Title: RNA interference RNAi ChingPing Tseng, Ph'D' Associate Professor Graduate Institute of Medical Biote
1RNA interference (RNAi) Ching-Ping
Tseng, Ph.D.Associate ProfessorGraduate
Institute of Medical Biotechnology Chang Gung
University
22006 Nobel Prize in Physiology and Medicine RNA
interference
Craig C. Mello
Andrew Fire
3Learning objectives
The discovery of RNA interference The origin and
nature of small interfering RNA The mechanism of
RNA interference in gene silencing Application of
RNA interference technique The origin and nature
of micro RNA The mechanism of micro RNA in gene
silencing Micro RNA and human diseases
4RNA interference (RNAi)
Initially defined as a technique in which
experimental introduction in C. elegans of dsRNA
homologous to a target mRNA led to degradation of
the targeted mRNA. More broadly defined as
degradation of target mRNAs by dsRNA
5Small interfering RNAs (siRNAs) 22 to 25 nt
RNAs derived from processing of linear dsRNA.
siRNAs assemble in complexes termed RISCs
and target homologous RNA sequences for
endonucleolytic cleavage. Synthetic siRNAs
also incorporate in RISCs and cleave
homologous RNA sequences MicroRNAs (miRNAs) are
endogenous 22 nt RNAs that can play
important regulatory roles in animals and
plants by targeting mRNA for cleavage or
translation repression
6The discovery of RNA interference
7Rich Jorgensen DNA Plant Technology Oakland,
CA University of Arizona Tucson, AZ
Chalcone synthase Anthocyanin pigments production
Co-suppression
Plant Cell 2279-289, 1990
8David Baulcombe Norwich
Express gene from the potato virus X in tobacco
plants
EMBO J 163675-3684, 1997
9Giuseppe Macino and Carlo Cogoni La Sapienza
University Rome, Italy
Neurospora crassa Expression of gene for orange
carotenoid pigment
Qde-1, qde-2, and qde-3 are crucial to the
quelling process
Leeuwenhock 65205-209, 1994
102006 Nobel Prize in Physiology and Medicine RNA
interference
Craig C. Mello
Andrew Fire
11Double-stranded RNA was more effective at
producing interference than was either strand
individually
- unc22 encodes an abundant but nonessential
myofilament protein - Decreases in unc-22 activity produce an
increasingly severe twitching phenotype
- dsRNA segments corresponding to various intron
and promoter sequences did not produce
detectable interference - consistent with interference at a
post-transcriptional level
12Injection of dsRNA produces a pronounced decrease
or elimination of the endogenous mRNA transcript
in situ hybridization of mex-3
Post-transcriptional gene silencing (PTGS) by
double-stranded RNA (dsRNA, or RNA interference
(RNAi))
13Richard Carthew University of Pittsburgh Pittsburg
, PA
Drosophila embryos Microinjection, gene gun Carry
a stretch of DNA containing an inverted repeat of
the gene to be silenced frizzled2 and wingless
for wing development
Cell 951017-1026, 1998
14Double-stranded RNA can be introduced
experimentally to silence target genes of
interest.
Gregory J. Hannon NATURE 2002,418244-251
15The pathways begins with long dsRNA, either a
biomolecular duplex or an extended hairpin, that
either is artificially introduced into the cell
or animal during a gene knockdown experiment or
is naturally generated from sense and antisense
genomic transcripts, or perhaps from the activity
of a cellular RNA-dependent RNA polymerase (found
in plants, fungi, and nematodes, but not flies or
mammals) or as an intermediate of viral
replication
16Origins of dsRNA
17RISC (RNA-induced silencing complex) DICER
18RNase III family members are among the few
nucleases that show specificity for dsRNA
- Dicer, Drosophila CG4792 contains two RNase III
signatures and an amino-terminal helicase domain - Drosha, a Drosophila enzyme that contains two
RNase III motifs and a dsRBD - Homeless, DExH box helicase
- DCR-2 processing long dsRNA
- DCR-1 processing miRNA
- Drosha nucleus, process to pre-miRNA
Bernstein et al. Nature 2001, 409363-366.
19RISC (RNA-induced silencing complex) Multisubunit
nuclease that directs target RNA destruction in
RNA interference. The core components of RISCs
are siRNAs and Argonaute proteins. Argonaute
Family of proteins that contain two conserved
domains termed PAZ and PIWI. Multiple paralogs
are typically present in each organsim. Argonaute
proteins are crucial for the maturation and
function of miRNA and are essential for RNA
interference. PAZ domain bind small RNAs PIWI
domain homology to RNAse H,
catalyzes mRNA cleavage in human
20The actions of small silencing RNAs
21RNA interference enters the nucleus
RNAi and DNA methylation/chromatin remodeling
22RNA interference enters the nucleus
RNAi and DNA methylation/chromatin remodeling
23Amplification of RNA interference
Nishikura et al. Cell 2001,107415-418
24RNA interference can spread systemically through
a plant
25RNA interference in mammalian cells
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28Focus on RNA interference
Well before RNA interference would be used in
mammalian systems, it was used to enhance the
color of petunias. An artistic interpretation of
RNA interference by Erin Boyle.
29Delivery of siRNAs
Exogenous delivery of synthetic siRNA Endogenous
expression of sh/siRNA Electroporation Microinje
ction Transfection Plasmid- or virus-based
expression cassettes
30DNA vector-based RNAi technology
31DNA vector-based RNAi technology
32miRNA-based vector
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36Genome-wide screening for gene function
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40RNase III family members are among the few
nucleases that show specificity for dsRNA
ATP-dependent for Drosophila but not human
Bernstein et al. Nature 2001, 409363-366.
41A Model for the RNAi Pathway
Nykanen et al. Cell 2001,107309-321