An Introduction to RNA Interference Liang Huang An Introduction to RNAi > Home

Part I: The discovery and mechanism of RNAi 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 Petunias, try to deepen the purple color of the flowers. Both endogenous and exogenous copies are silenced. Neurospora. 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

to the wor A The Guo and Kemphues’ s experiment (1995) Antisense RNA In vitro synthesized RNA Sense RNA (Control) Inject into the worm gonads to the wor Antisense RNA Sense RNA (Control) dsRNA A mG AAAAAAAAA mG AAAAAAAAA Par-1 mRNA Par-1 mRNA Embryonic lethality Embryonic lethality ??? An Introduction to RNAi > Part I > 1. History

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. Random and ectopic transcripts. 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 Dicer 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) Many organisms have several Dicers: processing dsRNA from various sources Involves in siRNA incorporation into RISC Dicer adaptor proteins: PAZ containing proteins; dsRNA-recognition proteins Number of Dicers: Drosophila: 2 C. elegans: 1 Mammals: 1 Arabidopsis: 4 An Introduction to RNAi > Part I > 2. Mechanism Figure adapted from: Dykxhoorn D.M., et al., 2003; Meister G. & Tuschl T., 2004. R2D2 picture courtesy: Lucas films.

RNA-induced Silencing Complex 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 Incorporation of antisense siRNA activates RISC Number of Argonatues: Drosophila: 5 C. elegans: >20 Human: 8 Arabidopsis: 10 S. pombe: 1 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 Unc-22 GFP dsRNA GFP dsRNA Unc-22 dsRNA GFP dsRNA Endogenous unc-22 silenced 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 Specific but also flexible: One specific gene/allele or a gene family Can be used in genome-wide studies Advantages Loss of function Less informative Less effective in some tissues Residue gene expression Based on known sequence and gene structure Disadvantages 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 Solution: Use siRNA instead activation activation Interferon activation PKR 2’, 5’-OAS P eIF2α eIF2α 2’, 5’ A ATP RNase L RNase L Inhibition of Translation non-specific mRNA degradation An Introduction to RNAi > Part II > 1. RNAi as a tool Figure adapted from: Hannon et al., 2003

Producing the initiator 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

mRNA secondary structure RNA biding Proteins Base composition Homology 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 Plants Mammalian cells 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 FITC-labelled E. coli or S. aureus Trypan blue Fluorescence-activated cell sorting (FACS) S2 cell An Introduction to RNAi > Part II > 1. RNAi as a tool

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 An Introduction to RNAi > Part II > 2. Medical applications of RNAi Table: Dykxhoorn D.M., et al., 2003.

Functions of siRNA / miRNA Inducible RNAi Future directions RNAi mechanism Functions of siRNA / miRNA Inducible RNAi Sysmetic RNAi in other organisms Practical RNAi therapies An Introduction to RNAi > Outlook

RNAi animation on Nature reviews: 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.

My Labmates and our Lab. Technician 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 Min Liang RNAi in mammals May 10 Yanli Ding RNAi in Drosophila May 17 Martin Schmerr Animal miRNAs