RNA interference: new tool,
ancient immune system
Bergen lecture – April 21st 2005
Torgeir Holen, CMBN
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Aims of this talk
Part I: To give a short introduction to the newly discovered
immune system RNA interference (RNAi) in various species
Part II: To look at some practical limitations to knocking
down mRNA gene expression by RNAi & siRNA, and some
recent solutions...
Part III: siRNA is already old hat – what’s going on now...?
short RNA : DNA/chromatin interactions...
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Part I: Gene silencing exist in
many multicellular organisms
roundworm
Petunias
Transgenic Petunias C. elegans
Transgenic potatoes
resist virus infection
fungus
Neurospora
3
fruitfly
Drosophila
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Gene silencing go back to the
earliest epochs of life on Earth

This ancient immune
system seems conserved
over 1.5 billion years
...thus we can learn about
different aspects of gene
and RNA silencing wherever
it is easiest... plants...
roundworms... flies...
human cell lines...
Wang, Kumar & Hedges, 1999
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A brief history of RNA silencing

1998: Andrew Fire and colleagues
show that double-stranded RNA
induce gene silencing in C. elegans
• 1999: Hamilton & Baulcombe find
small RNA where genes are silenced in
plants
• 2000: Greg Hannon and colleagues
isolate Dicer, the siRNA producing
RNase-III enzyme in Drosophila
• 2001: Thomas Tuschl and
colleagues show that synthetic
small RNA introduced into human
cells can silence human genes
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Plasmid produced siRNA
•Brummelkamp, 2002; Paddision, 2002; Miyahishi, 2002; Paul, 2002; Sui, 2002;
Lee, 2002; Jacque, 2002; Yu, 2002; Yang, 2002
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The PRK/interferon response




immune-system virus-response system in
mammalian cells
activated by long dsRNA
mechanism: shuts down all translation by by
phosphorylating eIF2-alpha (see Williams,
Oncogene, 1999)
the siRNA break-through of Thomas Tuschl
consisted of the PKR/interferon response not
being activated (Elbashir et al, Nature, 2001;
also shown by Natasha Caplen, PNAS, 2001)
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Part II: Limits to siRNA activity
- some other limitations to these great, but very new, tools
1.
siRNA exhibit position effects – some target positions superior to others
2.
siRNA activity fades out – in our case 3-4 days after transfection
3.
siRNA activity can be blocked by competition with less active siRNA
4.
Some siRNA have great tolerance for chemical modifications
5.
Some siRNA tolerate mutations
6.
Main problem of RNAi-field today: delivery, in vitro & in vivo
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Finding good target positions:
TF-luciferase-reporter assay
- Holen et al (Nucleic Acids Research, 2002).
- Tissue Factor (TF), is the principal coagulation trigger and
has been implicated in cardiac diseases. TF is also involved in
angiogenesis and metastasis of cancer.
Fusion Reporter Construct
...
TF (47-951)
LUC (37-2255)
pTRE
...





pTRE
Fen1, Aqp4 and GlnS Constructs
pTRE
Fen1/Aqp4/GS
LUC (37-2255)
pTRE
...





...
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Why the position effects?
hTF562i
hTF478i
hTF372i
hTF167i
PSK314i
HeLa
mRNA
AGGUGGCCGGCGCUUCAGGCACUACAAAUACUGUGGC
hTF158i
AGGUGGCCGGCGCUUCAGGTT
hTF161i
UGGCCGGCGCUUCAGGCACTT
hTF164i
CCGGCGCUUCAGGCACUACTT
hTF167i
hTF562i
hTF478i
hTF372i
hTF167i
PSK314i
293
GCGCUUCAGGCACUACAAATT
hTF170i
CUUCAGGCACUACAAAUACTT
hTF173i
CAGGCACUACAAAUACUGUTT
hTF176i
hTF562i
hTF478i
hTF372i
hTF167i
PSK314i
GCACUACAAAUACUGUGGCTT
Cos-1
Cotransfections with siRNAs targeting sites around hTF167
hTF929i
hTF562i
hTF478i
hTF459i
hTF372i
hTF256i
hTF167i
hTF77i
PSK739i
PSK566i
PSK546i
PSK314i
HaCaT
TF-LUC/RLUC
Normalised
Re lativ e hTF-Fluc/Rluc
120
100
80
60
40
20
0
PSK314i
20
40
60
80
hTF158i
hTF161i
hTF164i
hTF167i
hTF170i
hTF173i
100 120 140
Normalised TF-LUC/RLUC
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hTF176i
Knock-downs must be verified...
PSK739i
PSK566i
PSK546i
PSK314i
hTF562i
hTF478i
hTF372i
hTF167i
mock
- position effect consistent also in Northern assays, protein
assays and coagulation assays…
Inhibition of TF mRNA, procoagulant activity
Protein
&andCoagulation
antigen by siRNA
TF
GAPDH
TF
hTF176i
hTF173i
hTF170i
hTF167i
hTF164i
hTF161i
hTF158i
mock
Northerns
Normalised TF expression
120
100
80
mRNA
procoag
antigen
60
40
20
0
mock
hTF167i
hTF372i
PSK314i
Legend: mRNA (filled bars), procoagulant activity
(dotted bars) and TF protein (hatched bars)
GAPDH
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Some recent computational
solutions to position effects:






Khvorova, Cell, 2003: 5’end of active antisense
unstable...
Ui-Tei et al, Nucleic Acids Research, 2004:
(i) A/U at the 5' end of the antisense strand
(ii) G/C at the 5' end of the sense strand
(iii) at least five A/U residues in the 5' terminal one-third of the
antisense strand
(iv) the absence of any GC stretch of more than 9 nt in length.
siRNAs opposite in features with respect to the first three
conditions give rise to little or no gene silencing in mammalian
cells
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RNAi silencing sets in
slowly…
…why?
Normalised TF expression
Normalised TF/GAPDH mRNA
Time-dependence of hTF167i effect
100
procoag
80
mRNA
60
LUC
40
20
0
48 h
72 h
96 h
120 h
Measurements at 4 h, 8 h, 24 h, 48 h
…and fades out even more slowly,
but steadily…
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Tolerance for chemical modifications
P1+1
5’-g*c-gcuucaggcacuaca-a-a-u*a-3’
3’-g*c-c-g-cgaaguccgugaugu-u*u-5’
P0+2
5’-g-c-gcuucaggcacuaca-a-a*u*a-3’
3’-g*c*c-g-cgaaguccgugaugu-u-u-5’
P2+2
5’-g*c*gcuucaggcacuaca-a-a*u*a-3’
3’-g*c*c-g-cgaaguccgugaugu*u*u-5’
P2+4
5’-g*c*gcuucaggcacuaca*a*a*u*a-3’
3’-g*c*c*g*cgaaguccgugaugu*u*u-5’
M1+1
5’-GcgcuucaggcacuacaaauA-3’
3’-GccgcgaaguccgugauguuU-5’
M0+2
5’-gcgcuucaggcacuacaaaUA-3’
3’-GCcgcgaaguccgugauguuu-5’
M2+2
5’-GCgcuucaggcacuacaaaUA-3’
3’-GCcgcgaaguccgugauguUU-5’
M2+4
5’-GCgcuucaggcacuacaAAUA-3’
3’-GCCGcgaaguccgugauguUU-5’
Normalized TF/GAPDH mRNA
Normalised TF/GAPDH mRNA
120
100
80
60
40
20
0
mock
wt
A1+1
5’-GcgcuucaggcacuacaaauA-3’
3’-GccgcgaaguccgugauguuU-5’
A0+2
5’-gcgcuucaggcacuacaaaUA-3’
3’-GCcgcgaaguccgugauguuu-5’
P1+1 P0+2 M1+1 M0+2 A1+1 A0+2
P2+2 P2+4 M2+2 M2+4
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Inhibition of cholesterol
synthesis by siRNA: A recent
study by Alnylam Inc

"...Administration of chemically modified
siRNAs resulted in silencing of the apoB
messenger RNA in liver and jejunum,
decreased plasma levels of apoB protein,
and reduced total cholesterol. "
Soutschek et al, Nature, 2004
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An siRNA mutation study
**** * ** * *
5'-GCGCUUCAGGCACUACAAAUA
GCCGCGAAGUCCGUGAUGUUU-5'
Normalized
TF/GAPDH mRNA
mRNA
TF/GAPDH
Normalised
120
100
80
60
40
20
s1
6
s1
3
s1
1
s1
0
s7
s4
s3
s2
s1
t
w
ds
7/
1
ds 0
10
/1
ds 1
10
/1
ds 3
10
/1
6
m
oc
k
0
Amarzguioui M, Holen T, Babaie E, Prydz H. Tolerance for
mutations and chemical modifications in a siRNA. Nucleic
Acids Res. 2003 Jan 15;31(2):589-95.
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Other genes possibly
targeted by laminB2
CLSTN2
5'-AAGAGGAGGAAGAAGCCGAGG-3'
|||||||||| |||||||||
3'-UUCUCCUCCUCCUUCGGCUCA-5'
HS6ST3
5'-AAGAGGAGGAGGAAGACGAGC-3'
||||||||||||||| ||||
3'-UUCUCCUCCUCCUUCGGCUCA-5'
NM_015897
5'-AAGAGGAGGAGGAAGACGAGG-3'
||||||||||||||| ||||
3'-UUCUCCUCCUCCUUCGGCUCA-5'
The dangers of siRNA in
functional genomics:
“…the two other lamins, B1 and B2,
are now identified as essential
proteins” (Harborth et al, JCS, 2001)
NM_015355
5'-ACUCGGCCUCCUCCUCCUCCU-3'
||||||| ||||||||||| |
3'-UGAGCCGAAGGAGGAGGAGAA-5'
ATBF1
5'-AAGAGGAGGAGGAAGACGAGG-3'
||||||||||||||| ||||
3'-UUCUCCUCCUCCUUCGGCUCA-5'
SPTB
5'-AAGAGGAGGAGGAAACAGAGU-3'
|||||||||||||| | ||||
3'-UUCUCCUCCUCCUUCGGCUCA-5'
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The delivery problem
- comparing different transfection agents
Lipofectamine2000, Lipofectamine, Oligofectamine and RNAifect…
140
120
100
80
60
40
20
as
-1
6
hT 7
F1
67
as
-P
i
SK
41
1
as
-1
6
hT 7
F1
67
as
-P
i
SK
41
1
as
-1
6
hT 7
F1
67
as
-P
i
SK
41
1
as
-1
6
hT 7
F1
67
as
-P
i
SK
41
1
0
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Indirect delivery in vivo
- Tissue Factor in cancer metastasis
Day 10

Day 15
set-up: metastatic B16 cells transfected
with siRNA against TF, then injected into
tail-vein of mice
tumors will then colonize lungs of mouse
mTF223i
D
mTF223i
hTF167i

mock
hTF167i
mTF
GAP
Day 20
mTF321i
mTF223i
hTF167i
20
Mohammed Amarzguioui, Qian
Peng, Torgeir Holen, Vlada
Vasovic, Eshrat Babaie, Jahn
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M. Nesland
& Hans Prydz
RNAi delivery by virus




Lentiviruses delivery especially interesting for
delivery to neuronal cells.
An et al, Human Gene Theraphy, 2003
Stewart et al, RNA, 2003
Paddison PJ, Silva JM, Conklin DS, Schlabach M, Li
M, Aruleba S, Balija V, O'Shaughnessy A, Gnoj L,
Scobie K, Chang K, Westbrook T, Cleary M,
Sachidanandam R, McCombie WR, Elledge SJ,
Hannon GJ. A resource for large-scale RNAinterference-based screens in mammals.
Nature. 2004 Mar 25;428(6981):427-31.
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Transgenic RNAi: mice
expressing siRNA

Carmell, Rosenquist et
al, Nature Structural &
Molecular Biology,
2003
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Regulatable knock-downs
What of it? Can’t the Cre/LoxP system or other
conditional knock-outs do the same?

From Matsukura et al (Nucleic Acids Research, 2003)
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Part III: The possible link between RNA
and DNA silencing in higher organisms
1)
2)
3)
RNA silencing, more than siRNA
DNA & chromatin regulation: still unsolved...
Three cases of RNA-DNA silencing link
a)
b)
c)
d)
e)
dsRNA-induced DNA methylation in plants
transgene DNA silencing in Drosophila
centromere silencing in budding yeast (S. pombe)
X-chromatin silencing in humans...
recently: chromatin silencing induced by short RNA in
human cells
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More small RNA exist
• stRNA (short temporal RNA, also called micro-RNA,
miRNA) inhibit mRNA translation (Lee & Ambros,
1993; Reinhart, 2000; Lee, 2001; Lau, 2001; LagosQuintana, 2001; Llave, 2002; Lim, 2003)
• microRNA comes from microGenes, transcripted
as ~70 nt hairpins, and processed (as is shRNA) to
short ~21-mer RNAs...
• latest research hotspot: heterochromatic siRNA
might affect centromeres in budding yeast
(S.pombe) (Reinhart, 2002; Volpe, 2002)
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Double-stranded RNA
cause silencing in plants

PTSVd viroids (Wassenegger, Cell, 1994)
• PTSVd can lead to methylation of a
target down to 30 bp of DNA (Pelissier
& Wassenegger, RNA, 2000)
HOW???
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Lehninger,
1993
X-chromosome inactivation centre (Xic)

Xic (450 kb region) can be transferred to chromosome 12, resulting in
gene silencing, hypoacetylation, histone methylation (His3mLys9),
delayed DNA replacation and RNA coating...
(Lee & Jaenisch, Nature, 1997)
Lee, Cell, 2000
27Lewin, 200029/05/2016
Summary: What is the
mechanism?
Is the proposed mechanism
right?
...and does it exist in
higher organisms?
Allshire, Science, 2002
Nobody knows... yet
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Two recent papers find
chromatin modifications in
mammalian cells


Morris KV, Chan SW, Jacobsen SE, Looney DJ.
Small interfering RNA-induced transcriptional
gene silencing in human cells. Science. 2004
Aug 27;305(5688):1289-92. Epub 2004 Aug 05.
Kawasaki & Taira. Induction of DNA methylation
and gene silencing by short interfering RNAs in
human cells. Nature. 2004 Sep
9;431(7005):211-7. Epub 2004 Aug 15.
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