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    • RNAi Nick Yagoda Stockwell Lab October 15, 2004
    • 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.
    • 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 (>30n.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.
      • RNAi—regulation of endogenous gene expression
    • RNAi Pathway Hannon GJ (2004) Nature . 431: 371-378
    • 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.
    • 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’
    • 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
    • siRNA: 3’ two-nucleotide overhang Characteristic of Ribonuclease III degradation
    • RNAi Pathways siRNA miRNA Dykxhoorn D, Novina CD, Sharp PA (2003) Nature Reviews . 4: 457-467.
    • RNAi is ATP-dependent
      • CP—creatine phosphate
      • CK—creatine 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.
    • RNAi Pathways siRNA miRNA Dykxhoorn D, Novina CD, Sharp PA (2003) Nature Reviews . 4: 457-467.
    • Generating short RNAs Long hairpin RNA yields population of several sequence specificities. Processed by Dicer. Tandem promoters expressing sense and antisense strands of siRNA in trans . Short hairpin RNA (shRNA), sense and antisense associating in cis . Processed by Dicer. Dykxhoorn D, Novina CD, Sharp PA (2003) Nature Reviews . 4: 457-467. In vitro generation, chemically synthesized DNA-vector mediated in vivo generation with RNA Polymerase family Imperfect duplex hairpin based on pre-miRNA structure. Processed by Dicer.
    • DICER
      • RNase III family characteristic cleavage
        • 2n.t. 3’ overhang
        • Recessed 5’ phosphate
      www.nature.com/focus/animations/animation/animation.htm Blaszczyk J, Tropea JE, Bubunenko M, Routzahn KM, Waugh DS, Court DL, Ji X (2001) Structure . 9: 1225-1236
    • RISC
      • RNA-Induced Silencing Complex
        • siRNA must be 5’- phosphorylated
        • Double-stranded siRNA is unwound; antisense strand complexes with RISC proteins.
      • Slicer—Argonaute
        • RNA binding domain (PAZ)
        • RNase domain (PIWI)
      www.nature.com/focus/animations/animation/animation.htm
    • Argonaute Structure Song, et al. (2004) Science . 305: 1434-1441. Song J, Smith SK, Hannon GJ, Joshua-Tor L (2004), Science . 305: 1434-1441.
    • Sharp P.A., Novina C.D. (2004) Nature . 430: 161-164. siRNA Pathway
    • 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.
    • miRNA Pathway
      • Examples of miRNA function:
        • Worms—an entire class of genes ( lin-4 and let-7 ): transitions between larval stages
        • Plants—developmental transitions
        • Flies—cell division and cell death
        • Humans—miRNAs identified recently; little known about their activity
      Sharp P.A., Novina C.D. (2004) Nature . 430: 161-164.
    • RNAi Suppresses Protein Expression in HeLa Cells
      • Lamin A/C—nuclear envelope proteins
      • NuMA—nuclear mitotic aparatus protein
      • Hoechst—stains nuclear chromatin
      • GL2 Pp-luc—reporter 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.
    • RNAi in Mammalian cell lines
      • S2—Drosophila
      • NIH/3T3—mouse fibroblast
      • COS-7—monkey cells (containing SV-40)
      • HeLa S3—human cells (containing HPV)
      • 293—human embryonic kidney
      • a, c, e, g, i—cotransfection of pGL2 with indicated siRNA
      • b, d, f, h, j—cotransfection 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.
    • 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/animation.htm
    • 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
    • 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
      • Pools—multiple 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.
    • 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, O’Farrlel 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
    • Chemical Genetic Approach Stockwell, B.R. (2000) Nature Reviews Genetics 1 , 116-125. Chemical Genetic Screening add compounds select clone of interest large bud identify protein target protein LB1
    • Compounds that Selectively Kill Tumor Cells normal human cells engineered tumor cells cmpd8 cmpd8
    • Erastin-induced Cell Death in Tumor Cells Erastin
      • 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.
    • Engineered Human Tumor Cells hTERT (protein component of telomerase) RAS V12 SV40 Large T oncoprotein SV40 Small T oncoprotein In collaboration with William Hahn and Stephen Lessnick, Dana-Farber Cancer Institute primary cell tumor cell
    • Sources of Small Molecules 1. Combinatorial Library - 20,000 compounds 2. NCI diversity set - 1,990 compounds 3. Annotated Compound Library - 2,036 compounds 24,026 compounds Test for > 50% inhibition in tumor cells 380 compounds > 4-fold tumor cell selectivity vs normal cells 9 compounds IC 50 primary IC 50 tumor
    • An Automated Process for Cell-Based Screening Chemical Libraries Plate Barcoding Plate Replication Sterile Cell Dispensing Assay Execution Data Analysis Root DE, Kelley BP Stockwell BR (2003) Detection of Systematic Errors in Spatial Arrays. J Biomolecular Screening ,8 (4) 393-398
    • Genotype-Selective Lethality
    • Identification of Erastin-Binding Proteins B1 A6 B1 = immobilized inactive analog A6 = immobilized active analog erastin-specific binding protein In collaboration with Prolexys
      • Voltage-Dependent Anion Channel
      • Prohibitin
      • Ribophorin 1
    • 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)
    • Erastin-induced Cell Death in Tumor Cells Erastin
      • 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.
    • Acknowledgements Collaborators William Hahn & Steve Lessnick Prolexys Pharmaceuticals Funding Burroughs Wellcome Fund National Cancer Institute Erastin Project Brent Stockwell Sonam Dolma Steve Flaherty Allison Martino