg: IPTG (isopropylthiogalactoside) – lactose analog
- 31. • Repression inhibition of gene expression by repressor eg: inhibition of ALA synthase by heme Catabolite repression: Catabolite of a molecule inhibit gene expression
- 32. Types of gene expression regulation • Positive regulation increased gene expression mediated by positive regulator / enhancer / activator • Negative regulation reduced gene expression mediated by negative regulator / silencer / repressor
- 33. Regulation of Gene Expression
- 34. Regulation of Gene Expression • Gene expression can be modulated by • Control of transcription • Post transcriptional modifications • Gene amplification • Gene rearrangements • Control of translation • Protein modification / stabilization • Gene regulation is influenced by hormones, heavy metals and chemicals
- 35. Regulation of Gene Expression in prokaryotes • Regulation occurs at transcription • Operon Genes involved in a metabolic pathway when present in a linear array • Cistron Smallest unit of gene expression • Polycistronic mRNA: A single mRNA that encodes more than one separately translated protein. eg - lac operon mRNA
- 36. Operon concept of gene regulation • Francois Jacob and Jacques Monod, 1961 • Lactose metabolism in E.Coli (intestinal bacteria) • Lac Operon structural gene inhibitor gene promoter / operator areas • Repression – derepression mechanism
- 38. • Structural gene lacZ : β- galactosidase (lactase) lacY: galactoside permease lacA: thiogalactoside transacetylase • Inhibitor gene lacI: LacI (lac operon repressor protein) • Promoter site transcription of structural genes • Operator site binding of lac repressor
- 39. Regulation lac operon • Absence of lactose – reduced synthesis of lactose metabolizing enzymes • presence of lactose – increased enzyme synthesis • Presence of lactose and glucose – reduced synthesis of enzymes
- 40. Repression of Lac operon (absence of lactose)
- 41. Derepression of Lac operon (presence of lactose)
- 42. Catabolite repression (both glucose and lactose present)
- 43. Catabolite repression • Repression of lac operon by catabolite of glucose (catabolite activator protein) CAP + c AMP facilitate binding of RNAP to the promoter transcription of structural gene
- 44. Discovered in 1953 by Jacques Monod and colleagues, the trp operon in E. coli was the first repressible operon to be discovered. This operon contains five structural genes: trp E, trp D, trp C, trp B, and trp A, which encodes tryptophan synthetase. It also contains a promoter which binds to RNA polymerase and an operator which blocks transcription when bound to the protein synthesized by the repressor gene (trp R) that binds to the operator
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- 46. Amount of trp operon affect transcription
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- 48. Eukaryotic gene expression • Eukaryote genome is complex DNA is extensively folded and packed into chromatin (protein – DNA complex) Differs from prokaryotic gene regulation • Genes are not organized into operon • Separation of transcription and translation • RNA processing : capping at 5’ ends, addition of poly A tail at 3’ end splicing
- 49. Regulation of eukaryotic gene expression Regulation at transcription level Chromatin remodeling Enhancers Trans acting molecules – DNA binding proteins function as transcriptional factors Cis-acting regulatory elements eg: Hormone response elements Regulation by post transcriptional process alternative mRNA splicing mRNA editing Gene amplification Gene rearrangement
- 50. Eukaryotic DNA: DNA + Histones HIGHLY CONDENSED LESS CONDENCED EUCHROMATINS HETEROCHROMATINS NOT TRANSCRIPTED TRANSCRIPTED CHROMATINS
- 51. Mechanism Histone acetylation recruitement of remodelling complex conformational changes in DNA exposure of binding sites RNAP binds to the site transcription
- 52. • Chromatin remodeling Heterochromatin / Euchromatin. eg, Β-globin gene in reticulocytes and muscle Importance: 1) increases the accesibility of promoters and regulatory regions 2) increases binding of RNA polymerase
- 54. Enhancers • DNA elements which facilitate / enhance the initiation of transcription at the promoter Features • Is active only when it exists within the same DNA molecule (cis to the promoter) • Work when located long distances from the promoter • Work when upstream or downstream from the promoter • Work when oriented in either direction
- 55. Mechanism of action Enhancer work by : • binding one or more proteins • facilitating binding of the basal transcription complex to the promoter • recruiting chromatin-modifying co regulatory complexes • Insulators: prevents the enhancer function
- 56. DNA binding motifs • Mediate DNA-protein interactions Features • Binds with high affinity to the specific site • Maintained by hydrogen bonds, ionic interactions and van der Waals forces • Involved in providing: trans -activation domains / ligand binding sites surfaces for interaction with co-activators/repressors
- 57. Types of motifS Helix turn Helix Leucine zipper Zinc finger
- 58. Types of motiFs Binding motif Organism Regulatory protein Helix-turn-Helix E.Coli mammals Lac repressor CAP Pit 1, Oct 1 Zinc finger E.Coli mammals Gene 32 protein Steroid receptor family Leucine Zipper Yeast mammals GCN4 Fos, jun, c-myc
- 59. Gene amplification • Increases the number of genes available for transcription • Eg: resistance to methotrexate by cancer cells cancer cells increase the number of genes for DHFR drug resistance
- 60. Gene rearrangement • Occurs during immunoglobulin synthesis • IgG light chain mRNAs are coded by different segments/domains variable (VL) =300 coding sequences joining (JL) = 5 constant (CL) = 10 • Difficult for immune cells to achieve immunological specificity
- 62. Alternative RNA processing • Primary transcript is processed differently in different tissues Achieved by • Alternative transcription start sites • Alternative polyadenylation site • Alternative splicing and processing
- 63. Regulation of mRNA stability • Ends of mRNA 5’ cap structure: prevents attack by 5’ exonuclease 3’ poly (A) tail: prevents attack by 3’ exonuclease • Ribonucleoprotein particles
- 64. Different forms of tropomyosin by alternative gene splicing.
- 65. GTT CAA CAA UAA Apo protein B100 (4563 aa) Apo protein B48 (2153 aa) mRNA transcript Apo B gene mRNA C U Translation Liver Intestine Example of RNA Editing mRNA
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