Lecturer: 林志隆 ( IMB&RNAi Core)

05/14/2009/AS

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/content.cfm?pageid=10088

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 plant phenomenonplant phenomenon

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

IFN response in mammalian system---global gene silencingIFN response in mammalian system---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 Length of siRNA: 21 nts to 23 nts. 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

Biogenesis of Biogenesis of RNAiRNAi

DRCR8

Long dsRNALong dsRNA

siRNAsiRNA

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

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. 2003Reynolds et al. 2003

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; mRNA secondary structure?mRNA secondary structure?

Other considerationsOther considerations

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 80,249 80,249 16,020 16,020 17,48417,484 33503350 29,86129,861#1#1

MouseMouse 78,01778,017 15,92015,920 591591 156156 21,04521,045#2#2

Total Total 158,351 158,351 31,940 31,940 18,07518,075 35063506 25,41625,416

#1#1 Targeting 5,534 genes Targeting 5,534 genes #2#2 Targeting 4,335 genes Targeting 4,335 genes

• with different selection/ fluorescence markerswith different selection/ fluorescence markers

(11,466 TRC shRNAs targeting 1,956 genes)

Library Library performance-Iperformance-I

0

1,000

2,000

3,000

4,000

5,000

<10% <20% <30% <50% no KD

Clone Performance (% Expression)

# c

lon

es

0%

20%

40%

60%

80%

100%

Cu

mu

lati

ve %

clo

nes

44%44%

0

100

200

300

400

500

100%5/5

≥80%4/5

≥60%3/5

≥40%2/5

≥20%1/5

<20%0/5

% "good" clones/gene

# g

en

es

0%

20%

40%

60%

80%

100%

Cu

mu

lati

ve %

gen

es

60%

75%

60%

75%

TRC reportTRC report

0

200

400

600

800

1000

1200

1400

1600

1800

2000

≤10% ≤20% ≤30% ≤40% ≤50% >50%KD bin (% remaining)

# sh

RN

As

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

cum

ula

tive

% s

hR

NA

s

A549MCF7JurkatHepa

A549MCF7JurkatHepa

(11,466 TRC shRNAs targeting 1,956 genes)

Library performance-Library performance-IIII

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

GFP and shRNA-Luc virusesGFP and shRNA-Luc viruses

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

http://www.systembio.com/lenti_vectors.htm

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 geneticsForward and reverse genetics

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.

Configuration of TRC shRNA Configuration of TRC shRNA constructconstruct

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

GAPDH

high(>3800), medium (z2500–2800), or low (<350) SCFsin the HeLa transcriptome (z10 siRNAs for each group) Khvorova A. RNA (2008),14:853-861.

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)

Screens for essential genes in 12 cancer cell linesScreens for essential genes in 12 cancer cell lines

NSCLC glioblastoma SCLC leukemia

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/