RNAi

1
RNAi

• Nick Yagoda
• Stockwell Lab
• October 15, 2004

2
RNA Interference

• Sequence Specific Gene Silencing
• Conserved pathway critical to the stability of
  genome
• Implicated in silencing of transposons,
  repetitive sequences, and viruses
• Related to Post-Transcriptional Gene Silencing,
  PTGS (plants) and quelling (fungi).
• mRNA Dimmer Potent biological tool for
  targeted knockdown
• Functional Genomics
• Chemical Genetics
• Drug-Target Validation
• Highly specific disruption of clinically relevant
  genes.
• HIV, HPV, oncogenes, tumor suppressors,
  cell-surface receptors.

3
A few of the roles RNA plays in protein regulation

• mRNA template to protein translation
• Protective measures
• Conserved defense against transposition, viral
  infection, gene duplication.
• Mammalian cells RNA (gt30n.t.) will illicit
  Interferon Response
• Indirect activation of non-specific degradation
  of RNA (via RNase I)
• Global inhibition of mRNA translation via
  initiation factor phosphorylation.
• RNAiregulation of endogenous gene expression

4
RNAi Pathway
Hannon GJ (2004) Nature. 431 371-378
5
Discovery and Background

• Injection of dsRNA into c. elegans leads to
  sequence-specific gene silencing (Fire, Mello, et
  al. 1998)
• Short RNAs are found in plants
• Effector molecules of PTGS
• Long dsRNA is found to be cleaved into shorter
  dsRNA in Drosophila.
• These short RNAs were subsequently found in fly
  embryos.
• RNAi can be activated by endogenous miRNAs and
  transposition, and serves in some organisms as
  antiviral defense.

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RNAi Effectiveness of Long vs. Short RNA

• Long RNA
• Effective in silencing translation in c. elegans
  and Drosophila.
• Is cleaved into short RNAs (Zamore, Tuschl, et
  al. 2000)
• Limited applications in mammalian cells
• Interferon!
• Short RNA
• in vitro/in vivo production small enough to dodge
  Interferon radar

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Short RNAs

• Short Interfering RNA (siRNA)
• MicroRNA (miRNA)
• Tiny non-coding RNA (tncRNA)
• Small Modulatory RNA (smRNA)

21-25n.t Exogenous Insertion via
transfection Double-Stranded Exact sequence
compliment to target mRNA sequence
22n.t Endogenous regulatory RNA Up to 40,
000/cell Single-stranded Partial sequence
compliment to target mRNA
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siRNA 3 two-nucleotide overhang
Characteristic of Ribonuclease III degradation
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RNAi Pathways
siRNA
miRNA
Dykxhoorn D, Novina CD, Sharp PA (2003) Nature
Reviews. 4 457-467.
10
RNAi is ATP-dependent

• CPcreatine phosphate
• CKcreatine kinase
• Drosophila Lysate
• Both CP CK are required to convert ADP to ATP.
  Deficiency in either halts phosphorylation.
• Endogenous ATP sufficient to support RNAi

Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000)
Cell. 101 25-33.
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RNAi Pathways
siRNA
miRNA
Dykxhoorn D, Novina CD, Sharp PA (2003) Nature
Reviews. 4 457-467.
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Generating short RNAs
In vitro generation, chemically synthesized
Long hairpin RNA yields population of several
sequence specificities. Processed by Dicer.
Tandem promoters expressing sense and antisense
strands of siRNA in trans.
DNA-vector mediated in vivo generation with RNA
Polymerase family
Short hairpin RNA (shRNA), sense and antisense
associating in cis. Processed by Dicer.
Imperfect duplex hairpin based on pre-miRNA
structure. Processed by Dicer.
Dykxhoorn D, Novina CD, Sharp PA (2003) Nature
Reviews. 4 457-467.
13
DICER

• RNase III family characteristic cleavage
• 2n.t. 3 overhang
• Recessed 5 phosphate

www.nature.com/focus/animations/animation/animatio
n.htm
Blaszczyk J, Tropea JE, Bubunenko M, Routzahn KM,
Waugh DS, Court DL, Ji X (2001) Structure. 9
1225-1236
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RISC

• RNA-Induced Silencing Complex
• siRNA must be 5- phosphorylated
• Double-stranded siRNA is unwound antisense
  strand complexes with RISC proteins.
• SlicerArgonaute
• RNA binding domain (PAZ)
• RNase domain (PIWI)

www.nature.com/focus/animations/animation/animatio
n.htm
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Argonaute Structure
Song, et al. (2004) Science. 305 1434-1441.
Song J, Smith SK, Hannon GJ, Joshua-Tor L (2004),
Science. 305 1434-1441.
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siRNA Pathway
Sharp P.A., Novina C.D. (2004) Nature. 430
161-164.
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Not all siRNAs are created equal

• Factors limiting efficiency of knockdown
• Single nucleotide changes in siRNAs abrogate
  interference.
• 2-structure of mRNA (loops, hairpins, etc.)
• mRNA sequence involved in protein binding

Brummelkamp TR, Bernards R, Agami R (2002),
Science. 296 550-553.
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miRNA Pathway
Sharp P.A., Novina C.D. (2004) Nature. 430
161-164.

• Examples of miRNA function
• Wormsan entire class of genes (lin-4 and let-7
  ) transitions between larval stages
• Plantsdevelopmental transitions
• Fliescell division and cell death
• HumansmiRNAs identified recently little known
  about their activity

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RNAi Suppresses Protein Expression in HeLa Cells

• Lamin A/Cnuclear envelope proteins
• NuMAnuclear mitotic aparatus protein
• Hoechststains nuclear chromatin
• GL2 Pp-lucreporter plasmid linked to luciferase
• Control to monitor specificity of siRNA activity
• siRNA 25nM
• increased concentrations did not enhance
  silencing).
• Silencing effect only vanishes at extremely low
  siRNA concentrations 0.05nM (only 2- to 20-fold
  more concentrated than cotransfected DNA
  plasmids)

Elbashir SM, Harborth J, Lendeckel W, Yalcin A,
Weber K, Tuschl T (2001) Nature. 411 494-498.
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RNAi in Mammalian cell lines

• S2Drosophila
• NIH/3T3mouse fibroblast
• COS-7monkey cells (containing SV-40)
• HeLa S3human cells (containing HPV)
• 293human embryonic kidney
• a, c, e, g, icotransfection of pGL2 with
  indicated siRNA
• b, d, f, h, jcotransfection of pGL3 with
  indicated siRNA
• RESULTS highly-specific knockdown of target mRNA

Elbashir SM, Harborth J, Lendeckel W, Yalcin A,
Weber K, Tuschl T (2001) Nature. 411 494-498.
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RNAi Amplification(In organisms with endogenous
RNAi mechanisms, including Plants, Fungi, Worms,
Mammals)

• Dicer produces siRNAs en mass.
• siRNAs, not associated with RISC, serve as
  primers for RNA-dependent RNA Polymerase (RdRP)
  activity.
• RdRP assembles antisense strand based on mRNA
  template.
• Dicer produces more siRNA.
• Cycle Repeats itself

www.nature.com/focus/animations/animation/animatio
n.htm
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siRNA Delivery Vehicles

• Transfection, Lipid-mediated
• siRNA
• DNA-vector-mediated
• Nucleofection
• Swift delivery of cDNA into nucleus
• Viral Infection
• Pro introduce nucleic acids into
  non-transfectable cells.
• Con Safety Risks

Amaxa Biosystems
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Pros and Cons of siRNA Vector

• Vector-Mediated
• Pros
• Can produce stable cell lines
• Induceable Constructs
• Avoids Interferon Response in Mammals
• Clone multiple specificities
• Delivery with virus in to non-transfectable
  cells.
• Cons
• Delayed effect (transcription, nuclear export)
• Inefficient transfection results

• siRNA
• Pros
• Poolsmultiple specificities
• Immediate effect (cytoplasmic)
• Higher transfection efficiency compared to DNA,
  generally.
• Cons
• Non-renewable, transient effect in mammals.
• Difficult to knockdown proteins with long
  half-life.
• Dilution with cell doubling.

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Applications

• RNAi has the potential to identify functions of
  each gene in a cell-type or pathway-specific
  manner.
• Functional Genomics
• determining gene function
• Berns K, et al. (2004) A large-scale RNAi screen
  in human cells identifies new components of the
  p53 pathway. Nature. 428 431-437
• Foley E, OFarrlel PH. (2004) Functional
  dissection of an innate immune response by a
  genome-wide RNAi screen. PLoS Biology. 2
  1091-1106.
• Chemical Genetics
• Identifying proteins underlying biological
  processes with chemical tools
• Drug-Target Validation
• Validating therapeutic benefit of
  depletion/amplification of target protein

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Chemical Genetic Screening
Chemical Genetic Approach
Stockwell, B.R. (2000) Nature Reviews Genetics 1,
116-125.
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Compounds that Selectively Kill Tumor Cells
27
Erastin-induced Cell Death in Tumor Cells

• Chemical Genetic Screen
• Stage 1 Identification of small molecules that
  induce
• genotype-specific lethality (large-scale
  screen).
• Stage 2 Identification of targets within cell
• Pull-down analysis ? putative targets
• Monitor cell sensitivity to drug treatment after
• Putative Target Knockdown (RNAi)
• Putative Target Overexpression (cDNA
  transfection)
• Stage 3 Drug Development, etc.

Erastin
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Engineered Human Tumor Cells
hTERT (protein component of telomerase)
SV40 Small T oncoprotein
SV40 Large T oncoprotein
RASV12
In collaboration with William Hahn and Stephen
Lessnick, Dana-Farber Cancer Institute
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Sources of Small Molecules
1. Combinatorial Library - 20,000 compounds
2. NCI diversity set - 1,990 compounds
3. Annotated Compound Library - 2,036 compounds
380 compounds
Test for gt 50 inhibition in tumor cells
24,026 compounds
gt 4-fold tumor cell selectivity vs normal cells
IC50primary IC50tumor
9 compounds
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An Automated Process for Cell-Based Screening
Chemical Libraries
Plate Barcoding
Plate Replication
Assay Execution
Sterile Cell Dispensing
Data Analysis
Root DE, Kelley BP Stockwell BR (2003) Detection
of Systematic Errors in Spatial Arrays. J
Biomolecular Screening,8 (4) 393-398
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Genotype-Selective Lethality
32
Identification of Erastin-Binding Proteins
In collaboration with Prolexys
B1 A6
B1 immobilized inactive analog A6 immobilized
active analog

• Voltage-Dependent Anion Channel
• Prohibitin
• Ribophorin 1

erastin-specific binding protein
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Effect of Putative Target mRNA Knockdown on
Erastin Sensitivity in Human Tumor Cells

• Day1
• BJeLR (engineered human tumor line) cells seeded
  at 200,000 cells/well in 384-well plates.
• Day2
• Anti-VDAC2 siRNA transfected via liposome complex
  with Oligofectamine.
• Day3
• RNAi Analysis
• Total RNA isolation.
• Real-Time RT-PCR.
• Erastin Treatment
• Day4
• Viability Assay (Alamar Blue)

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Erastin-induced Cell Death in Tumor Cells

• Chemical Genetic Screen
• Stage 1 Identification of small molecules that
  induce
• genotype-specific lethality (large-scale
  screen).
• Stage 2 Identification of targets within cell
• Pull-down analysis ? putative targets
• Monitor cell sensitivity to drug treatment after
• Putative Target Knockdown (RNAi)
• Putative Target Overexpression (cDNA
  transfection)
• Stage 3 Drug Development, etc.

Erastin
35
Acknowledgements
Erastin Project Brent Stockwell Sonam Dolma Steve
Flaherty Allison Martino
Collaborators William Hahn Steve
Lessnick Prolexys Pharmaceuticals Funding Burroug
hs Wellcome Fund National Cancer Institute