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

2. 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

3. 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

4. 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

5. 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

6. 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

7. PTGS or co-suppression in plants is also mediated by dsRNA
An Introduction to RNAi > Part I > 1. History
Figure: Montgomery & Fire A., 1998

8. RNA interference a common phenomena
An Introduction to RNAi > Part I > 1. History
Table: Cogoni C. et al., 1998

9. 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

10. 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

11. 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.

12. 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., R2D2 picture courtesy: Lucas films.

13. 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.

14. 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

15. 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

16. 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

17. 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.

18. Biological functions of RNAi
An Introduction to RNAi > Part I > 5. Function
Table: Denli A.M. & Hannon G. J., 2003

19. Part II: Technical and medical applications of RNAi
An Introduction to RNAi > Part II

20. 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

21. 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

22. 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

23. 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.

24. 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.

25. 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

26. 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

27. 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.

28. 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

29. 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.

30. 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

31. RNAi animation on Nature reviews:
RNAi Resources
RNAi animation on Nature reviews:
Company websites
e.g. Ambion: siRNA and RNAi resource
An Introduction to RNAi > RNAi Resources
Figure: Nature website.

32. 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

33. Thank you
An Introduction to RNAi > The end

34. 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