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Welcome To SeminarOn

RNA Interference: An approach for

Sequence- Specific Knockdown of

mRNA

Monday, September 22, 2008

Tripti Jain(Ph. D. Scholar)

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IntroductionIntroduction

Historical AspectHistorical Aspect

Mechanisms of RNAi (RNAi pathway)Mechanisms of RNAi (RNAi pathway)

Component of RNAi PathwayComponent of RNAi Pathway

ApplicationsApplications

Conclusion and future prospectsConclusion and future prospects

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IntroductionIntroduction

What is RNAi ?????What is RNAi ?????

SynonymsSynonyms

Historical AspectHistorical Aspect

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RNAi is a process of gene silencing in which dsRNA induces

sequence- specific knockdown of mRNA either by mRNA-

degradation or by translation inhibition.

(Grunweller & Hartmann, 2005; Jens Kurreck, 2006)

RNA interference is an evolutionarily highly conserved

fundamental process present in all eukaryotes from yeast to

mammals.

Form of primitive immunity to protect from nucleic acids

introduced by viruses and transposons.

What is RNA Interference (RNAi) ???What is RNA Interference (RNAi) ???

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What is RNAi ?What is RNAi ? (continued.)

Today RNA interference (RNAi), is a technique in which exogenous,

double-stranded RNAs (dsRNAs) that are complimentary to known

mRNA's, are introduced into a cell to specifically destroy that

particular mRNA, thereby diminishing or abolishing gene

expression. The technique has been proved effective in Drosophila,

Caenorhabditis elegans, plants, and recently, in mammalian cell

culture.

Highly selective if perfect homology with target mRNA is provided.

High potency to silence genes in a sequence-specific manner.

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PTGS(Post Transcriptional Gene Silencing)- Plants

VIGS(Virus Induced Gene Silencing) Plants

Co suppression- Plants.

Quelling- Fungi.

RNAi- Animals.

Some alternate terms (Synonyms)

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Historicalaspectof RNAi

PTGS was observed first in Petunia, when Napoli et al.

(1990) discovered that introduction of a pigment-

producing gene under control of a powerful promoter

suppressed expression of both the introduced gene and

the homologous endogenous gene, a phenomenon theycalled cosuppression.

Similar effects seen in N. crassa

Quelling [Cogoni et al., 1996]

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FinalDiscovery

Andrew Fire , Craig Mello 1998 announced

RNAi discovery- (Awarded Nobel Prize 2006.)

The crucial 1998 discovery by Fire et al., that injection ofdsRNAa mixture of both sense and antisense strands of the

target mRNA, rather than either strand in the nematode C.

elegans resulted in extremely potent silencing

unequivocably identified dsRNA as the inducer ofRNAinterference.

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Their Experiment

C. elegans unc-22 inactivation

Injected sense, antisense, or both into c. elegans gut

dsRNAwas orders of magnitude more effective than ssRNA

Effective even in tiny amounts Inactivation was due to degradation of target mRNA

The unc-22 gene encodes a myofilament protein. Decreased unc-22activity produce twitching movements. Only dsRNA inducedtwitching in progeny.

Mello argued that mechanism could not just be a pairing ofantisenseRNA to mRNA, and he coined the term RNAinterference for the unknown mechanism.

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2.2. RNAi PathwayRNAi Pathway2.2. RNAi PathwayRNAi Pathway

Initiation step (siRNA or miRNA generation)

Processing of long dsRNA or shRNAby enzyme ofRNase III

family nucleases Dicer into small dsRNAmolecules, siRNA

and miRNA. (Zamore et al., 2000)

Effector step ((Degradation of mRNA orDegradation of mRNA orinhibition of translation)

siRNAs are bound by multi-protein complex RISC RNA

Induced silencing complex (with RNase activity), unwounded and

guided targeted mRNA to degradation. (Hammond et al., 2000)

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First step - processing large ds

RNA

s to 2123 nt siRNA

molecules.(Zamore et al.2000, Elbashir et al. 2001)

RNase-III enzyme Dicer found responsible for processing.

(Bernstein et al. 2001)

ATP-dependent translocation of Dicer along dsRNA cause cleavage

sequentially, starting at termini producing small dsRNA (siRNA)

fragments of defined length. (Ketting et al.2001, Zhang et al. 2002)

siRNAs has 3hydroxyl, a 3 overhang of two nts on each strand & 5

phosphate added by Kinase. (Elbashir et al. 2001)

Longer the dsRNA greater the amount of siRNA produced hence more

potent silencing effect. (Bernstein et al. 2001)

Initiation step (siRNA generation)Initiation step (siRNA generation)Initiation step (siRNA generation)Initiation step (siRNA generation)

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InitiationInitiation StepStepInitiationInitiation StepStep

ATP

ADP + ppi

DICERDICERDICERDICER

dsRNA trigger

ATP

ADP + ppi

KINASEKINASEKINASEKINASE

siRNA DICERDICER

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Second stage mediated by RISC which is guided by siRNA to

target mRNA causing its degradation. (Hammond et al. 2000)

ATP-dependent step involved in unwinding of the siRNA duplex,

RISC converted to RISC*. (Nykanen et al., 2001)

RISC complex hasATP-dependent helicase & RNase activity.

(Bantounas et al., 2004)

RNAdependentRNApolymerase (RdRP), uses target mRNA as

template to produce new dsRNA. (Bantounas et al., 2004)

SeveralRdRPs participating in RNAi have been identified in fungi,

plants and invertebrates (Sijen et al., 2001; Martens et al.,

2002).

Evidence for presence of a similar amplification mechanism in

mammalian cells has not yet been found (Bantounas et al., 2004)

Effector step (Degradation of mRNA)Effector step (Degradation of mRNA)

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RdRPRdRPRdRPRdRP

ATP

ADP + ppi

siRNA binding

siRNA unwinding

RISC activation

Effector StepEffector StepEffector StepEffector Step

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He and Hannon, 2004

Initiation

Effector

A model for the processing and mode of action of

Pri-miRNAs and dsRNAs

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Translational Inhibition

Imperfect match between siRNA or miRNA in RISC andtarget mRNA

RISC usually binds 3 UTR

Mechanism of inhibition... ????

He and Hannon, 2004

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mRNA Degradation

Perfect complementarity between

siRNA or miRNA in RISC and the

target mRNA

Cleavage by RISC Slicer (nuclease )activity

Other endo/exonucleases?

Novina and Sharp, 2004

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1. RNA components

siRNA

miRNA

2. Protein componentsDrosha

Dicer

RdRPRISC

3. Components of RNAi

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The RNA Components of RNAi pathway

siRNA:

short-interfering RNA, 21-25 nt.

Mostly exogenous origin.

dsRNA precursors

miRNA:

microRNA, 21-25 nt.

Encoded by endogenous genes.

Hairpin precursors

How do they arise ?

What are their characteristics ?

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Small interfering RNA (Small interfering RNA (siRNA)siRNA)

Small interfering RNA (siRNA), sometimes known as short

interfering RNA or silencing RNA, have a well definedstructure

Short (usually 21 nucleotide) double-strand of RNA (di-RNA)

Each strand has a 5' phosphate group and a 3' hydroxyl (-

OH) group.

This structure is the result of processing by Dicer, an enzymethat converts either long dsRNAs or hairpin RNAs intosiRNAs.

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siRNA Biogenesis

Dicer cleaves long dsRNA

into siRNA 21-25nt

dsRNA from exogenous

sources

Symmetric 2nt 3 overhangs,

5 phosphate groups

Evidence for amplification

in C. elegans and plants

Allows persistence of RNAi ?Novina and Sharp, 2004

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What About Mammals???

Evidence ofRNAi in mammals was harder to establish

Methods for RNAi not a straight forward

Critical observation [Elbashir et al., 2001]

Size do matter

In mammalian cells, introduction of long dsRNA (>30 nt)initiates a potent antiviral response, exemplified by nonspecificinhibition of protein synthesis and RNA degradation.

The mammalian antiviral response can be bypassed, however,by the introduction or expression of siRNAs.

siRNA (21-22nt) mediate mammalian RNAi

No RdRP activity identified

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Rational Design of siRNA

Arising from research on RISC assembly

RISC contains one strand of the siRNA duplex[Martinez et al., 2002]

Needs to be the antisense strand to find right target

Can we direct preferential incorporation of the antisense strand

into RISC ? Observation: less stable 5 end of an siRNA strand is incorporated

into RISC most efficiently

[Schwarz et al., 2003]

Empirical siRNA Design Rules 21nt long, with 2nt 3 overhangs

Avoid introns and UTRs

Avoid regions >50% GC content

PreferA-U instead of GC at 5 end of antisense strand

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Rational Design Points

Mittal, 2004

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Still Not Too Efficient

-Usually need to design several siRNAs toget an effective one

Increased possibility of non-specific targeting

Dont know which siRNA is most potent

Limitations:

Inability to interact with RISC

Target inaccessibility (structural constraints ?)Instability of the siRNA

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Stratagies for siRNA generationStratagies for siRNA generationStratagies for siRNA generationStratagies for siRNA generation

Chemical synthesisChemical synthesis

In vitroIn vitro transcriptiontranscription

In vivoIn vivo transcription (Vectors expressing siRNAs)transcription (Vectors expressing siRNAs)

Plasmid-based vectors

Transient nature

Low and variable transfection efficiencyViral-based vectors

Highly efficient

Retroviral vectors

Adenovirus vectors

Adenoassociated viral vectorsLentiviral vector

Pol III promoter- U6 & H1. (Paddison et al.,2002;Brummelkamp etal.,2002)

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U6

H1

si

Promoters.

U6 (Paddison et al.,2002)

H1 (Brummelkamp etal.,2002)

Two promoter sites for

RNA pol III each

transcribes either a

sense or antisense

which anneal in the

nucleus to create

an siRNA de novo.(Tuschl, 2002)

siRNA expression cassettessiRNA expression cassettessiRNA expression cassettessiRNA expression cassettes

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shRNA expression cassettesshRNA expression cassettes

U6

Sense

sequence

Anti-sense

sequence

Hair-pin

loop

s RNA

Sequence encoding bot sense & t e anti-sense strand, separated by a

air-pin loop.

Single promoter site for RNA Pol III. Transcribed strand folds

over and anneals to itself creating s RNA

.

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RNAi by In Vivo Transcribed siRNA(shRNA)

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Endogenous siRNAs

Endogenous siRNAs have been identified in plants, fungi.

In many cases, endogenous siRNAs originate fromrepetitive elements within the genome, such as

heterochromatic regions at centromeres and telomeres,and are therefore known as repeat-associated siRNAs(rasiRNAs).

rasiRNAs appear to function, through an RNA-induced

transcriptional silencing (R

ITS) complex, in maintainingthe heterochromatin, and hence transcriptionallyrepressed, state of the region that encodes the rasiRNA.

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Micro RNA (miRNA)

MicroRNAs (miRNAs) are a large class of highly conservedRNAs

found in plants and animals. Transcribed from endogenous gene as

pri-miRNA.

These small RNAs have been shown to play critical roles indevelopmental timing, hematopoietic cell differentiation, cell death,

cell proliferation, and oncogenesis . The tissue-specific expression

patterns of miRNAs are providing a few hints about their possible

functions like difference between differentiated cells.

miRNAs may represent 23% of the total number of genes in humans

, and estimates of the number of miRNA target binding sites indicate

that miRNAs may play a role in regulating as many as 30% of

mammalian gene.

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miRNA Biogenesis

Transcribed from endogenous gene as pri-

mRNAPrimary miRNA: long with multiple hairpins

Imperfect internal sequence complementarity

Cleaved by Drosha into pre-miRNA

Precursor miRNA: ~70nt imperfect hairpins

Exported from nucleus

Cleaved by Dicer into mature miRNA

21-25nt

Symmetric 2nt 3 overhangs, 5 phosphate groups

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microRNA target

recognition

Nucleus (seed) critical for

target recognition on mRNA

Nucleus is typically 7bp long

Usually located at the 5 end

of microRNA

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Differences in miRNA Mode of Action

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miRNA-Mediated Gene Silencing

A)Post-Transcriptional Gene Silencing

mRNA degradation

Translation block

B) Transcriptional Gene Silencing Histone methylation

Heterochromatin formation

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miRNA Vs siRNA

miRNA siRNA

Size 18-24 nt 18-24 nt

Origin Endogenous ssRNA Endogenous or

with a imperfactly base-pairedstem-loop structure

(miRNA genes-All are believed perfectly bas

be transcribed by RNA polII) dsRNA

(no siRNA genes)

Conservation Phylogenetically Conserved non conserved

Mode of action Translation inhibition mRNA cleavage

(or cleavage) Chromatin silencing

Complimentarity Not 100% 100%

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The Protein Components of RNAi

Pathway

What are they?

How do they function?

Drosha

Dicer

RdRP

RISC

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Drosha

Processes pri-miRNA into pre-miRNALeaves 3 overhangs on pre-miRNA

Nuclear RNase-III enzyme [Lee at al., 2003]

Not yet found in plants

May be Dicer does its job?

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Dicer

Cleaves dsRNA or pre-miRNA

Cytoplasmic RNase-III enzyme

Has the ability to produce dsRNA fragments, 21 nts long with 3

overhangs of 2 nts

Functional domains in Dicer [Bernstein et al., 2001]

Putative helicase/ ATPase domain

PAZ domain

TandemRNase-III domains

dsRNA binding domain

These nucleases are evolutionarily conserved in worms, flies, fungi,plants and animals

Multiple Dicer genes in Drosophila and plants [He and Hannon, 2004]

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RNA Dependent RNA Polymerase (RdRP)

RdRP activity found in plants and C. elegans

May explain efficiency ofRNAi

Required for RNAi?

Not found in mammals or Drosophila

[Lipardi et al., 2001]

Proposed mechanism

siRNA acts as primer for elongation on targetmRNA.

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RNA Induced Silencing Complex

(RISC)

RNAi Effector complex Approx 500 kDa nuclease complex Sequence specific nuclease activity resulting in ablation of target mRNA.

Critical for target mRNA degradation or translation inhibition

Not well characterized: 4 subunits? More?

Activities associated with RISC

HelicaseEndonuclease and exonuclease Slicer

Preferentially incorporates one strand of unwound RNA

Antisense

Argonaute is the catalytic engine ofRISC in mammals and responsiblefor the cleavage of the target mRNA by its RNase H like domain

Argonaute also contains PAZ domain.

The strand with less 5 stability usually incorporated into RISC due toeasier unwinding from one end.

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TECHNOLOGICAL APPLICATION OF

RNA INTERFERENCE

GENE KNOCK DOWN- TO STUDY FUNCTION OF

PROTEIN-

* Whole genome RNAi screening

* Done in C. elegans

* 19 757 protein coding genes (predicted)

* 16 757 inactivated using RNAi

FUNCTIONAL GENOMICS THERAPEUTIC APPLICATION-

* Control of viral replication in mammals and

to knock down the disease causing gene.

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Is being used to elucidate role of particular protein insignal transduction pathway.

Reveal function of specific protein in development process.

To study role of different protein in cell cycle regulation.

Cell death : RNAi has recently been used to establish rolefor specific gene in apoptotic & pro -apoptotic pathways.

For example role of p73 & p53-mediated apoptosis wasdemonstrated by showing that depletion of p73 usingsiRNA prevents cell death.

GENE KNOCKDOWN

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THERAPEUTIC APPLICATION OF RNAi

RNAi provides novel approach for disease therapy.Themost obvious clinical uses ofRNAi are for diseases inwhich selective depletion of one or few protein s would beexpected to slow or halt disease process. It may be possibleto exploit RNA interference in therapy of following

diseases- CANCER

NEURODEGENERATIVE DISEASES

INFECTIOUS DISEASE

AUTOIMMUNE DISEASE

Among the first applications to reach clinical trials were

in the treatment ofmacular degeneration and respiratory

syncytial virus.

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POTENCIAL THERAPEUTIC TARGET OF RNA

INTERFERENCE

CELL CYCLE PROTEIN CYCLIN, CYCLIN DEPENDENTPROTEINKINASE, TELOMERASE.---Depletion of these proteincritical for cell cycle, might be effective in treatment of cancer.

INHIBITIONOF ANTI-APOPTOTIC PROTEIN :Blocking theproduction of anti apoptotic protein such as Bcl-2, inhibitor ofapoptosis protein may be used to kill cancer cells.

INHIBITION OF APOPTOTIC PROTEIN- P53, CASPASES, IN

DEGENERATIVENEUROLOGICALAND AUTOIMMUNEDISORDERS may slow or stop the degenerative processes

PATHOGEN SPECIFIC PROTEIN:Bacterial and Viral gene areobvious targets for RNAi based therapeutic intervention in infectiousdiseases.

OXIDA

TIVE

STRE

SSRELA

TE

D PR

OTE

IN

S:

genes that codeproteins involved in oxidative stess and inflammation might be

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Conclusion and future prospectsConclusion and future prospects

When Science nominated RNAi as the break through ofthe year 2002, it was already clear that RNAi willrevolutionize biomedical research during the next fewyears.

RNAi is a tool to study gene function & to interfere withpathogenic gene expression in various diseases.

With the knowledge of genome sequence of many species ,RNAi can contribute to a more detailed understanding ofcomplicated physiological processes, and also to developmany more new drugs, since it connects genomics,proteomics & functional genomics.

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