Gene silencing is generally defined as an epigenetic modification of gene expression leading to inactivation of previously active individual genes or larger chromosome regions..pptx

Gene silencing is generally defined as an epigenetic modification of gene expression leading to inactivation of previously active individual genes or larger chromosome regions.
Flavr Savr tomatoes is an example of gene silencing. They are made more resistant to rotting by the introduction of an antisense gene that interferes with the production of polygalacturonase enzyme that degrades pectin in the cell walls.

1. Gene silencing

2. Gene silencing is  the regulation of gene expression in a cell to prevent the expression of a certain gene . Gene silencing can occur during either transcription or translation and is often used in research.

3. Before RNAi… Ribosome mRNA Protein synthesis mRNA X No protein synthesis antisense mRNA ‹#› Ribosome can’t bind Antisense RNA inhibits protein synthesis by blocking Translation

4. ‹#› What is RNAi? 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

5. RNAi Petunia hybrida L . (Napoli et al. 1990) was discovered in plant transgenic phenomenon 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) 4

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7. ‹#› RNA silencing works on at least three different levels in plants viz. Cytoplasmic silencing by dsRNA results in cleavage of mRNA, Endogenous mRNAs are silenced by micro-RNAs ( miRNAs ), which negatively regulate gene expression by base-pairing to specific mRNAs, resulting in either RNA cleavage or blocking protein translation called PTGS. RNA silencing is associated with sequence-specific methylation of DNA and the consequent suppression of transcription [transcriptional gene silencing TGS.

8. ‹#› There are two main RNAi pathway: small interfering RNAs ( siRNAs ) generated via processing of longer dsRNA and microRNAs ( miRNAs ) that are generated via processing of stem loop precursors Multiple pathways for RNAi

9. ‹#› There are four component for siRNAs generation Dicer small Interfering RNA (siRNA ) RNA-Induced Silencing Complex (RISC) RNA-Dependent RNA Polymerase (RdRP) Main component of RNAi machinery for siRNA generation

10. Dicer 3/6/2014 ‹#› First discovered by Bernstein et al. (2001) in Drosophila. Enzyme belonging to the RNase III ribonuclease family. Cutting mechanism of the Dicer enzyme is ATP-independent (Kuznetsov 2003). Responsible for the processing of dsRNA into siRNAs Cleaves dsRNA or pre-miRNA-Leaves 3’ overhangs and 5’ phosphate groups. Initiates RNAi.

11. 2001 Functional domains in Dicer (Bernstein et al., Putative helicase PAZ domain Tandem RNAse-III domains dsRNA binding domain Multiple Dicer genes in Drosophila and plants (He and Hannon, 2004) ‹#›

12. 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 RNA-Induced Silencing Complex (RISC) 3/6/2014 ‹#›

13. 3/6/2014 ‹#› RNAi effector complex Critical for target mRNA degredation or tranlslation inhibition Activities associated with RISC Helicase Endonuclease and exonuclease “Slicer” (or is it Dicer?) “homology seeking”/RNA binding Preferentially incorporates one strand of unwound RNA [Khvorova et al., 2003] Antisense How does it know which is which?

14. RNA-dependent RNA Polymerase (RdRP) 3/6/2014 ‹#› RdRP – Triggering and amplifying the silencing effect Systemic nature of RNAi RdRP activity found in plants and C. elegans May explain efficiency of RNAi Required for RNAi? Not found in mammals or drosophila RdRP deficient plants and worms... Results not decisive

15. siRNA 3/6/2014 ‹#› 21-23 nucleotide dsRNA that mediate PTGS Produced in vivo by cleavage of dsRNA Amplification by an RNA-dependent RNA polymerase (RdRP) may occur Incorporated into the RISC guiding it to mRNA Complementary to a specific sequence of target mRNA for degradation.

16. 3/6/2014 ‹#› Mechanism of RNAi Initiation phase Maintenance phase Signal amplification and spreading phase

17. dsRNA cut by Dicer Initiation phase 3/6/2014 ‹#›

18. Features of efficient and specific siRNAs 3/6/2014 ‹#›

19. siRNA incorporated into RISC Maintenance phase ‹#›

20. Amplified signal 3/6/2014 ‹#›

21. Transitive RNAi by Secondary siRNA 3/6/2014 ‹#›

22. Mechanism of RNAi Vagner et al., 2004 3/6/2014 ‹#›

23. Jagtap et. al., 2011 3/6/2014 ‹#›

24. 3/6/2014 ‹#› Cell to cell movement of RNA silencing….. a. Short distance movement : Exogenous ds RNA is processed by Dicer like enzymes into 21nt and 24nt Primary SiRNAs. 24nt SiRNA was shown to be dispensable for local movement. 21nt SiRNA may spread over 10-15 adjacent cells in the absence of relay amplification.

25. 3/6/2014 ‹#› b. Extensive silencing movement: Perception of 21nt Si RNAs in recipient cell triggers de nova synthesis of dsRNA The dsRNA further processed by dicer resulting in 21nt secondary siRNA This further Spread over 10-15 cells.

26. 3/6/2014 ‹#› Main component of RNAi machinery for miRNA generation There are six component for miRNAs generation Drosha Dicer Argonaute (Ago) small Interfering RNA (siRNA) RNA-Induced Silencing Complex (RISC) RNA-Dependent RNA Polymerase (RdRP)

27. 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 How does it identify pri-miRNA? Hairpin terminal loop size Stem structure Hairpin flanking sequences Not yet found in plants Maybe Dicer does its job? 3/6/2014 ‹#›

28. RISC Preference for Antisense RNA 3/6/2014 ‹#› Helps ensure specificity for target 5’ stability of siRNA and miRNA duplex strands often different The strand with less 5’ stability usually incorporated into RISC [Schwarz et al., 2003] If strand stability is similar (rare), strands incorporated at similar frequency [He and Hannon, 2004]

29. Argonaute (Ago) 3/6/2014 ‹#› Consistently co-purifies with RISC [Hammond et al., 2001] “Homology seeking” activity? Binds siRNA and miRNA [Ekwall, 2004] Distinguishes antisense strand [Novina and Sharp, 2004 ] Multiple Ago family proteins Different RISCs? Tissue specific? Developmentally regulated? Evidence for different RISCs [Tijsterman et al., 2004] Drosophila Dicer1 vs Dicer2/R2D2 Inhibition vs. degradation [Lee et al., 2004]

30. 3/6/2014 ‹#› miRNA Biogenesis Transcribed from endogenous gene as pri-miRNA Primary miRNA: long with multiple hairpins Imperfect internal sequence complementarity Cleaved by Drosha into pre-miRNA Precursor miRNA: ~70nt imperfect hairpins Exported from nucleus Cleaved by Dicer into mature miRNA – 21-25nt Symmetric 2nt 3’ overhangs, 5’ phosphate groups

31. miRNA Biogenesis Novina and Sharp, 2004 3/6/2014 ‹#›

32. Translational Inhibition Imperfect match between siRNA or miRNA in RISC and target mRNA RISC usually binds 3’ UTR Mechanism of inhibition... ???? He and Hannon, 2004 3/6/2014 ‹#›

33. mRNA Degradation Perfect complementarity between siRNA or miRNA in RISC and the target mRNA Cleavage by RISC activity Could be Dicer? Other endo/exonucleases? Recruitment of components? Slicer other Novina and Sharp, 2004 3 c 2 3/6/2014

34. RNAi: Two Phase Process Initiation Generation of mature siRNA or miRNA Execution Silencing of target gene Degradation or inhibition of translation 3/6/2014 33

35. Initiation 1. hpRNA structure Williams et al. 2004 3/6/2014 ‹#›

36. 2. Processing of inactivate pri-miRNA Williams et al. 2004 3/6/2014 ‹#›

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

38. RNA Gene-silencing constructs commonly used in plant genetic modification 3/6/2014 ‹#›

39. 3/6/2014 ‹#›

40. DNA directed RNAi Williams et al. 2004 3/6/2014 39

41. Cecilia et. al., 2010 3/6/2014 ‹#›

42. Application of RNAi 3/6/2014 ‹#›

43. 3/6/2014 ‹#› 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.

44. 3/6/2014 ‹#› 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 Arabidop sis 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

45. 3/6/2014 ‹#› High degree of specificity - It is highly specific to the mRNA Highly potent and effective- Only a few dsRNA molecules per cell are required for effective interference Systemic silencing- The interfering activity can cause interference in cells and tissues far from the site of introduction Advantages of RNAi

46. Non specific effect - Nonspecific effects can also occur in response to siRNA. But these effects are concentration dependent , siRNA used at lower concentrations, can reduce the concentration-dependent nonspecific side effects. Off target effect - siRNA have a similar cellular machinery with miRNA , for the loose homology requirements for activation of miRNA function, siRNA can works as a miRNA even in a low concentration. Limitations of RNAi 3/6/2014 ‹#›