Monday, September 22, 2008

Welcome To Seminar
On

RNA Interference: An approach for

Sequence- Specific Knockdown of
mRNA

Tripti Jain
(Ph. D. Scholar)
Introduction
Historical Aspect
Mechanisms of RNAi (RNAi pathway)
Component of RNAi Pathway
Applications
Conclusion and future prospects
Introduction

 What is RNAi ?????

 Synonyms
 Historical Aspect
 What is RNA Interference (RNAi) ???
• 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 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.

Some alternate terms (Synonyms)
PTGS (Post Transcriptional Gene Silencing)- Plants

VIGS (Virus Induced Gene Silencing) – Plants

Co suppression- Plants.

Quelling - Fungi.

RNAi - Animals.
Historical aspect of 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 they called
“cosuppression.”

 Similar effects seen in N. crassa

“Quelling” [Cogoni et al., 1996]
Final Discovery

Andrew Fire , Craig Mello 1998 announced

RNAi discovery- (Awarded Nobel Prize 2006.)

The crucial 1998 discovery  by Fire et al., that injection of

dsRNA—a 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 of RNA
interference.
Their Experiment…
• C. elegans unc-22 inactivation

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

• dsRNA was 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-22

activity produce twitching movements. Only dsRNA induced
twitching in progeny.

• Mello argued that mechanism could not just be a pairing of antisense

RNA to mRNA, and he coined the term RNA interference for the
unknown mechanism.
2. RNAi Pathway
Initiation step (siRNA or miRNA generation)

Processing of long dsRNA or shRNA by enzyme of RNase III

family nucleases “Dicer” into small dsRNA molecules, ‘siRNA’
and ‘miRNA’. (Zamore et al., 2000)

Effector step (Degradation of mRNA or inhibition 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)
Initiation step (siRNA generation)
 First step - processing large dsRNAs to 21–23 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 3’ hydroxyl, 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

dsRNA trigger

ATP
DICER
ADP + ppi

ATP
KINASE
ADP + ppi

DICER
siRNA
Effector step (Degradation of mRNA)
 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 has ATP-dependent helicase & RNase activity.

(Bantounas et al., 2004)

 RNA dependent RNA polymerase (RdRP), uses target mRNA as
template to produce new dsRNA. (Bantounas et al., 2004)
 Several RdRPs 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
RdRP

• siRNA binding ATP

• siRNA unwinding ADP + ppi
• RISC activation
RNA interference
A model for the processing and mode of action of
Pri-miRNAs and dsRNAs

Initiation

Effector

He and Hannon, 2004

Translational Inhibition
• Imperfect match between siRNA or miRNA in RISC and
target mRNA

• RISC usually binds 3’ UTR

• Mechanism of inhibition... ????

He and Hannon, 2004

 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

3. Components of RNAi

1. RNA components
siRNA
miRNA

2. Protein components
Drosha
Dicer
RdRP
RISC
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 ?
Small interfering RNA (siRNA)
• Small interfering RNA (siRNA), sometimes known as short
interfering RNA or silencing RNA, have a well defined structure

• 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 enzyme

that converts either long dsRNAs or hairpin RNAs into siRNAs .
 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 Novina and Sharp, 2004
– Allows persistence of RNAi ?
What About Mammals???
• Evidence of RNAi 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 nonspecific inhibition 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

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
• Prefer A-U instead of GC at 5’ end of antisense strand
Rational Design Points

Mittal, 2004
Still Not Too Efficient
-Usually need to design several siRNAs to
get an effective one
– Increased possibility of non-specific targeting
– Don’t know which siRNA is most potent
• Limitations:
– Inability to interact with RISC
– Target inaccessibility (structural constraints ?)
– Instability of the siRNA
Stratagies for siRNA generation
Chemical synthesis
In vitro transcription
In vivo transcription (Vectors expressing siRNAs)
Plasmid-based vectors
Transient nature
Low and variable transfection efficiency
Viral-based vectors
Highly efficient
Retroviral vectors
Adenovirus vectors
Adenoassociated viral vectors
Lentiviral vector
Pol III promoter- U6 & H1. (Paddison et al.,2002;Brummelkamp et al.,2002)
 siRNA expression cassettes

Promoters.
U6
U6 (Paddison et al.,2002)
H1 (Brummelkamp et al.,2002)

Two promoter sites for

RNA pol III – each siRNA
transcribes either a
sense or antisense
which anneal in the
nucleus to create
an siRNA de novo. H1
(Tuschl, 2002)
 shRNA expression cassettes
U6
Sense
shRNA
sequence

Anti-sense Hair-pin
sequence loop

Sequence encoding both sense & the anti-sense strand, separated by a

hair-pin loop.
Single promoter site for RNA Pol III. Transcribed strand folds
over and anneals to itself – creating shRNA.
RNAi by In Vivo Transcribed siRNA(shRNA)
Endogenous siRNAs
• Endogenous siRNAs have been identified in plants, fungi.

• In many cases, endogenous siRNAs originate from

repetitive 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 (RITS) complex, in maintaining
the heterochromatin, and hence transcriptionally
repressed, state of the region that encodes the rasiRNA.
 Micro RNA (miRNA)
• MicroRNAs (miRNAs) are a large class of highly conserved RNAs
found in plants and animals. Transcribed from endogenous gene as pri-
miRNA.

• These small RNAs have been shown to play critical roles in

developmental 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 2–3% 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.
miRNA Biogenesis
• Transcribed from endogenous gene as pri-mRNA
– Primary 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
microRNA target
recognition
• Nucleus (“seed”) critical for
target recognition on mRNA

• Nucleus is typically 7bp long

• Usually located at the 5’ end

of microRNA
Differences in miRNA Mode of Action
miRNA-Mediated Gene Silencing
A)Post-Transcriptional Gene Silencing
 mRNA degradation
 Translation block

B) Transcriptional Gene Silencing

 Histone methylation
 Heterochromatin formation
 miRNA Vs siRNA
miRNA siRNA

Size 18-24 nt 18-24 nt

Origin Endogenous ssRNA Endogenous or

with a imperfactly base-paired

stem-loop structure exogenous
(miRNA genes-All are believed perfectly base paired to

be transcribed by RNA pol II) dsRNA

(no siRNA genes)

Conservation Phylogenetically Conserved non conserved

Mode of action Translation inhibition mRNA cleavage

(or cleavage) Chromatin silencing
The Protein Components of RNAi
Pathway
•Drosha
•Dicer
•RdRP
•RISC

 What are they?

 How do they function?
Drosha

• Processes pri-miRNA into pre-miRNA

– Leaves 3’ overhangs on pre-miRNA
• Nuclear RNase-III enzyme [Lee at al., 2003]

• Not yet found in plants

– May be Dicer does its job?
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
– Tandem RNase-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]
 RNA Dependent RNA Polymerase (RdRP)
• RdRP activity found in plants and C. elegans
• May explain efficiency of RNAi
• Required for RNAi?
– Not found in mammals or Drosophila
[Lipardi et al., 2001]
• Proposed mechanism
– siRNA acts as primer for elongation on target
mRNA.
 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
– Helicase
– Endonuclease and exonuclease “Slicer”
• Preferentially incorporates one strand of unwound RNA
– Antisense
• Argonaute is the catalytic engine of RISC in mammals and responsible for
the cleavage of the target mRNA by it’s RNase H like domain
• Argonaute also contains PAZ domain.
• The strand with less 5’ stability usually incorporated into RISC due to
easier unwinding from one end.
 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.
 GENE KNOCKDOWN
• Is being used to elucidate role of particular protein in signal
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 role

for specific gene in apoptotic & pro -apoptotic pathways.
For example role of p73 & p53-mediated apoptosis was
demonstrated by showing that depletion of p73 using
siRNA prevents cell death.
THERAPEUTIC APPLICATION OF RNAi

• RNAi provides novel approach for disease therapy.The

most obvious clinical uses of RNAi are for diseases in
which selective depletion of one or few protein s would be
expected to slow or halt disease process. It may be possible
to 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 of macular degeneration and respiratory
syncytial virus.
 POTENCIAL THERAPEUTIC TARGET OF RNA
INTERFERENCE
• CELL CYCLE PROTEIN– CYCLIN, CYCLIN DEPENDENT PROTEIN
KINASE, TELOMERASE.---Depletion of these protein critical for cell
cycle, might be effective in treatment of cancer.

• INHIBITION OF ANTI-APOPTOTIC PROTEIN :Blocking the production

of anti apoptotic protein such as Bcl-2, inhibitor of apoptosis protein may
be used to kill cancer cells.

• INHIBITION OF APOPTOTIC PROTEIN- P53, CASPASES, IN

DEGENERATIVE NEUROLOGICAL AND AUTOIMMUNE DISORDERS
may slow or stop the degenerative processes

• PATHOGEN SPECIFIC PROTEIN: Bacterial and Viral gene are obvious

targets for RNAi based therapeutic intervention in infectious diseases.
• OXIDATIVE STRESS RELATED PROTEINS: genes that code proteins
involved in oxidative stess and inflammation might be targeted in
autoimmune and infectious diseases.
 Conclusion and future prospects

• When ‘Science’ nominated RNAi as the break through of

the year 2002, it was already clear that RNAi will
revolutionize biomedical research during the next few
years.

• RNAi is a tool to study gene function & to interfere with

pathogenic gene expression in various diseases.

• With the knowledge of genome sequence of many species ,

RNAi can contribute to a more detailed understanding of
complicated physiological processes, and also to develop
many more new drugs, since it connects genomics,
proteomics & functional genomics.