lecturer: 林志隆 ( imb&rnai core) 04/13/2010 resources and applications of trc rnai reagents...
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Lecturer: 林志隆 ( IMB&RNAi Core)
04/13/2010
Resources and applications of Resources and applications of TRC RNAi reagents in TRC RNAi reagents in
National RNAi Core Facility National RNAi Core Facility
The Nobel Prize in physiology The Nobel Prize in physiology /medicine 2006 /medicine 2006
RNAi: A gene silencing by RNAi: A gene silencing by dsRNAdsRNA
Professor Andrew Z Fire, Ph.D.Professor Andrew Z Fire, Ph.D.Stanford University School of MedicineStanford University School of MedicineStanford, CA. USAStanford, CA. USA
Professor Craig C. Mello, Ph.D.Professor Craig C. Mello, Ph.D.University of Massachusetts Medical SchoolUniversity of Massachusetts Medical SchoolWorcester, MA, USAWorcester, MA, USA
Goes to
RNA interference (RNAi)
RNAi does not result in stable genetic changes; RNAi does not result in stable genetic changes; but in lower animal or plants, RNAi effects can be but in lower animal or plants, RNAi effects can be inherited for one or two generations.inherited for one or two generations.
A form of A form of post-transcriptionalpost-transcriptional gene silencing, gene silencing, mimicking the effect of loss-of-gene-function.mimicking the effect of loss-of-gene-function.
Timeline of RNAi achievements
Adapted from: http://www.invitrogen.com/etc/medialib/en/images/mainbody/Thing/Data/Diagram.Par.12505.Image.-1.0.1.gif
Dr. R Jorgensen’s Experiment
C Napoli, C Lemieux, and R Jorgensen Plant Cell. 1990 April; 2(4): 279.
• Attempts to overexpress chalcone synthase by inserting multiple copies of that gene into the plant’s genome.
• Purple plants should become purpler...
• Co-suppression: both endogenous and introduced genes silenced.
• PTGS – but what is the causative factor?
PTGSPTGS= = PPost-ost-TTranscriptional ranscriptional GGene ene SSilencingilencing
PTGS in plants is due to small PTGS in plants is due to small dsRNAdsRNA
dsRNA hypothesis explained this dsRNA hypothesis explained this phenomenonphenomenon
Andrew J. Hamilton and David C. Baulcombe Science 1999 286: 950-52
Nature 391, 806-811 (19 February 1998)
Long dsRNAs trigger non-specific Long dsRNAs trigger non-specific silencing in mammalian silencing in mammalian
dsRNAdsRNA C. elegansC. elegans DrosophilaDrosophila
Long dsRNA initiate IFN response in mammalian Long dsRNA initiate IFN response in mammalian ---global gene silencing---global gene silencing
In In mammal?mammal?
dsRNA-induced translation dsRNA-induced translation inhibition inhibition
in mammalianin mammalian
Cytokine Growth Factor Rev. 2007 18:363-71.
How to Apply RNAi to Mammalian SystemHow to Apply RNAi to Mammalian System
??
Effector of RNAiEffector of RNAi長雙股長雙股RNA (long RNA (long dsRNAdsRNA))
cytosolcytosol
--small-interfering RNA; small-interfering RNA; siRNAsiRNA
Length of siRNA: 21 nts to 23 nts. Length of siRNA: 21 nts to 23 nts.
Gregory Hannon identified the “Dicer” – an enzyme that chops double-stranded RNA into little pieces.
Nature. 2000;404:293-6; Nature. 2001;409:363-6
Nature 411, 494-498 (24 May 2001)
What Does siRNA DoWhat Does siRNA Do
Guide/ antisense strand Guide/ antisense strand http://www.nature.com/focus/rnai/animations/index.htmlhttp://www.pbs.org/wgbh/nova/sciencenow/3210/02.html
RISCRISC: RNAi-induced silencing complex : RNAi-induced silencing complex
Mechanism of RNAi in mammalian cellsMechanism of RNAi in mammalian cells
Antonin et. al ., 2007 Nature Reviews Drug Discovery 6, 443-453
Rational siRNA design Rational siRNA design
Low stability in this region enhances RISC/AS-mediated cleavage of mRNA and promote RISC complex release.U at position 10 at SS is recommended.
Sense strandSense strand Antisense strandAntisense strand
1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 12 13 14 1513 14 15 16 17 18 1916 17 18 19
3’’- OH
3’’- OH
P- 5’’
5’-P5’-P U AAA xG
Reynolds et al. 2004Reynolds et al. 2004
High thermal stability of the 5’ sense strand (SS) blocks incorporation of SS into RISCG or C is preferred.
Low thermal stability of the 5’anti-sense strand (AS) promotes Incorporation of AS into RISC.AU rich is suggested.
no high GC content (35-65%);no high GC content (35-65%); no inverted repeat sequence;no inverted repeat sequence; no consecutive 3 Gs or 3 Cs;no consecutive 3 Gs or 3 Cs; no consecutive 4 Ts if use polIII promoter;no consecutive 4 Ts if use polIII promoter;
Other considerationsOther considerations
Rational siRNA design Rational siRNA design
Reynolds et al. 2004Reynolds et al. 2004
These characteristics are used by rational siRNA design algorithm to evaluate potential targeted sequences and assign scores to them. Sequences with higher scores will have higher chance of success in RNAi.
Criteria
Description Score
Yes No
1 Moderate to low (30%-52%) GC Content 1 point
2 At least 3 A/Us at positions 15-19 (sense) 1 point /per A or U
3 Lack of internal repeats (Tm*<20¡ãC) 1 point
4 A at position 19 (sense) 1 point
5 A at position 3 (sense) 1 point
6 U at position 10 (sense) 1 point
7 No G/C at position 19 (sense) -1 point
8 No G at position 13 (sense) -1 point
The RNAi Consortium (TRC)The RNAi Consortium (TRC) The RNAi CoreThe RNAi Core
Phase I (May/2004 to Apr/2007)Phase I (May/2004 to Apr/2007) Jun/2005 to Apr/2008Jun/2005 to Apr/2008
Phase II (Oct/2007 to Sept/2011) Phase II (Oct/2007 to Sept/2011) May/2008 to Apr/2011May/2008 to Apr/2011
Phases of TRC ProgramPhases of TRC Program
Vector Used by RNAi CoreVector Used by RNAi Core
http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Functional_Genomics_and_RNAi/Product_Lines/shRNA_Library.html
Name Description
U6 Promoter
RNA generated with four uridine overhangs at each 3' end
Cppt/CTECentral polypurine tract /constitutive transport element
hPGK Human phosphoglycerate kinase eukaryotic promoter
puroR Puromycin resistance gene for mammalian selection
SIN/LTR 3' self inactivating long terminal repeat
f1 ori f1 origin of replication
ampR Ampicillin resistance gene for bacterial selection
pUC ori pUC origin of replication5' LTR 5' long terminal repeatPsi RNA packaging signalRRE Rev response element
Configuration of TRC shRNA Configuration of TRC shRNA constructconstruct
Materials Received from TRCMaterials Received from TRC
shRNA constructs and knockdown information:shRNA constructs and knockdown information:
35 shRNA expression vectors (some are 35 shRNA expression vectors (some are intermediates).intermediates).
TRC-ITRC-I TRC-IITRC-II
Clone #Clone # Gene #Gene # Clone #Clone # Gene #Gene # Knockdown Knockdown InformationInformation
Human Human 83,117 83,117 16,026 16,026 53,07053,070 10,14910,149 41,77441,774#1#1
MouseMouse 79,20079,200 15,97615,976 44,04244,042 9,0279,027 34,32534,325#2#2
Total Total 162,317 162,317 32,002 32,002 97,11297,112 19,17619,176 76,09976,099
#1#1 Targeting 7,074 genes Targeting 7,074 genes #2#2 Targeting 6,738 genes Targeting 6,738 genes
• with different selection/ fluorescence markerswith different selection/ fluorescence markers
04-11-2010 updated
Highest quality data: 10,711 genes
TRC library TRC library performanceperformance
Good hairpins/gene coverage
0%
5%
10%
15%
20%
25%
all H
PS
~4/
5
~3/
5
~2/
5
~1/
5
~0/
5
% g
enes
"good" clones/gene
0
2000
4000
6000
8000
10000
12000
14000
16000
100-90 90-80 80-70 70-50 No KD
#s
hR
NA
%KD
TRC reportTRC report
Throughput: 1,200 2,000 genes/month
TRC validation (QRT-TRC validation (QRT-PCR)PCR)
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
Sep
tem
ber
Feb
ruar
yA
pril
May
June
July
Aug
ust
Sep
tem
ber
Oct
ober
Nov
embe
rD
ecem
ber
Janu
ary
Feb
ruar
yM
arch
Apr
ilM
ayJu
neJu
lyA
ugus
tS
epte
mbe
rO
ctob
erN
ovem
ber
Dec
embe
rJa
nuar
yF
ebru
ary
Mar
chA
pril
May
June
July
Aug
ust
Sep
tem
ber
Oct
ober
Nov
embe
rD
ecem
ber
Janu
ary
2006 2007 2008 2009 2010
# g
enes
(cu
mu
lati
ve)
successful
attempted
1,200 Genes / month(6,000 shRNAs)
Processupgrade
Resume
Tests on 30,000 hairpin panels (~150,000 tests) Released data on 14K distinct genes
TRC reportTRC report
Available Lentivirus Available Lentivirus Item Item ((arrayedarrayed, , pooledpooled, , individualindividual))
human kinase and phosphatase (hKP) subsethuman kinase and phosphatase (hKP) subset
Mouse kinase subset / Mouse phosphatase subsetMouse kinase subset / Mouse phosphatase subset
Human/ Mouse tumor suppressor subsetHuman/ Mouse tumor suppressor subset
Human / Mouse transcription Factor subsetHuman / Mouse transcription Factor subset
Human deubiqutinating enzymes subsetHuman deubiqutinating enzymes subset
Control virusesControl viruses
Pooled hKPPooled hKP
Pooled mouse kinase / Pooled mouse phosphatasePooled mouse kinase / Pooled mouse phosphatase
Pooled Human/ Mouse tumor suppressor setPooled Human/ Mouse tumor suppressor set
Pooled Human / Mouse transcription Factor subsetPooled Human / Mouse transcription Factor subset
Pooled Human deubiqutinating enzymes subsetPooled Human deubiqutinating enzymes subset
Customized VSV-G pseudotyped lentivirus
80K Human shRNA Pool (16,026 genes)80K Human shRNA Pool (16,026 genes)
Ctl M 1 2 3 4 5 6 7 8 9 10 MPool #
10K6K
4K
3K
2K
Migration Profile of hTRC1 Migration Profile of hTRC1 Pooled pDNAPooled pDNA
86723 Clones 17745 Genes80264 TRC1 Human Clones 16026 TRC1 Human Genes4713 TRC1 Mouse Clones 1377 TRC1 Mouse Genes
1661TRC2 New Human Clones
342TRC2 New Human Genes
85 Control Clones
Transcription initiation of DNA-dependent Transcription initiation of DNA-dependent RNApol III promoters (U6 or H1) are well RNApol III promoters (U6 or H1) are well characterized. RNApol III transcription uses a characterized. RNApol III transcription uses a well-defined termination signal (TTTTT) and well-defined termination signal (TTTTT) and the products have no extra sequence.the products have no extra sequence.
Transcription from these promoters is very Transcription from these promoters is very efficient in various tissues.efficient in various tissues.
Expression of Hairpin RNA (shRNA) Expression of Hairpin RNA (shRNA) Using Pol III PromotersUsing Pol III Promoters
Configuration/Structure of hU6 PromoterConfiguration/Structure of hU6 Promoter
DSEDSE PSEPSE
≈≈ 250bp250bp
TATATATTATATAT
+1+1
--2323
--4848
+1+1
Structure of VSV-G-Pseudotyped Structure of VSV-G-Pseudotyped LentivirusLentivirus
Modified from http://www.washington.edu/alumni/columns/dec00/cells4.html
Replication of Retrovirus Replication of Retrovirus
http://www.accessexcellence.org/RC/VL/GG/retrovirus.html
Genome Organization of Lentiviral VectorGenome Organization of Lentiviral Vector(Improved biosafety by eliminating non-essential genes or sequences)(Improved biosafety by eliminating non-essential genes or sequences)
pCMVΔR8.91:
CMV promoter
SD Gag PolSA SASD
Tat SV40 PolyA
ΔΨ
Rev
RRE
pLKO.1-puro:
R-U5
RSV promoterPsi signal
RRE U6 promoter
AgeI
EcoRI
hPGK promoter
Puro
U3-R
SV40 PolyAPPTpbs
SIN
CMV promoterSV40 PolyA
VSV-G pMD.G:
Procedures:Procedures:
Day1: seeding cellsDay1: seeding cells
Day2: co-transfectionDay2: co-transfection
Day3: re-fresh media Day3: re-fresh media
Day4: harvest viruses/Day4: harvest viruses/
re-add media re-add media
Day5: harvest virusesDay5: harvest viruses
HEK293T as Packaging CellsHEK293T as Packaging Cells
From genome sequence to gene functionFrom genome sequence to gene functionFunction GenomicsFunction Genomics
What does the gene mean?What does the gene mean?
Forward and reverse genetics Forward and reverse genetics approach to study gene functionapproach to study gene function
Forward Genetics:Forward Genetics: Reverse Genetics:Reverse Genetics:
start with a phenotype, find the gene.start with a phenotype, find the gene. naturally occurring mutants can be used.naturally occurring mutants can be used.
start with a gene, determine its phenotype.start with a gene, determine its phenotype. identify the phenotypes resulting from the identify the phenotypes resulting from the disruption of a particular gene.disruption of a particular gene.
Applications ofApplications of RNAi librariesRNAi libraries
RNA RNA Interference Interference
CancerCancer Basic ResearchBasic Research Viral Viral InfectionInfection
• Host factors required for viral replication• Biological pathways• And more
• Gene functions
• Tumor biology
Approaches to large-scale RNAi Approaches to large-scale RNAi screen/selectionscreen/selection
Arrayed RNAi library/ScreenArrayed RNAi library/Screen Pooled RNAi libraryPooled RNAi library /Selection/SelectionsiRNAsiRNA Plasmid VectorPlasmid Vector Viral VectorViral Vector
TransfectionTransfection TransductionTransduction
High Throughput AssayHigh Throughput Assay
for for Altered Phenotype(s)Altered Phenotype(s)
Hits identification:Hits identification:Barcode microarray/Barcode microarray/RT-PCR sequencingRT-PCR sequencing
22ndnd assay to validate hits assay to validate hits
TransductionTransduction
Viral VectorViral Vector
Selective screen for Selective screen for
Altered Phenotype(s)Altered Phenotype(s)
RNAi pooled screening: positive selectionRNAi pooled screening: positive selection
R&D in RNAi CoreR&D in RNAi Core
Search for cellular factors that support Search for cellular factors that support primary human primary human small airway epithelial cell (SAEC) growth using RNAismall airway epithelial cell (SAEC) growth using RNAi pooled selectionpooled selection, 17 genes , 17 genes that support SAEC growing in that support SAEC growing in soft agar are identified.soft agar are identified.
SAEC/ ControlSAEC/ Control
SAEC/ SAEC/ RNAiRNAiAnchorage-dependent growth assayAnchorage-dependent growth assay
> 0.45 mm
SAEC/ ControlSAEC/ Control
SAEC/ SAEC/ RNAiRNAi
RNAi pooled screening: negative selectionRNAi pooled screening: negative selection
How to ensure that hits aren't off-targetHow to ensure that hits aren't off-target
◙ ◙ Off-Target:Off-Target:
◙ ◙ How/ How/ CriterionCriterion::
Phenotype change is caused by two or more independent Phenotype change is caused by two or more independent shRNAs that target the same geneshRNAs that target the same gene
◙ ◙ Why:Why:
Degradation of mRNA can occur by Degradation of mRNA can occur by two separate pathways in RNAitwo separate pathways in RNAi
Khvorova A. RNA (2008),14:853-861.
3’ UTR hexamer frequency in human 3’ UTR hexamer frequency in human genome genome
SCF: seed complementary frequency
high(>3800), medium (z2500–2800), or low (<350) SCFs in the HeLa transcriptome
Khvorova A. RNA (2008),14:853-861.
Microarray signatures of GAPDH- and Microarray signatures of GAPDH- and PPIB-targeting siRNAs PPIB-targeting siRNAs
Same seed sequences in different target genes:GAPDH H15 sense: 5-GAAGUAUGACAACAGCCUC PPIB H17 sense: 5-CGACAGUCAAGACAGCCUG
One nt shift in seed sequence: GAPDH M1 sense: 5-GGCUCACAACGGGAAGCUU
GAPDH M8 sense: 5-GCUCACAACGGGAAGCUUGSeed region not static
Khvorova A. RNA (2008),14:853-861.
Seed sequence plays major role in off-Seed sequence plays major role in off-targettarget
Khvorova A. RNA (2008),14:853-861.
Configuration of TRC shRNA Configuration of TRC shRNA constructconstruct
How are the TRC library shRNAs processed into short dsRNAs?
Implications: hairpin design, off-target effects
A
CGGGTCGAGCTGGACGGCGACGTACT
GTTTTTCAGCTCGACCTGCCGCTGCATG
Which strand goes into RISC?(Strand that goes into RISC is more stable/abundant)
Where does DICER cut?polIII transcription start and stop;evidence for DROSHA processing?
shRNA processing
TRC: Jen Grenier, Andrew Grimson, Ozan Alkan
22 nts
22mer 22mer 18,28518,285 4% 4% 5% 5% 21mer 21mer 39,09539,095 9% 9% 10%10%20mer 20mer 6,7606,760 2% 2% 2%2%
23mer 23mer 45,61045,610 10% 10% 11%11%22mer 22mer 205,249 205,249 46% 46% 51%51%21mer 21mer 40,44440,444 9%9% 10%10%
23mer23mer 23,26323,263 5%5% 6%6%
r4Tsr5Ts
GGGGGG
23mer 23mer 5,2175,217 1% 1% 11%11%22mer 22mer 32,27932,279 7% 7% 67%67%21mer 21mer 8,0298,029 2% 2% 17% 17%20mer 20mer 1,029 1,029 <1%<1% 2%2%
GG21merSenseStrandSeqncCG21merAntisenseStrandSTTTTT
AATT
CCGG
LengthLength#reads#reads % shRNA% shRNA % strand% strand
r
eeee
3Ts
5Ts4Ts
GG
ee3Ts
mm4Ts
}} 17%17%
}} 72%72%
(5)
(3)(3)
(4)(4)
Small RNA sequencing: all 26 shRNAs
Highly parallel identification of Highly parallel identification of essential genes in cancer cells essential genes in cancer cells
Biao Luo etc, Biao Luo etc,
Proc Natl Acad Sci U S A, Proc Natl Acad Sci U S A, 2008, 105: 20380–20385. 2008, 105: 20380–20385.
Pooled RNAi screening (45K lentiviruses) Pooled RNAi screening (45K lentiviruses)
Pooled RNAi screening (45K lentiviruses) Pooled RNAi screening (45K lentiviruses)
Involed in FAS induced apoptosis
Screens for essential genes in 12 cancer cell linesScreens for essential genes in 12 cancer cell lines
NSCLC glioblastoma SCLC leukemia
Commonly essential gene
Cell lineage-specific essential gene
Cell line-specific essential gene(Cancer specific gene dependency)
Time course analysis for the top Time course analysis for the top 100 essential genes in K562 cells100 essential genes in K562 cells
Integration of functional and structural Integration of functional and structural genomicsgenomics
(Case in NSCLC)(Case in NSCLC)
Integration of functional and structural Integration of functional and structural genomicsgenomics
(Case in NSCLC)(Case in NSCLC)
Integration of functional and structural Integration of functional and structural genomicsgenomics
(Case in NSCLC)(Case in NSCLC)
References:
Review paper & original paper
1. Oncogene (2004) 23:8346-8352; 8376-8383; 8384-8391; 8392-8400; 8401-8409.2. Moffat J & Sabatini DM 2006. Building mammalian signaling pathways with
RNAi screens. Nature Rev. 7:177-187.3. Focus on RNA interference (a user guide). Nature Methods 2006 Sep 3(9):669-
719.4. Paddison PJ (2008) RNA interference in mammalian cell systems. Curr Top
Microbiol Immunol. 2008;320:1-19.5. Recent reviews on RNAi. Curr Top Microbiol Immunol 2008, volume 320:1-201.6. Luo Biao et al. 2008. Highly parallel identification of essential genes in cancer
cells. Proc Natl Acad Sci U S A. 105(51): 20380–20385.
National RNAi Core website
http://rnai.genmed.sinica.edu.tw/