RNA Interference

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RNA Interference

• Elaine Jane Chua

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From DNA to Protein

• Transcription
• process where information held in the DNA is
  transferred to RNA
• Translation
• conversion of RNA to amino acid sequence of
  protein

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Types of RNA

• Messenger RNA (mRNA)
• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)

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DNA
mRNA
Protein
transcription
translation
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RNA Interference (RNAi)

• Post-transcriptional gene silencing (PTGS)
• Process by which double-stranded RNA (dsRNA)
  directs sequence-specific degradation of mRNA

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Remarkable Properties of RNAi

• RNAi is highly gene-specific
• dsRNA appears to move freely across cell
  boundaries
• Mechanism of RNAi is not well understood
• RNAi works in flies, mice, and plants

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Gene-Silencing in Some Species
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Applications of RNAi

• Fight spread of diseases
• HIV
• Cancer
• Polio
• Hepatitis
• Drug Development
• Aging
• Functional gene analysis

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How does RNAi work?

• Initiation step
• dsRNA digested into 21-23 nt siRNAs (guide RNAs)
  with the help of Dicer
• Effector step
• siRNA assemble into RISC
• siRNA guide the RISC, and cleave and destroy
  complementary RNA

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Major Actors in RNAi

• Dicer
• Small interfering RNA (siRNA)
• RNA-Induced Silencing Complex (RISC)

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Dicer

• Enzyme belonging to the RNase III ribonuclease
  family
• Responsible for the processing of dsRNA into
  siRNAs
• Initiates RNAi

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Small Interfering RNA (siRNA)

• 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

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RNA-Induced Silencing Complex (RISC)

• Nuclease complex composed of proteins and siRNA
• Targets and destroys endogenous mRNAs
  complementary to the siRNA

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Double-Stranded RNA (dsRNA)

• Sense and Antisense strands

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Double-Stranded RNA (dsRNA)
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Double-Stranded RNA (dsRNA)

• Exogenous
• injection
• soaking
• Endogenous
• folding of RNA to form double helix (hairpin loop)

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How long does RNAi last?

• Silencing caused by injection (synthetic siRNA)
  lasts for several days and appear to be
  transferred to daughter cells, but eventually
  diminish
• Cells transfected with siRNA expression vector
  experience steady, long-term mRNA inhibition

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RNAi Tools and Products

• siRNA Delivery
• GeneSuppressor RNA Interference Kit
• Targefect-siRNA Transfection Kit
• TransIT-TKO Transfection Reagent
• siRNA localization
• Label IT siRNA Tracker Kuts
• siRNA expression
• PCR-Based siRNA Expression Systems

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References

• Gregory Hannon, RNA Interference, Nature, 418
  244-251, July 11, 2002
• Leonid Gitlin, Sveta Karelsky, Raul Andino,
  Short Interfering RNA Confers Intracellular
  Antiviral Immunity in Human Cells. Nature,
  418430-434, July 25, 2002
• Jean-Marc Jacque, Karine Triques, Marlo
  Stevenson, Modulation of HIV-1 Replication by
  RNA Interference. Nature, 418 435-438, July
  25, 2002
• Roger J. Pomerantz, RNA Interference Meets
  HIV-1 Will Silence be Golden?. Nature
  Medicine, 8,7659-660, July 2002
• Small RNAs Make Big Splash, Science,
  2982296-2297, December 20, 2002
• Gordon G. Carmichael, Silencing Viruses with
  RNA. Nature, 418379-380, July 25, 2002
• Julia M. Bosher Michel Labouesse, RNA
  Interference Genetic Wand and Genetic Watchdog.
  Nature Cell Biology, 2E31-E36, February 2000
• Deborah Mitchell, RNA Interference May Provide
  New Therapeutic Approach to HIV Infection
  (February 12, 2003) http//www.aidsmeds.com/news/
  20030212scie002.html

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References (cont.)

• Hilla Giladi, RNA Interference of HBV Gene
  Expression http//www.hadassah.org.il/departments
  /gen_ther/sca_rnai_hbv.htm
• Duanne Hewitt, Studying Aging with RNA
  Interference (January 5, 2003)
  http//www.immortality.org/RNAi.html
• Anne Rieger, Isabelle Guillemin, Hans Gerd
  Nothwang, Functional Gene Analysis by RNA
  Interference (RNAi)
• RNA Interference Cuts Hepatitis Down to Size
  http//focus.hms.harvard.edu/2003/Feb21_2003/genet
  ics.html
• New Method of Turning Off Viruses May Help
  Control HIV infection, Says Jefferson Scientist
  http//www.sciencedaily.com/releases/2002/07/02070
  5091216.htm
• RNA Interference and Gene Silencing History
  and Overview (May 20, 2002) http//www.ambion.com
  /hottopics/RNAi/rnai_may2002_print.html
• Mirus Gene Transfer Specialists
  http//www.genetransfer.com/rnainterference.php
• Antisense RNA http//users.rcn.com/jkimball.ma.u
  ltranet/BiologyPages/A/AntisenseRNA.html
• Double-Stranded RNA Interference with Gene
  Expression RNAi http//avery.rutgers.edu/WSSP.St
  udentScholars/project/introduction/RNAi.html

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References (cont.)

• The Rest is Silence http//www.hhmi.ucla.edu/par
  asite_course/rna20interference/subchapters/discov
  ery.htm
• RNA Interference Silencing Tools and
  Therapeutics, Beyond Genome Solutions for the
  Post-Genomic Era. http//www.beyondgenome.com/rni
  .asp
• IMGENEX Innovations in Functional Genomics
  http//www.imgenex.com/products_genesuppressor.htm
  l
• Heres How RNA Interference Works (October
  2000) http//www.findarticles.com/cf_dls/m0DED/3_2
  1/66520543/p1/article.jhtml
• RNA Structure http//www-biol.paisley.ac.uk/Cour
  ses/StFunMac/glossary/RNA.html
• Targeted Gene Silencing by RNA Interference,
  Benitec Technology. http//www.benitec.com.au/gen
  e-silencing.htm
• The Small World of Small RNAs
  http//www.hhmi.ucla.edu/parasite_course/rna20int
  erference/subchapters/small20rnas.htm
• New Products Targefect-siRNA Transfection Kit
  for RNA Interference Studies
• RNA Transcription and Processing
  http//ntri.tamuk.edu/cell/rna.html

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Thank you.