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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.
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MicroRNAs
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Biological Functions: Anti-viral, genome defense,
heterochromatin, gene regulation (miRNA).
Part II: Technical and medical applications of RNAi
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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
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Increasing gene dosage causes silencing (Co-supperssion)
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Virus induced PTGS
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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
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Secondary siRNA
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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
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RNA-dependent RNA Polymerase (RdRP)
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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