Cambridge University Press 0521836778 - RNA Interference ...€¦ · Model of RNAi pathway in...

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Cambridge University Press0521836778 - RNA Interference Technology: From Basic Science to Drug DevelopmentEdited by Krishnarao AppasaniExcerptMore information

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d-siRNAs

dsRNAs

7MeG AAA

7MeG

7MeG

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target mRNA

productsDicersubstrates

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target gene

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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

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RNaseIII

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1917 aa

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1845 aa

1374 aa

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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.






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.






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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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.






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).






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.






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).






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.






Cambridge University Press 0521836778 - RNA Interference ...€¦ · Model of RNAi pathway in...

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