Figu
re1.
1.M
odel
ofRN
Aip
athw
ayinC.elegans.
Tran
smem
bran
epr
otei
nSI
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allo
ws
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ente
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ece
ll.In
the
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plas
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oces
sed
byD
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istin
gin
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ithRD
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,RD
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and
DRH
-1.
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lting
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show
nbo
und
toRD
E-1.
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ble
stra
nded
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unw
ound
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ase,
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7m
ight
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ngto
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erta
rget
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RNA
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te)b
ound
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ISC
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ded
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ades
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ense
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asa
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hich
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ate
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ate
and
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uces
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ean
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sesi
RNA
s(o
rang
e).S
ense
siRN
As
(blu
e)do
not
accu
mul
ate
inC.elegans.
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d-siRNAs
dsRNAs
7MeG AAA
7MeG
7MeG
7MeG7MeG
7MeG
7MeG
AAA
AAA
AAAAAA
AAA
AAA
+ siRNA-dependent DNA methylation
large dsRNA
miRNAprecursor
tncRNAprecursor
shRNA
tncRNA
siRNA
miRNA
siRNA pool
RdRP
RISC
RISC
RISCRISC
RISCRISC
RISCRISC
Dicer
Dicer
Dicer
Dicer
Dicer
Silencing mechnisms
target mRNA
productsDicersubstrates
siRNA amplification
hairpinexpressionconstruct
chemically-synthesizedor in vitro-transcribedsiRNA
pol II + RdRP
_
genomicsequence
pri-miRNA miRNAprecursor
Drosha
largedsRNA
tncRNAprecursor
shRNA
pol II or III
target gene
pol II etc.
Figure 2.1. Sequence dependent regulation of gene expression. Expression of particular genomicsequences produces various dsRNAs. Dicer is responsible for processing the dsRNAs into small RNAs.The small RNA is then incorporated into RISC, guiding the protein complex to a specific mRNA. RISCenforces specific gene suppression either by cleavage of the mRNA or inhibition of translation. Thedegree of complementarity between the small RNA and the mRNA determines the fate of the mRNA;perfect or near perfect complementarity generally results in cleavage of the mRNA, whereas, a fewmismatches results in suppression of translation. In a sequence specific manner, small RNAs alsoregulate chromatin structure and hence gene expression. Some of the processes depicted here maynot be present in all organisms or cell types. From a practical standpoint small RNAs can be usedto investigate gene function; several types of small dsRNAs can either be introduced into the cell orproduced inside the cell. In all cases the small RNA is funneled into the RNAi pathway and triggersspecific gene suppression.
PAZH.s. Dicer
M.m. Dicer1
D.m. Dicer1
D.m. Dicer2
C.e. DCR-1
S.p. C188.13c
A.t. Carpel Factory
HelicaseDUF283
RNaseIII
RNaseIII
dsRNABD
1922 aa
1917 aa
2249 aa
1722 aa
1845 aa
1374 aa
1909 aa
Figure 2.2. Alignment of Dicer homologs. Most Dicer proteins contain six conserved domains:a DExH helicase domain; a domain of unknown function (DUF283); a PAZ domain; two RNaseIII catalytic domains; and finally a double stranded RNA binding domain. Spacing between thedomains is different for the homologs, which may explain the different size classes of small RNAsfound in some species.
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ATP
RISC mRNA
DICER
RDE-4
ATP
Figure 3.1. Model for mRNA degradation in the cytoplasm by RNAi. Introduced dsRNAs (red) arerecognized by RDE-4/R2D2, a dsRNA binding protein. These dsRNAs are then processed by Dicerinto 21-23nt duplexes that can associate with an enzyme complex called RISC. After unwinding ofthe siRNAs, RISC becomes competent to target homologous mRNA transcripts for degradation.
Figure 3.2. Amplification of dsRNA by an RNA-dependent RNA polymerase. In certain organisms,new dsRNAs can be generated by RDRPs, primed by siRNAs on mRNA targets. The new dsRNAscan be used subsequently by Dicer to create more siRNAs, which can lead to additional rounds ofamplification.
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Figure 3.3. Model for gene silencing in the nucleus by RNAi. RNAi can also silence the transcrip-tion of targeted genes in certain organisms. In this model a signal can direct a putative nuclearRNAi silencing complex (NRISC), composed of chromatin modifying proteins, to the targeted locus,silencing gene expression at the level of transcription.
Figure 5.4. Microtubule Associated Protein 2 (MAP2) suppression in primary cortical neurons bycognate 21nt-siRNAs. A. Double fluorescence staining of neurons transfected with non-specificsiRNA or with MAP2-siRNA. Upper panels-staining with MAP2 monoclonal antibody (green); lowerpanels-staining with actin-bound toxin phalloidin (red). B. Distribution of MAP2 expression levelsin control and targeted cells, two different siRNA (siRNA1 and siRNA2) show a very similar effect.In each experiment, at least 70 random neurons per experimental condition were analyzed andgene expression was quantified in both control and targeted cells. The figure is reprinted from:Krichevsky, A. M. and Kosik, K. S. “RNAi functions in cultured mammalian neurons.” Proc Natl AcadSci U S A., 99(18):11926–9 (2002).
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Figu
re6
.2.
Am
odel
for
the
siRN
A-m
edia
ted
RNA
im
echa
nism
depi
ctin
gm
ajor
step
sin
(1)
initi
aldu
plex
reco
gniti
onby
the
pre-
RISC
com
plex
;(2
)AT
P-de
pend
ent
RISC
activ
atio
nan
dsi
RNA
unw
indi
ng,(
3)
targ
etre
cogn
ition
,(4
)ta
rget
clea
vage
and
prod
uct
rele
ase.
The
desi
red
outc
ome
isdi
rect
edby
antis
ense
stra
nden
try∗
into
the
RISC
topr
oduc
e“o
n-ta
rget
”sile
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gef
fect
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ndes
irab
leof
f-ta
rget
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cts
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ght
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cted
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nse
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ndid
entit
yto
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late
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quen
ces
but
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ized
usin
gra
tiona
lde
sign
coup
led
with
com
preh
ensi
vese
quen
cean
alys
es.
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45
97
35 38
57
83
66
42
100
0
20
40
60
80
100
120
38 39 40 48 NT
Clone Name
% o
f Non
-Tra
nsfe
cted
Real Time PCRImmunofluorescence
HeLa Clone 39Non-transfected HeLa Clone 48
Figure 10.3. Long Term Silencing of GAPDH with CMV Puro Plasmid. HeLa cells were transfectedwith a CMV puro plasmid expressing GAPDH-specific siRNAs. The cells were cloned, and clonalpopulations were selected in 2.5 µg/ml puromycin. Three weeks after selection, GAPDH expressionwas analyzed by (A) RT-PCR or (B) immunofluorescence. Expression levels of several cell clones areshown. Green: GAPDH. Blue: DAPI stained nuclei.
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Figure 11.4. Method for identifying effective shRNA sequences. To screen for siRNAs that are ef-fective against a gene of interest, the gene to be targeted is cloned into an expression vector asa translational fusion to a fluorescent protein. This construct is then co-transfected with test andcontrol siRNA sequences against the gene. If the siRNA sequence is effective, then expression of thefusion protein will be reduced, resulting in a loss of fluorescence.
Figure 15.4. RNAi in the neuroepithelium of E10 mouse embryos. E10 mouse embryos wereinjected, into the lumen of the telencephalic neural tube, with the two reporter plasmids pEGFP-N2 (for GFP) and pSVpaXD (for βgal), either without (a–c and g, Control) or with (d-f and g,siRNA) βgal-directed esiRNAs, followed by directional electroporation and whole embryo culturefor 24 hours. (a-f) Horizontal cryosections through the targeted region of the telencenphalon wereanalysed by double fluorescence for expression of GFP (green; a and d) and βgal immunoreactivity(red; b and e). Co-expression of GFP and βgal in neuroepithelial cells appears yellow in the merge(c and f, arrowheads). Note the lack of βgal expression in neuroepithelial cells in the presenceof βgal-directed esiRNAs. Upper and lower dashed lines indicate the lumenal (apical) surface andbasal border of the neuroepithelium, respectively. Asterisks in (b and e) indicate signal due to thecross-reaction of the secondary antibody used to detect βgal with the basal lamina and underlyingmesenchymal cells. Scale bar in (f), 20 µm. (g) Quantitation of the percentage of GFP-expressingneuroepithelial cells that also express βgal without (Control) or with (siRNA) application of βgal-directed esiRNAs. Data are the mean of three embryos analyzed as in (a-f); bars indicate S.D.(Reprinted figure with permission from PNAS).
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A
B
Figure 16.1. Chicken embryos are a good model system for developmental studies due to theiraccessibility. Chicken embryos can be accessed in ovo (A) through a window in the eggshell thatcan be resealed after manipulations with a coverslip and melted paraffin. As an alternative approach,chicken embryos can be used as ex ovo cultures (B). With both methods embryos can be kept alivethroughout embryonic development.
Figure 18.2. The pZJM RNAi vector. The tet operator (TetOp), dual T7 terminators (red octagons), tet-inducible T7 promoters (T7 arrows), ribosomal DNA spacer (rDNA), actin poly(A) addition sequence(ACT polyA), phleomycin resistance gene (BLE), splice acceptor site (SAS), aldolase poly(A) additionsequence (ALD polyA). The plasmid is shown in linearized form, after cleavage in the rDNA spacer,and is not drawn to scale.
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Figure 20.1. (a) Albino and wild type (yellow) colonies obtained by transformation of the wildtype strain with carB sequences. Segregation of albino (b) and wild-type (c) transformants aftera cycle of vegetative growth. Colonies showing different phenotypes (arrows) are obtained fromspores of the original transformants. Photographs were taken after illumination with blue light for24 hours.
Figure 21.1. ACMV-[CM]-infected N. benthamiana showing recovery phenotype. N. benthamianaplants imaged at 2-weeks post inoculation [(WPI) (control-A-left; Infected-A-right)] and at 5-WPI(control-B-left; Infected-B-right).
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Figure 21.2. ACMV-[CM]-infected GFP silenced GFP-transgenic N. benthamiana (line 16C). Plant photographedusing dissecting microscope (A) Normal light and (B) UVfilter. Symptom-less recovered leaves appeared red underUV light.
Figure 21.3. Effect of anti-PTGS activity of AC2 gene of EACMCV and ICMV; and AC4 gene ofACMV-[CM] and SLCMV. Leaf of GFP-transgenic N. benthamiana (line 16C) plant agroinfiltratedwith pBin-GFP alone (A), or bacterial mixture harboring pBin-GFP along with the following viralgene constructs, P1/HC-Pro of TEV (B); AC4 of ACMV-[CM] (C), AC2 of EACMCV (D), AC4 of SLCMV(E) and AC2 of ICMV (F). Leaves were photographed 7 days after infiltration using a dissectingmicroscope.
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