Gene Silencing

1. 3/6/2014 1 Cenorhabditis elegans WELCOME

2. 3/6/2014 2 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Gene Silencing Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola Department of Botany, Biotechnology centre Petunia Chalcone Synthase (sense strand) Surbhaiyya Shobha Devidas PhD Scholar Dr. PDKV, Akola .

3. Contents Introduction Short history of gene silencing Gene silencing methods Methods for PTGS Case study Application of RNAi Conclusion References 26/9/2016 3

4. 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. 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. Introduction 26/9/2016 4

5. There are so many approaches for gene silencing Gene Knockout Gene Knockdown Gene silencing and degradation of gene using RNA technology - Antisense RNA Technology - RNAi Technology 26/9/2016 5 Cont….

6. 26/9/2016 6 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. Resulted in degradation of the endogenous and transgene mRNA. 1995 Guo and Kemphues : I njection 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. The discovery of the mechanism of RNA interference by ds RNA by prof . Andrew Fire and prof . Craig Mello in 1998, gave them the Nobel prize in 2006. Short history of gene silencing

7. 26/9/2016 7 G enes 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 Transcriptional gene silencing Post transcriptional gene silencing Types of Gene silencing Transcriptional gene silencing Post transcriptional gene silencing 1. Genomic Imprinting 1. Antisense RNA technology 2. Paramutation 2. RNAi technology 3. Transposon silencing - mi RNA 4. Transgene silencing - sh RNA 5. Position effect - si RNA 6. RNA-directed DNA methylation

8. 26/9/2016 8 It 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.). Genomic imprinting: Genomic imprinting or parental imprinting occurs when a gene inherited from either the father or the mother has been permanently inactive in that parent and passed on to offspring in the same inactivated condition. The inactivated genes has not mutated – the DNA sequence is normal. Transcriptional gene silencing

9. Inactivation caused by methylation of DNA in the regulatory sequences of the inactivated gene This results in the silencing of an allele in either male (paternally imprinted) or female (maternally imprinted) gametes. For example- Insuline like growth factor gene(Igf2) in mice The product of these gene is required for normal growth in the embryo If maternal allel is highly methylated and silence, only paternal allele is expressed, results shows that dwarfing mutant mice produced. 26/9/2016 9 Cont….

10. 26/9/2016 10 Paramutation : P aramutation 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. This change may be in the pattern of DNA methylation For example – Anthocyanin pigment in corn plant B allele – Anthocyanin pigment coded

11. Paramutagenic allele at this locus(B’) cause reduced pigment production B allele is silenced by the B’ allele in the first generation In next generation, the newly silence B allele is paramutagenic and silence. 26/9/2016 11 Cont….

12. 26/9/2016 12 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). For example eye colour (w+ shows red pigment) in Drosophila

13. 26/9/2016 13 The ability of exogenous or sometimes endogenous RNA to supress the expression of the gene which corresponds to the m-RNA sequence. Post transcriptional gene silencing Anti sense RNA technology: 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. Antisense RNA inhibits protein synthesis by blocking Translation

14. RNAi (Interference)Technology 26/9/2016 14

15. 26/9/2014 15 Cenorhabditis elegans Discovery of RNA interference (1998) - silencing of gene expression with dsRNA

16. 26/6/2014 16 What is RNA interference (RNAi)? “ The Process by which dsRNA silences gene expression...” Degradation of mRNA or translation inhibition

17. Cont..... RNA interference (RNAi): Cellular process by which an mRNA is targeted for degradation by a dsRNA with a strand complementary to a fragment of such mRNA. A selective gene knock-down phenomenon. Specific terms for gene silencing Post-transcriptional gene silencing ( PTGS ) - Plants Quelling - Fungi RNA interference ( RNAi ) – Animals RNAi operates and its natural role for virus defence and endogenous gene regulation in plants 26/9/2016 17

18. RNAi phenomenon was discovered in transgenic plant Petunia hybrida L . ( Napoli et al. 1990) They want to enhance anthocyanin pigments Unexpectedly, transgenic plants producing white or chimeric flowers were obtained instead of dark purple flowers due to the silencing of endogenous homologous gene and this phenomenon was termed as “ co-suppression ”. Petunia Chalcone Synthase (sense strand) 26/9/2016 18

19. 26/9/2016 19

20. 26/9/2016 20 Nobel prize winners in the C. elegans field Sidney Brenner John Sulston Robert Horvitz Andrew Fire Craig Mello

21. 26/9/2016 21 RNAi was found to work in many diverse species Fungi Trypanosomes Insects Zebrafish Mice

22. Over view of RNAi : During RNA i , Double-stranded RNAs cut into short double-stranded RNAs i.e.s (small) i (interfering) RNAs, by an enzyme called Dicer. These then base pair to an mRNA through a dsRNA -enzyme complex(RISC). This will either lead to degradation of the mRNAstrand Highly specific process Very potent activity So far only been seen in eukaryotes Evidence 30% of genome is regulated by RNA I RNA i pathway guided by, There are three types of dsRNA produed and they leads to RNAi pathway: small interfering RNAs ( siRNAs ) generated via processing of longer dsRNA microRNAs ( miRNAs ) that are generated via processing of stem loop precursors 3. short hairpin RNAs ( shRNA ) that are generated via hair pin structure 26/9/2016 22

23. Main component of RNAi machinery for siRNA , miRNA and shRNA generation 26/9/2016 23

24. 26/9/2016 24 There are five component for siRNA , miRNA and shRNA generation Drosha Dicer RNA-Induced Silencing Complex (RISC) Argonaute (Ago) RNA-Dependent RNA Polymerase ( RdRP )

25. 26/9/2016 25 DROSHA Processes pri-miRNA into pre- miRNA Leaves 3’ overhangs on pre- miRNA Nuclear RNAse -III enzyme [Lee at al., 2003] Tandem RNAse -III domains Pri -mRNA look like, Hairpin terminal loop size Stem structure Hairpin flanking sequences Not yet found in plants 25

26. Dicer 26/9/2016 26 RNAse III-like dsRNA -specific ribonuclease • It is able to digest dsRNA into uniformly sized small RNAs ( si RNA) Dicer family proteins are ATP- dependent nucleases. 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 potein -protein interaction

27. First discovered in Drosophila, by Hammond et al. (2000) Nuclease complex composed of proteins and siRNA Targets and destroys endogenous mRNAs complementary to the siRNA RISC consists of both protein and RNA Activities associated with RISC Slicer Argonaute “homology seeking”/RNA binding Preferentially incorporates one strand of unwound RNA [ Khvorova et al., 2003] Antisense RNA-Induced Silencing Complex (RISC) 26/9/2016 27

28. 26/9/2016 28 Argonaute (Ago) Consistently co-purifies with RISC [Hammond et al., 2001] “Homology seeking” activity Binds siRNA , miRNA and shRAN [ Ekwall , 2004] Distinguishes antisense strand [ Novina and Sharp, 2004 ] Multiple Ago family proteins Different RISCs Tissue specific, Dvelopmentally regulated Evidence for different RISCs [ Tijsterman et al., 2004] Drosophila Dicer1 vs Dicer2/R2D2

29. siRNA Pathway 26/9/2016 29

30. There are four component for siRNAs generation Dicer small Interfering RNA ( siRNA ) RNA-Induced Silencing Complex (RISC ) Argonaute (Ago) RNA-Dependent RNA Polymerase ( RdRP ) Main component of RNAi machinery for siRNA generation 26/9/2016 30

31. siRNA - Short Interference RNA 21-23 nucleotide dsRNA that mediate PTGS Produced in vivo by cleavage of dsRNA Incorporated into the RISC guiding it to mRNA Each strand of Si RNA has: a. 5’-phosphate termini b. 3’-hydroxyl termini and 2/3-nucleotide 3’ overhangs Complementary to a specific sequence of target mRNA for degradation Amplification by an RNA-dependent RNA polymerase ( RdRP ) may occur 26/9/2016 31

32. Mechanism of siRNA Initiation phase Maintenance phase Signal amplification and spreading phase 26/9/2016 32

33. 3/6/2014 33 33 siRNA Mediated RNA i Pathway: dsRNA are chopped into short interfering RNAs ( siRNA ) by Dicer. The siRNA -Dicer complex recruits additional components to form an RNA- Induced Silencing Complex (RISC). The siRNA unwinds. The unwound siRNA base pairs with complementary mRNA, thus guiding the RNAi machinery to the target mRNA. The target m RNA is effectively cleaved and subsequently degraded–resulting in gene silencing.

34. 26/9/2016 34 miRNA Pathway

35. 26/9/2016 35 There are seven component for siRNAs generation Drosha /DGCR8 Exportin 5 Dicer small Interfering RNA ( miRNA ) RNA-Induced Silencing Complex (RISC ) Argonaute (Ago) RNA-Dependent RNA Polymerase ( RdRP ) Main component of RNAi machinery for miRNA generation

36. 26/9/2016 36 m iRNA - micro RNA 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 .

37. 26/9/2016 37 Mechanism of miRNA Initiation Generation of mature siRNA or miRNA Execution Silencing of target gene Degradation or inhibition of translation

38. He and Hannon, 2004 Initiation Execution 3/6/2014 38

39. 26/9/2016 39 Difference between miRNA and siRNA Function of both species is regulation of gene expression. Difference is in where they originate. siRNA originates with dsRNA . miRNA originates with ssRNA that forms a hairpin secondary structure. siRNA is most commonly a response to foreign RNA (usually viral) and is often 100% complementary to the target. miRNA regulates post-transcriptional gene expression and is often not 100% complementary to the target.

40. 26/9/2016 40 siRNA miRNA

41. 26/9/2016 41 41 sh RNA Pathway

42. 42 There are five component for siRNAs generation Dicer Short hair pin RNA ( shRNA ) RNA-Induced Silencing Complex (RISC ) Argonaute (Ago) RNA-Dependent RNA Polymerase ( RdRP ) Main component of RNAi machinery for shRNA generation 26/9/2016

43. 26/9/2016 43

44. RNA Gene-silencing constructs commonly used in plant genetic modification 26/9/2016 44

45. Cecilia et. al., 2010 26/9/2016 45

46. 3/6/2014 46 Trait Target Gene Host Application Enhanced nutrient content Lyc Tomato Increased concentration of lycopene ( carotenoid antioxidant) DET1 Tomato Higher flavonoid and bcarotene contents SBEII Wheat, Sweet potato, Maize Increased levels of amylose for glycemic anagement and digestive health FAD2 Canola, Peanut, Cotton Increased oleic acid content SAD1 Cotton Increased stearic acid content ZLKR/SDH Maize Lysinefortified maize Reduced production of lachrymatory factor synthase lachrymatory factor synthase gene Onion “Tearless” onion Examples of novel plant traits engineered through RNAi.

47. 26/9/2016 47 Reduced alkaloid production CaMXMT1 Coffee Decaffeinated coffee COR Opium poppy Production of nonnarcotic alkaloid, instead of morphine CYP82E4 Tobacco Reduced levels of the carcinogen nornicotine in cured leaves Heavy metal accumulation ACR2 Arabidopsis Arsenic hyperaccumulation for phytoremediation Reduced polyphenol production scadinene synthase gene Cotton Lower gossypol levels in cottonseeds, for safe consumption Ethylene sensitivity LeETR4 Tomato Early ripening tomatoes ACC oxidase gene Tomato Longer shelf life because of slow ripening Reduced allergenicity Arah2 Peanut Allergen free peanuts Lolp1, Lolp2 Ryegrass Hypo-allergenic ryegrass

48. 26/9/2016 48 Case study

49. 26/9/2016 49

50. 50 26 /9/2016 Sunilkumar et al., 2006 - Cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol, making them unsuitable for human consumption 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. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta- cadinene synthase (key enzyme in gossypol production), without affecting the enzymes production in other parts of plants.

51. Advantages of RNAi 26/9/2016 51 Downregulation of gene expression simplifies " knocdown " analysis. Easier than use of antisense oligonucleotides . Si RNA more effective and sensitive at lower concentration. Cost effective 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.

52. Disadvantages of RNAi 26/9/2016 52 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.

53. 26/9/2016 53 Application of RNAi 53

54. 26/9/2016 54 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. Protection from insect pests : Baum et al. 2007. showed that silencing of a vacuolar ATPase gene (V-type ATPase A gene) in midgut cells of western corn rootworm (WCR) led to larval mortality and stunted growth.

55. Nematode resistance : Huang et al., 2006. showed that Arabidopsis plants expressing dsRNA for a gene involved in plant–parasite interaction (16D10) had suppressed formation of root galls by Meloidogyne nematodes and reduced egg production. Bacterial and fungal risistance : Escobar et al . 2001. showed that silencing of two bacterial genes ( iaaM and ipt ) could decrease the production of crown gall tumors ( Agrobacterium tumefaciens ) to nearly zero in Arabidopsis , suggesting that resistance to crown gall disease could be engineered in trees and woody ornamental plants. 26/9/2014 55

56. 26/9/2016 56 Conclusion 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.

57. 26/9/2016 57 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).

58. 3/6/2014 58 THANK YOU