RNA interference (RNAi)

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RNAi is a powerful, conserved biological process through which the small, double-stranded RNAs specifically silence the expression of homologous genes, largely through degradation of their cognate mRNA.

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RNA interference (RNAi)

  1. 1. RNA interference RNAi BY Kamlesh Kumar Chandel Ph.D. Scholar Department of Genetics and Plant Breeding
  2. 2. Content I. Overview view / History II. Mechanism / Process III. Function IV. Applications V. RNAi Glossary VI. References Nobel Prize in 2006
  3. 3. RNAi  RNAi is a powerful, conserved biological process through which the small, double-stranded RNAs specifically silence the expression of homologous genes, largely through degradation of their cognate mRNA.  its responsible for post-transcriptional gene silencing of the gene from which it was derived  Endogenous cellular mechanisms  Effecter molecules for functional genomics  Great potential as therapeutic agents for treatment of human disease
  4. 4. RNA interference Technology  RNAi is used to block the expression of genes and create phenotypes that can potentially yield clues about the function of these genes.  In the post-genomic era, the elucidation of the physiological function of genes has become the rate-limiting step in the quest to develop ‘gene-based drugs’ and RNAi could potentially play a pivotal role in the validation of such novel drugs. RNAi is a fantastic discovery , all the RNAi idea will be describing in these video (1) http://www.youtube.com/watch?v=H5udFjWDM3E&feature=related (2) http://www.youtube.com/watch?v=A-l8tqjm4Vg&feature=related (3) http://www.youtube.com/watch?v=3kdhYCJFmZc&feature=related (4) http://www.youtube.com/watch?v=kCxQdXX0Dbk (5) http://www.youtube.com/watch?v=h1kayIVEfcY&feature=related
  5. 5. Time - Line
  6. 6. Discovery of RNAi or PTGS (Post transcriptional gene silencing) 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 Also genes called became Co-suppression suppressed! Suppression was mostly due to increased degradation of the mRNAs (from the endogenous and introduced genes) Flowers from 3 different transgenic petunia plants carrying copies of the chimeric DFR gene above. The flowers had low DFR mRNA levels in the non-pigmented areas, but gene was still being transcribed.
  7. 7. RNA interference also reported in •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 Fire and Mello 1998 Injection of dsRNA into C. elegans RNA interference (RNAi) or gene silencing Ambros 1993 (2000) Identification of small RNA in C. elegans (micro RNA)
  8. 8. RNAi discovered in Nematode {Caenorhabditis elegans} 1998 (first animal) while attempting to use antisense RNA in vivo Nobel Prize in 2006 ( Field of Physiology & Medicine) Craig Mello & Andrew Fire Control “sense” RNAs also produced suppression of target gene! sense RNAs were contaminated with dsRNA. dsRNA was the suppressing agent. -RNAi can be induced in C. elegans in three simple ways: -Injection of dsRNA into the worm gonads -Soaking the worms in dsRNA solution -Feeding the worms engineered bacteria producing dsRNA
  9. 9. Double-stranded RNA (dsRNA) induced interference of the Mex-3 mRNA in the Nematode {Caenorhabditis elegans} 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.
  10. 10. Mechanism/Process  A cellular mechanism that degrades unwanted RNAs in the cytoplasm but not in the nucleus What happens ?  dsRNA is processed into shorter interfering (siRNAs) that guide the targeted cleavage of homologous RNA.
  11. 11. Mechanism of RNA interference (RNAi)  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.
  12. 12. A model for the mechanism of RNAi - Silencing triggers in the form of double-stranded RNA may be presented in the cell as synthetic RNAs, replicating viruses or may be transcribed from nuclear genes. - These are recognized and processed into small interfering RNAs by Dicer. - The duplex siRNAs are passed to RISC (RNA-induced silencing complex) - The complex becomes activated by unwinding of the duplex. - Activated RISC complexes can regulate gene expression at many levels: •Promoting RNA degradation •Translational inhibition •Chromatin remodelling - Amplification of the silencing signal in plants may be accomplished by siRNAs priming RNA-directed RNA polymerase (RdRP)-dependent synthesis of new dsRNA.
  13. 13. Mechanism of RNA interference
  14. 14. Mechanism of RNAi : Role of Dicer  Cells (plants and animals) undergoing RNAi contained small fragments (~25 nt) of the RNA being suppressed.  A nuclease (Dicer) was purified from Drosophila embryos that still had small RNA fragments associated with it, both sense and antisense.  The Dicer gene is found in all organisms that exhibit RNAi, and mutating it inhibits the RNAi Coenfcfleucsito.n: 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.
  15. 15. RNAi FUNCTIONS - To regulates expression of protein coding genes - To mediates resistance to both exogenous parasitic and exogenous pathogenic nucleic acid - To used experimentally to block gene expression
  16. 16. RNAi applications  Genome-wide RNAi screening  Done in C. elegans  19 757 protein coding genes (predicted)  16 757 inactivated using RNAi  New standard for systematic genome wide functional studies  RNAi as a solution for mammalian genetics  Defense against Infection by viruses  Potential therapeutic use  Prevents viral infection  Inhibits the expression of viral antigens  Suppresses the transcription of viral genome  Blocks viral replication  Silences viral accessory genes  Hinders the assembly of viral particles & Displays roles in virus-host interactions
  17. 17. Responses to Mechanical Stimuli HIV levels can be reduced by 30-50 fold by 17 siRNA!!!
  18. 18. Biotechnology & Agriculture  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 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.
  19. 19. Biotechnology & Agriculture  Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plants.  Although no plant products that use RNAi-based genetic engineering have yet passed the experimental stage, development efforts have successfully reduced the levels of allergens in tomato plants and decreased the precursors of likely carcinogens in tobacco plants.  Other plant traits that have been engineered in the laboratory include the production of non-narcotic natural products by the opium poppy, resistance to common plant viruses, and fortification of plants such as tomatoes with dietary antioxidants.
  20. 20. RNA interference characteristics  dsRNA needs to be directed against an exon, not an intron in order to be effective  Homology of the dsRNA and the target gene/mRNA is required  Targeted mRNA is lost (degraded) after RNAi  The effect is non-stoichiometric; small amounts of dsRNA can wipe out an excess of mRNA (pointing to an enzymatic mechanism)  ssRNA does not work as well as dsRNA
  21. 21. Advantage of RNAi  Downregulation of gene expression simplifies "knockout" analysis.  Easier than use of antisense oligonucleotides. siRNA more effective and sensitive at lower concentration.  Cost effective  High Specifity middle region 9-14 are most sensitive  With siRNA, the researcher can simultaneously perform experiments in any cell type of interest  Can be labelled  Ease of transfection by use of vector
  22. 22. Importance of RNAi  Powerful for analyzing unknown genes in sequenced genomes.   efforts are being undertaken to target every human gene via siRNAs  Faster identification of gene function  Gene therapy: down-regulation of certain genes/ mutated alleles  Cancer treatments  knock-out of genes required for cell proliferation  knock-out of genes encoding key structural proteins
  23. 23. http://www.rnaiweb.com/RNAi/RNAi_Web/
  24. 24. http://www.rnainterference.org/Sequences.html
  25. 25. RNAi Glossary  Dicer – Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs. Dicer processes long dsRNA into siRNA of 21-23 nt.  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  Ribozyme – Ribozymes are RNA molecules that act as enzymes in the absence of proteins.  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
  26. 26. References http://www.rna.com/ http://www.cambridge.org/catalogue/catalogue.asp?isbn=0511081316 http://www.youtube.com/watch?v=H5udFjWDM3E&feature=related http://www.youtube.com/watch?v=kCxQdXX0Dbk http://arabidopsis.info/students/rohan/mechanismrnai.html http://www.youtube.com/watch?v=h1kayIVEfcY&feature=related * Simple, Efficient Production of Short Double-Stranded RNA Using RNase III (Judith E. Meis, EPICENTRE).website : http://www.epibio.com/pdfforum/9_3dsrnarnaseiii.pdfmicroRNA formation and function http://www.youtube.com/watch?v=_-9pROnSD-A http://www.rnaiweb.com/RNAi/RNAi_Web_Resources/RNAi_Companies/RNAi_Therapeutics/index.html http://www.alnylam.com/Programs-and-Pipeline/Programs/Liver-Cancer.php (1)* Meister ,G., Tuschl ,T.. (2004). Mechanisms of gene silencing by double-stranded RNA. Natural. 431(7006). 343-9. (2)* Kedde ,M., Strasser ,M.J., Boldajipour ,B., Oude Vrielink ,J.A., Slanchev ,K., le Sage ,C., Nagel ,R., Voorhoeve ,P.M., van Duijse ,J., Ørom ,U.A., Lund ,A.H., Perrakis ,A., Raz ,E., Agami ,R.. (2007). RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. cell. 131(7). 1273-86. (3)*Klionov ,M.S., Stoliarenko ,A.D., Riazanskiĭ ,S.S., Sokolova ,O.A., Konstantinov ,I.N., Gvozdev ,V.A.. (2007). Role of short RNAs in regulating the expression of genes and mobile elements in germ cells. ONTOGENES. 38(3). 213-27. (4)*Aalto ,A.P., Sarin ,L.P., van Dijk ,A.A., Saarma ,M., Poranen ,M.M., Arumäe ,U., Bamford ,D.H.. (2007). Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase. RNA(New York .N.Y.). 13(3), 422-9. http://books.google.jo/books?id=bjAm2mTbnPoC&pg=PA56&lpg=PA56&dq=repeat+- associated+short+interfering+RNAs+(rasiRNAs)&source=bl&ots=ii34nFhrYx&sig=ABKvNRISLOdkGX0zwC4sW0i-qbU&hl=en&ei=SMXxSanVBcLm- Ab1wLidDw&sa=X&oi=book_result&ct=result&resnum=1#PPP5,M1
  27. 27. Thank you

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