RNA interference

Class presentation on RNA interference

1. RNA interference (RNAi) MBB601 Advances in Plant Molecular Biology Presented By Mr. Ekatpure Sachin 2016-21-022 1

2. Outline Introduction -RNA silencing -Definition of RNA interference -Discovery of RNAi Mechanism of RNA interference Generation of small interfering RNA Small interfering RNA delivery methods Applications of RNA interference -Therapeutic applications -Other applications Conclusion 2

3. Introduction 3

4. RNA silencing Several terms are used to described RNA silencing; usually there are three phenotypically different but mechanistically similar phenomena: Cosuppression or post-trascriptional gene silencing (PTGS) in plants Quelling in fungi RNA interference in animal kingdom 4

5. Quelling: The silencing in fungal system Quelling came to light during attempts to boost the production of an orange pigment made by the gene al1 of the fungus Neurospora crassa N. crassa (al1+) transformed with plasmid+al1 few transformants show albino phenotype al1-quelled strain had similar level of unspliced al1 mRNA to wilde-type Native al1 mRNA was highly reduced indicating that quelling and NOT the rate of transcription affected the level of mature mRNA in a homology-dependent manner 5

6. RNAi: Silencing in Cenorhabditis elegans dsRNA administrated to worms can permeate and affect the entire body causing a systemic RNA-interference RNAi studies represents a means of identifying partial or complete loss-of-function phenotypes, possibly leading to the identification of gene function 6

7. Definition RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes RNAi targets include RNA from viruses and transposons 7

8. Need for interference Defense Mechanism -Defense against Infection by viruses, etc -As a defense mechanism to protect against transposons and other insertional elements Genome Wide Regulation -RNAi plays a role in regulating development and genome maintenance -30% of human genome regulated 8

9. PTGS in plants: The discovery of Jorgensen and Napoli in 1990 They were trying to make petunias more purple Overexpression of petunia gene Entered homologous RNA Expected:more pigments Observed:white sectors Cosuppression: Loss of mRNAs of both endo-and transgene 9

10. Andrew Fire Craig C. Mellow 10

11. Discovery Inject worms with dsRNA corresponding to a gene ( important for muscle function) involved in wiggling ( unc-22 ) 11

12. Conclusion: dsRNA triggers potent and specific gene silencing Inject worms with dsRNA corresponding to a gene (important for muscle function) involved in wiggling ( unc-22 ) Discovery 12

13. Mechanism of RNAi 13

14. RNAi Overview During RNAi Double-stranded RNAs cut into short double-stranded RNAs, s(small) i (interfering) RNA's, by an enzyme called Dicer These then base pair to an mRNA through a dsRNA -enzyme complex. This will either lead to degradation of the mRNA strand Highly specific process Very potent activity So far only been seen in eukaryotes Evidence 30% of genome is regulated by RNAi 14

15. The components In Interference RNA -siRNA: dsRNA 21-22 nt. - miRNA : ssRNA 19-25nt. Encoded by non protein coding genome RISC : -RNA induced Silencing Complex, that cleaves mRNA Enzymes -Dicer : produces 20-21 nt cleavages that initiate RNAi -Drosha : cleaves base hairpin in to form pre miRNA ; which is later processed by Dicer 15

16. 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. Each strand of siRNA has: a. 5’-phosphate termini b. 3’-hydroxyl termini c. 2/3-nucleotide 3’ overhangs 16

17. miRNA Originate from capped & polyadenylated full length precursors ( pri-miRNA ) Hairpin precursor ~70 nt (pre- miRNA ) Mature miRNA ~22 nt ( miRNA ) 17

18. Difference between miRNA and siRNA Function of both species is regulation of gene expression. Difference is in where they originate. siRNA originates with dsRNA. siRNA is most commonly a response to foreign RNA (usually viral) and is often 100% complementary to the target. miRNA originates with ssRNA that forms a hairpin secondary structure. miRNA regulates post-transcriptional gene expression and is often not 100% complementary to the target. Also miRNA help to regulate gene expression, particularly during induction of heterochromatin formation serves to downregulate genes pre- transcriptionally (RNA induced transcriptional silencing or RITS ) 18

19. Dicer Loss of dicer → loss of silencing processing in vitro Dicer homologs exist in many organisms including C.elegans, Drosphila, yeast and humans ( Dicer is a conserved protein) RNase III-like dsRNA-specific ribonuclease Enzyme involved in the initiation of RNAi. It is able to digest dsRNA into uniformly sized small RNAs (siRNA) Dicer family proteins are ATP-dependent nucleases. Rnase III enzyme acts as a dimer 19

20. Dicer’s domains 1 4 3 2 2 Dicer is a ribonuclease (Rnase III family) with 4 distinct domains : 1. Amino-terminal helicase domain 2. Dual Rnase III motifs in the carboxy 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 20

21. RISC RISC is a large (~500-kDa) RNA- multiprotein complex, which triggers mRNA degradation in response to siRNA Unwinding of double-stranded siRNA by ATP independent helicase The active components of an RISC are endonucleases called argonaute proteins which cleave the target mRNA strand 21

22. Mechanism of RNA interference dsRNA are chopped into short interfering RNAs ( siRNA ) by Dicer . 2. The siRNA -Dicer complex recruits additional components to form an RNA-Induced Silencing Complex ( RISC ). The siRNA unwinds. 3. The unwound siRNA base pairs with complementary mRNA, thus guiding the RNAi machinery to the target mRNA. 4. The target mRNA is effectively cleaved and subsequently degraded – resulting in gene silencing 22

23. 23

24. Illustration of miRNA processing 24

25. Summary of components Drosha and Pasha are part of the “Microprocessor” protein complex (~600-650kDa) Drosha and Dicer are RNase III enzymes Pasha is a dsRNA binding protein Exportin 5 is a member of the karyopherin nucleocytoplasmic transport factors that requires GTP Argonautes are RNase H enzymes 25

26. Generation of siRNA’s 26

27. Generation of small interference RNA 27

28. siRNA Expression Vectors 28

29. siRNA design 21-23nt 2-nt 3' overhangs ( UU overhangs ) G/C content: 30-50%. No base pair mismatch Synthesized siRNA should not target introns, the 5′and 3′-end untranslated regions (UTR ) Sequences within 75 bases of the start codon (ATG ) BLAST : eliminate any target sequences with significant homology to other coding sequences. 29

30. siRNA delivery methods 30

31. Effective methods for the delivery of small RNA to allow a sufficient silencing effect in the target organ(s) and/or cells are yet to be developed In particular, toxicity and side effects of RNAi must be well characterized and limited Therefore, careful design and selection of target sequence and quantification of the effect on the expression of target protein and mRNA are essential for success of gene interfering approaches 31

32. High-pressure injection “ High-pressure injection ” was the first strategy to demonstrate successful delivery of siRNA in vivo A large volume (1–2mL) of saline containing unmodified siRNA is injected intravenously into the tail vein of mice within very short time (in less than 7 sec), which presumably results in the siRNA molecules being forced into several organs mainly the liver, kidney and to a lesser degree the lung Certainly, such an approach seems to be impossible in human subjects (1000 mL saline solution containing siRNA per 10 kg of weight) 32

33. Electroporation Electroporation of small RNA directly into target tissues and organs has also been developed to successfully silence gene function 33

34. Delivery of siRNA to tissue is a problem both because: The material must reach the target organ And it must enter the cytoplasm of target cells RNA cannot penetrate cellular membranes, so systemic delivery of siRNA is unlikely to be successful RNA is quickly degraded by RNAse activity in serum and even siRNA chemically modified to be more stable has a half-life of only a few hours at most Problem 34

35. For these reasons, other mechanisms to deliver siRNA to target cells has been devised These methods include: Viral delivery The use of liposomes or nanoparticles Bacterial delivery Chemical modification of siRNA to improve stability Solution 35

36. Viral delivery Viral delivery has been used extensively in gene therapy to deliver DNA to target cells There are 5 main classes of viruses used in the delivery of nucleotides to cells: Retrovirus Adenovirus Lentivirus Baculovirus Adeno -associated-virus (AAV). 36

37. Liposome's and nanoparticles Liposomes and nanoparticles have been known as an alternative to viral delivery systems. Unmodified siRNA has a half-life of less than 1 hour in human plasma and siRNA is rapidly excreted by the kidneys. Liposomes and nanoparticles can act as envelopes to protect the siRNA from metabolism and excretion, but can also carry specific molecules designed to target the siRNA to specific tissue types Liposomes such as Lipofectamine have been used to carry siRNA into cells Nanoparticles such as the cationic polymer , polyethyleneimine ( PEI ) have also been used to successfully deliver siRNA to target cells 37

38. Bacterial delivery Bacterial delivery using nonpathogenic bacteria has been used to silence genes in a process known as transkingdom RNA interference ( tkRNAi ) Generally, the shRNA is produced in bacteria that invade and release the RNA into eukaryotic cells (hence the term transkingdom ) The bacteria can also be engineered to carry shRNA encoding DNA plasmids The advantages of this system include: - Safety - Ability to control the vector using antibiotics 38

39. Chemical modification Finally, chemical modification of siRNA has been used to improve stability and prevent degradation by serum RNAase Importantly, these modifications must obviously not affect the RNA interference activity of the siRNA One of the most common modifications is the use of locked nucleic acid residues (LNA). A methylene bridge connects the 4.C with the 2.O in LNA residues. This modification increases the stability of oligonucleotides in serum, without reducing the gene silencing effect 39

40. Side effects of gene silencing by small RNA molecules Unspecific silencing Caused by the failure to identify similar sequences with only few nt. difference in other genes Activation of intracellular PKR and immune pathways that are linked to toll-like receptor activation PKR ( protein kinase R) is activated by dsRNA longer than 30 nt , which subsequently induces the production of cytokines These ultimately promote inflammatory responses. 40

41. Side effects of gene silencing… High pressure injection ” and electroporation can cause significant damage to the integrity of the normal tissues and organs and thus exclude the utilization in a clinical set-up Liposomes/cationic encapsulated siRNA may also be toxic to the host and may cause severe host immune responses Other emerging strategies includes chemical modification of siRNA molecules and encapsulated with different molecules are still in their initial stage and need to be thoroughly investigated before used in therapeutic applications 41

42. Applications of RNAi 42

43. Hematology (blood) Hematologic disorders result from Loss of gene function Mutant gene function Absent gene function RNAi May be used to create models of disease processes Could help to develop pharmacologic and genetic therapeutic targets 43

44. Oncology (cancer) Targeting of oncogenes Dominant mutant oncogenes, amplified oncogenes, viral oncogenes Define role of signaling molecules in tumor-creation Improvement efficacy of chemotherapy and radiotherapy Tumor regression through creation of potentially new mode of chemotherapy 44

45. Stem cell biology Mouse research -Knock out tumor-suppression gene in mouse embryonic stem cell -Observe tumor phenotype -Positive correlation between extent of Trp 53 (suppression gene) inhibition and severity of disease 45

46. Infectious Diseases Virus targeting -RNAi – inhibit cellular and viral factors of disease -RNA transcriptase is RNAi target -Inhibition of replication Main goal -Render cells resistant to infectious organisms 46

47. Biotechnology & Agriculture In the engineering of food plants that produce lower levels of natural plant toxins Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks For example, cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol , making them unsuitable for human consumption RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta- cadinene synthase , a key enzyme in gossypol production, without affecting the enzyme's production in other parts of the plant, where gossypol is important in preventing damage from plant pests 47