Transcript C. elegans
An Introduction to RNA Interference Liang Huang An Introduction to RNAi > Home Contents Part I: The discovery and mechanism of RNAi • • • History Mechanism: Current model, siRNA, Dicer, the RISC, other adapters. Systemic and inherent RNAi: RdRP, amplification of RNAi, secondary siRNA, SID-1. • MicroRNAs • Biological Functions: Anti-viral, genome defense, heterochromatin, gene regulation (miRNA). Part II: Technical and medical applications of RNAi • • RNAi as a research tool Therapeutic potentials of RNAi An Introduction to RNAi > Contents RNA interference phenomena in plants and fungi Introducing chalcone Synthase transgenes • Increasing gene dosage causes silencing (Co-supperssion) • Virus induced PTGS • Quelling in fungi: a diffusible trans-acting factor is responsible for PTGS An Introduction to RNAi > Part I > 1. History Figure: Jorgensen RA et al., 1996; Table: Cogoni C. & Macino G., 2000 The Guo and Kemphues’ s experiment (1995) Antisense RNA In vitro synthesized RNA Sense RNA (Control) Inject to intothe the worm wor gonads Antisense RNA A mG Sense RNA (Control) dsRNA AAAAAAAAA Par-1 mRNA Embryonic lethality An Introduction to RNAi > Part I > 1. History mG AAAAAAAAA Par-1 mRNA Embryonic lethality ??? Fire and his colleague's Discovery (1998) RNA interference • Gene silencing induced by dsRNA but not ssRNA • Extremely Specific • Can pass to the next generation in C. elegans • Has the ability to Cross cellular boundaries in C. elegans An Introduction to RNAi > Part I > 1. History Figure: Fire A. et al., 1998 Fire and his colleague's Explanation (1998) RNA produced by bacteriophage RNA polymerase in vitro contains double-stranded RNA. It is dsRNA, not ssRNA, that causes the interference. An Introduction to RNAi > Part I > 1. History Figure: Montgomery & Fire A., 1998 PTGS or co-suppression in plants is also mediated by dsRNA An Introduction to RNAi > Part I > 1. History Figure: Montgomery & Fire A., 1998 RNA interference a common phenomena An Introduction to RNAi > Part I > 1. History Table: Cogoni C. et al., 1998 Components of RNAi machinery Many components of RNAi machinery have been identified through genetic screening for RNAi defective mutants and through biochemical studies using cell extracts (e.g. Drosophila embryo extract). An Introduction to RNAi > Part I > 1. History Table: Cerutii H., 2003 Characteristics of RNAi • Epigenetical: No DNA sequence change is involved, even though the RNAi treats can be pass to the next generation • Post-transcriptional: RNA transcription is not affected (Exception to the rule: siRNA induced chromatin modification) • Induced by a trans-acting factor other than DNA or protein: RNA • Potent: Very few molecules is required. In C. elegans, it is estimated that 2 molecules of dsRNA is enough • Homology: Sequence is strictly complementary. Not a single mismatch is allowed. • Well conserved across kingdoms An Introduction to RNAi > Part I > 2. Mechanism Overview of current model for RNAi Aberrant RNA, shRNA or viral RNA RITS CH3 Chromatin modification (Methylation) Heterochromatin formation and TGS An Introduction to RNAi > Part I > 2. Mechanism Figure adapted from: Dykxhoorn D.M., et al., 2003. siRNA & Dicer siRNAs • About 21-26nt in length • 5’ Phosphate and 3’ OH groups • 2-3nt overhang at 3’ ends • Products of Dicer Dicer • RNase III family endonuclease • Most Dicers contain a PAZ domain: function as a RBD (PAZ: Piwi, Argonaute, Zwille) Number of Dicers: • Many organisms have several Dicers: processing dsRNA from various sources Drosophila: 2 C. elegans: 1 • Involves in siRNA incorporation into RISC • Dicer adaptor proteins: PAZ containing proteins; dsRNA-recognition proteins An Introduction to RNAi > Part I > 2. Mechanism Mammals: 1 Arabidopsis: 4 Figure adapted from: Dykxhoorn D.M., et al., 2003; Meister G. & Tuschl T., 2004. R2D2 picture courtesy: Lucas films. Assembly of RISC RNA-induced Silencing Complex • Components of RISC: e.g. Drosophila RISC - Ago2, VIG, dFXR, TSN • Argonaute protein contains a PAZ domain and a PIWI domain. • Assemble of RISC involving Dicer and Dicer adaptor • Putative RNA helicase is involved Number of Argonatues: Drosophila: 5 C. elegans: >20 Human: 8 Arabidopsis: 10 S. pombe: 1 • Incorporation of antisense siRNA activates RISC An Introduction to RNAi > Part I > 2. Mechanism Figure adapted from: Meister G. & Tuschl T., 2004. Systemic and inherent RNAi Systemic and inherent RNAi is observed in plants and C. elegans, i) RNAi induced in one place can spread to the other places of the body; ii) The RNAi treats can pass through several generations. • Amplification of RNAi (next two slides) • Transmission of the trans-acting factors (siRNA, or antisense RNA) • In C. elegans: SID-1 • Heterochromatin formation An Introduction to RNAi > Part I > 3. Systemic RNAi Secondary siRNA • Secondary siRNA • Transitive RNAi Unc-22 GFP GFP dsRNA Unc-22 dsRNA Endogenous unc-22 silenced GFP Unc-22 GFP dsRNA GFP dsRNA Endogenous unc-22 not silenced An Introduction to RNAi > Part I > 3. Systemic RNAi Figure adapted from: Hannon et al., 2003 Amplification of RNAi by RdRP • RNA-dependent RNA Polymerase (RdRP) • Random Degradative PCR An Introduction to RNAi > Part I > 3. Systemic RNAi Figure adapted from: Cerutii H., 2003, Hannon et al., 2003 MicroRNAs • Processed by Dicer and Drosha • Structurally similar to siRNA, partial complementary • RISC • More than 200 miRNA genes are identified. Play different roles in the cell. Dicer & Drosha An Introduction to RNAi > Part I > 4. MicroRNAs Figure adapted from: Dykxhoorn D.M., et al., 2003; Table: Ambros V., 2004. Biological functions of RNAi An Introduction to RNAi > Part I > 5. Function Table: Denli A.M. & Hannon G. J., 2003 Part II: Technical and medical applications of RNAi An Introduction to RNAi > Part II RNAi as a tool in Biological Sciences Researches • Effective and fast Advantages • Specific but also flexible: One specific gene/allele or a gene family • Can be used in genome-wide studies • Loss of function • Less informative Disadvantages • Less effective in some tissues • Residue gene expression • Based on known sequence and gene structure An Introduction to RNAi > Part II > 1. RNAi as a tool Problems in mammalian cells dsRNA >30 bp Problem: Long dsRNA induces non-specific cellular responses activation activation Interferon PKR activation 2’, 5’-OAS Solution: Use siRNA instead eIF2α eIF2α P 2’, 5’ A RNase L Inhibition of Translation ATP RNase L non-specific mRNA degradation An Introduction to RNAi > Part II > 1. RNAi as a tool Figure adapted from: Hannon et al., 2003 RNAi as a tool in Biological Sciences Researches (cont.) • siRNA Design • Producing the initiator • Delivering the initiator An Introduction to RNAi > Part II > 1. RNAi as a tool siRNA design • mRNA secondary structure • RNA biding Proteins • Base composition • Homology An Introduction to RNAi > Part II > 1. RNAi as a tool Figure: Dykxhoorn D.M., et al., 2003. siRNA synthesis B . In vivo generated A. In vitro synthesized An Introduction to RNAi > Part II > 1. RNAi as a tool Figure: Dykxhoorn D.M., et al., 2003. siRNA Delivery C. elegans & Drosophila • Long dsRNA: Injection, Soaking (Feeding) • In vivo expression (Pol II or Pol III vectors) Plants • Particle Bombardment • Transformation • Agroinfiltration • Virus Mammalian cells • siRNA: Transfection • Virus An Introduction to RNAi > Part II > 1. RNAi as a tool An example: Using RNAi for functional genomic studies cDNA lib. S2 cell dsRNA Incubate with FITClabelled E. coli or S. aureus Trypan blue S2 cell An Introduction to RNAi > Part II > 1. RNAi as a tool Fluorescence-activated cell sorting (FACS) An example: Using RNAi for functional genomic studies (Cont.) An Introduction to RNAi > Part II > 1. RNAi as a tool Figure:Rämet M., et al., 2002. Using RNAi for new drug discovery and Human therapies • A tool for study disease gene function • Direct degradation of RNA virus • Inhibit the synthesis of viral proteins • Control host proteins that are required for virus infection and survival • Specifically target oncogenes • Inducing TGS • Cell division arrest • Inducing cell death An Introduction to RNAi > Part II > 2. Medical applications of RNAi An Introduction to RNAi > Part II > 2. Medical applications of RNAi Table: Dykxhoorn D.M., et al., 2003. Future directions • RNAi mechanism • Functions of siRNA / miRNA • Inducible RNAi • Sysmetic RNAi in other organisms • Practical RNAi therapies An Introduction to RNAi > Outlook RNAi Resources • RNAi animation on Nature reviews: http://www.nature.com/focus/rnai/animations/index.html • Company websites e.g. Ambion: siRNA and RNAi resource http://www.ambion.com/techlib/resources/RNAi/index.html An Introduction to RNAi > RNAi Resources Figure: Nature website. Acknowledgements • My advisor: Dr. Tanda • Dr. Lee • My Labmates and our Lab. Technician • My wife An Introduction to RNAi > Acknowledgement Thank you An Introduction to RNAi > The end MCB 741 Spring 2005 Presentations Date Speaker Subject April 12 Liang Huang Introduction April 19 Raja Anupam Therapeutic RNAi April 26 Akwasi Agyeman RNAi in C. elegans May 3 RNAi in mammals Min Liang May 10 Yanli Ding RNAi in Drosophila May 17 Martin Schmerr Animal miRNAs