Post – transcriptional gene

silencing

 PTGS in plants (Co-suppression)

 Quelling in fungi

 RNAi in animals
Co-suppression
 What is RNAi
A novel way to regulate gene expression

2006
Nobel
Prize
 What is RNAi
RNA interference (RNAi) is a highly evolutionally
conserved process of post-transcriptional gene
silencing (PTGS) by which double stranded RNA
(dsRNA), when introduced into a cell, causes
sequence-specific degradation of homogolous
mRNA sequences.

It was first discovered in 1998 by

Andrew Fire and Craig Mello in
the nematode worm
Caenorhabditis elegans and later
found in a wide variety of
organisms, including mammals.
 Mechanism of RNA interference
 A. On entering the cell, long dsRNAs act
as a trigger of RNAi process.
 B. It is first processed by the RNAse III
enzyme Dicer in an ATP-dependent
reaction.
 C. Dicer processes dsRNAs into 21-23 nt
short interfering RNA (siRNA) with 2-nt 3'
overhangs. siRNA can also be synthesized
outside the cell and then be introduced into
a cell.
 D. The siRNAs are incorporated into the
RNA-inducing silencing complex (RISC)
which consists of an Argonaute (Ago)
protein as one of its main components.
Ago cleaves and discards the passenger
(sense) strand of the siRNA duplex leading
to activation of the RISC.
 E and F. The remaining guide (antisense)
strand of the siRNA guides RISC to its
homologous mRNA, resulting in the
endonucleolytic cleavage of the target mRNA
 1995
 First noticed that sense RNA was as
effective as antisense RNA for
suppressing gene expression in worm
 Guo S, and Kemphues KJ.
 C. elegans
 Guo S, and Kemphues KJ.
par-1, a gene required for establishing polarity in C. elegans embryos, enc
. Cell. 1995 May 19;81(4):611-20.
First report…. No clue
Cell lineages and early divisions
in C. elegans
Scoring for Embryonic lethality
in RNA Injections
 1998
 First described RNAi phenomenon in C.
elegans by injecting dsRNA into C.
elegans which led to an efficient
sequence-specific silencing and coined
the term "RNA Interference".
 Fire et al.
 C. elegans
 Fire et al. Potent and specific genetic interference by double-stranded RNA in

Caenorhabditis elegans. Nature. 1998 Feb 19;391(6669):806-11.

Effects of sense, antisense and mixed RNAs on
progeny of Injected animals
 Analysis of RNA-interference effects in individual cells

Fluorescence micrographs show progeny of injected animals from GFP-reporter strain PD4251. a–c, Progeny
of animals injected with a control RNA (double-stranded (ds)-unc22A). a, Young larva, b, adult, c, adult body
wall at high magnification. These GFP patterns appear identical to patterns in the parent strain, with prominent
fluorescence in nuclei (nuclear-localized GFP–LacZ) and mitochondria (mitochondrially targeted GFP). d–f,
Progeny of animals injected with ds-gfpG. Only a single active cell is seen in the larva in d, whereas the entire
vulval musculature expresses active GFP in the adult animal in e. f, Two rare GFPpositive cells in an adult:
both cells express both nuclear-targeted GFP–LacZ and mitochondrial GFP. g–i, Progeny of animals injected
with ds-lacZL RNA: mitochondrial-targeted GFP seems unaffected, while the nuclear-targeted GFP– LacZ is
absent from almost all cells (for example, see larva in g). h, A typical adult, with nuclear GFP–LacZ lacking in
almost all body-wall muscles but retained in vulval muscles. Scale bars represent 20 mm.
 Effects of mex-3 RNA interference on levels of the endogenous mRNA

Enbryo
Negative from
control
Un injected
parent

AS mex 3
RNA Ds mex 3
RNA

Interference contrast micrographs show in situ hybridization in embryos. The 1,262-nt mex-3 cDNA clone20 was divided into two
segments, mex-3A and mex- 3B, with a short (325-nt) overlap (similar results were obtained in experiments with no overlap
between interfering and probe segments). mex-3B antisense or dsRNA was injected into the gonads of adult animals, which
were fed for 24 h before fixation and in situ hybridization (ref. 5; B. Harfe and A.F., unpublished observations). The mex-3B
dsRNA produced 100% embryonic arrest, whereas .90% of embryos produced after the antisense injections hatched. Antisense
probes for the mex-3A portion of mex-3 were used to assay distribution of the endogenous mex-3 mRNA (dark stain). four-cell-
stage embryos are shown; similar results were observed from the one to eight cell stage and in the germ line of injected adults.
a, Negative control showing lack of staining in the absence of the hybridization probe. b, Embryo from uninjected parent (showing
normal pattern of endogenous mex-3 RNA20). c, Embryo from a parent injected with purified mex-3B antisense RNA. These
embryos (and the parent animals) retain the mex-3 mRNA, although levels may be somewhat less than wild type. d, Embryo from
a parent injected with dsRNA corresponding to mex-3B; no mex-3 RNA is detected
SOAKING C. elegans WORKS ALMOST
AS EFFECTIVELY AS INJECTING
 These images are from an
experiment performed by Jeff
Norman (in 1999), demonstrating
the results of mex-3 in situ
hybridization following an RNAi
soaking protocol (for original
methods, see Tabara et al. '98
Science 282: 430-31; specific
protocol used in this experiment was
obtained from K. Subrumaniam in
Geraldine Seydoux's lab). The left
panels show the wildtype pattern of
endogenous mex-3 mRNA in
untreated adults and embryos. The
right panels show loss of mex-3
staining following soaking of L4
hermaphrodites overnight in mex-3
dsRNA. Endogenous mex-3 RNA is
greatly reduced, although still faintly
detectable; this experiment resulted
in approximately 90% dead embryos.
Although not as effective as directly
injecting dsRNA, this approach is
VASTLY EASIER and may be good
enough for analysis of most
maternally acting genes. Soaking as
a delivery method, however, does
NOT work for many other nematode
species
 2000
 Reported processing of long dsRNA by
Rnase III (Dicer) into shorter fragments of
21-23-nt intervals in Drosophila extracts
 Zamore et al.
 Drosophila
 Zamore et al.
RNAi: double-stranded RNA directs the ATP-d
. Cell. 2000 Mar 31;101(1):25-33.
 RNAi Requires ATP

Denaturing agarose-gel analysis of 59-32P-radiolabeled Rr-luc mRNA

incubated for the times indicated in an in vitro RNAi reaction with or
without ATP, creatine phosphate (CP), or creatine kinase (CK), as
indicated below each panel.

Pretreated with hexokinase and glucose which converts ATP to ADP in lysate
 RNAi Does Not Require mRNA
Translation

A) Protein synthesis, as reflected by luciferase activity produced after incubation of

RrlucmRNAin the in vitroRNAi reaction for 1 hr, in the presence of the protein synthesis
inhibitors anisomycin, cycloheximide, or chloramphenicol, relative to a reaction without any
inhibitor.
(B) Denaturing agarose-gel analysis of 59-32Pradiolabeled Pp-luc mRNA after incubation for the
indicated times in a standard RNAi reaction with and without protein synthesis inhibitors. The
arrowhead indicates the position of full-length mRNA in the gel, and the bracket marks the
position of stable, 59 cleavage products.
 21–23 nt RNA Fragments Are Produced upon
Incubation of dsRNA in Drosophila Embryo Lysate

An enlargement of the 17 to 27 nt region of a gel showing the

products formed upon incubation of uniformly 32P-radiolabeled
dsRNAs in lysate without and with ATP.
 2001
 Cloned Dicer, the RNase III enzyme that is
evolutionarily conserved and contains
helicase and PAZ domains, as well as two
dsRNA-binding domains.
 C. elegans
 Bernstein et al.
Role for a bidentate ribonuclease in the initiatio
initiati
Nature. 2001 Jan 18;409(6818):363-6.
 2001
 First described RNAi in mammalian cells
 Mammals
 Elbashir et al.
Duplexes of 21-nucleotide RNAs mediate RNA
. Nature. 2001 May 24;411(6836):494-8.
 2003
 Short hairpin RNAs (shRNAs) induce sequence-
specific silencing in mammalian cells.
 Mammals
 Paddison et al. Genes Dev. 2002 Apr
15;16(8):948-58
Sui et al Proc Natl Acad Sci U S A. 2002 Apr
16;99(8):5515-20
Paul et al. Nat Biotechnol. 2002 May;20(5):505-8
　
 2003
 First reported that siRNAs can be used
therapeutically in whole animals.
 Mammals
 Song et al.
RNA interference targeting Fas protects mice
Nat Med. 2003 Mar;9(3):347-51. 　
Injection of siRNA duplex efficiently silences
Fas gene expression in mouse hepatocytes.
a, Hepatocytes harvested 24 h after 3 injections of
saline or Cy-5-labeled Fas(sequence
Fas(sequence 1)
siRNA were stained with albumin-FITC (alb-
FITC) and analyzed by flow cytometry. A high
proportion of hepatocytes take up the duplex
siRNA, as indicated. b, RPA for Fas mRNA
expression in hepatocytes from mice that
were untreated (lane 1) or were injected 24 h
earlier with saline (lane 2), GFP(sequence
GFP(sequence 1)
siRNA (lane 3) or Fas(sequence
Fas(sequence 1) siRNA
(lane 4). Silencing of Fas expression in mice
treated with Fas siRNA is maintained 5 (lane
5) or 10 d (lane 6) later. Expression of other
genes involved in the Fas pathway and
housekeeping genes (L32 (L32 and Gapd)
Gapd) were
unaffected (names of the corresponding
proteins are listed at right). Similar results
were obtained in 3 independent experiments.
The graph shows results of densitometric
quantification of the Fas/
Fas/Gapdh ratios in 3
mice per condition. Fas mRNA levels in
hepatocytes are significantly lower (*, P <
0.001) at all times in mice treated with Fas
siRNA mice than in control mice. c, Fas
immunoblot of lysates from hepatocytes
obtained from untreated mice (lane 1), or 24 h
after saline (lane 2), GFP siRNA (lane 3) or
Fas siRNA (lane 4) injection, and 5 (lane 5) or
10 d (lane 6) after Fas siRNA injection. Mouse
recombinant Fas and FasL proteins serve as
positive (P) and negative (N) controls,
respectively. Similar results were obtained in
3 independent experiments.
 Fas gene silencing protects mice from
fulminant hepatitis and hepatic fibrosis

a, Representative liver histology of ConA-induced hepatitis in

mice injected with saline, GFP(sequence 1) siRNA and
Fas(sequence 1) siRNA (n = 5 per group). Livers were
stained with H&E 20 h after ConA injection. Original
magnification 200.
C. Representative liver histology 1 week after 6 weekly
injections of ConA in mock-treated mice and mice injected
with GFP(sequence 1) siRNA and Fas(sequence 1) siRNA (n Survival advantage of mice
= 3 per group). Original magnification 100. Livers of mice injected with Fas siRNA as
treated with Fas siRNA mice did not develop bridging fibrosis. compared to saline or GFP
siRNA
 2004
 First observed that siRNA silences gene at
transcriptional level possibly through
directing de novo DNA methylation.
 Human
 Morris et al.
Small Interfering RNA-Induced Transcriptional

Science. 2004 Aug 5

 2004
 First phase I clinical trial of siRNA drug for
age-related macular degeneration (AMD)
 Human
 Acuity Pharmaceuticals
 2006
 Won Noble Prize in Physiology or
medicine for discovering RNAi
mechanism.
 Andrew Fire and Craig Mello
 Initiation Step
ATP
DICER
ADP + ppi

ATP
KINASE
ADP + ppi

RdRP
 Effector Step

• siRNA binding
• siRNA unwinding
• RISC activation
Dicer
 siRNA

5’ 3’

3’ 5’
 RISC
• 2 RNA binding
proteins
• RNA/DNA Helicase
• Translation
Initiation Factor
• RNA-Dependent
RNA Polymerase
(RdRP)
• Transmembrane
protein? C.
elegans and
plants – only
systemic spread
Vectors expressing siRNAs
U6

siRNA

H1
Vectors expressing siRNAs
U6

Sense sequence Sense

sequence

Anti-sense
Anti-sense
sequence
sequence
Hair-pin
loop
 Vectors expressing siRNAs
 Plasmid-based vectors
 Transient nature
 Low and variable transfection efficiency

 Viral-based vectors
 Highly efficient
 Better than plasmid-based

 Pol II promoter-based plasmid vectors

 Tissue specific
 siRNA Delivery
 in vitro
 Chemical transfection (Lipofectamine,
Oligofectamine, TransIT-TKO, Siport Amine,
Siport
 Electroporation into parasites

 in vivo
 Intramuscular injection
 Hydrodynamic transfection into mammals
 Problems
 Silencing efficiency
 Vector-based large dsRNA delivery

 Systemic silencing
 Therapeutic siRNAs
siRNA target gene Disease
p53 mutant
K-Ras
BCR-ABL
MDR1
C-RAF Cancer
Bcl-2
VEGF
PKC-α
Β-Catenin
(Sioud, 2004)
 Therapeutic siRNAs
siRNA target gene Disease
HIV-Tat
HIV-Rev
HIV-Vif, -Hef
HPV-E6 and –E7 Viral Infection
HBV-S1, -S2, -S, -X
CCR5, CXCR4
CD4
(Sioud, 2004)
 Therapeutic siRNAs

siRNA target gene Disease

Fas receptor
Acute Liver Failure
Caspase-8
TNF-α Sepsis
(Sioud, 2004)
The First Blue Rose (CSIRO)
How did they produce it?