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Credit SeminarCredit Seminar
05/25/1505/25/15 S. S. JenaS. S. Jena

Siddhartha Swarup
Jena
RAD/10-30
Ph.D. Mol. Bio &
Siddhartha Swarup
Jena
RAD/10-30
Ph.D. Mol. Bio &
05/25/1505/25/15

ContentsContents
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S. S. JenaS. S. Jena

What is RNAi?What is RNAi?
RNA interference (RNAi) is a phenomenonRNA interference (RNAi) is a phenomenon
triggered by either endogenous ortriggered by either endogenous or
exogenous dsRNA, and silencesexogenous dsRNA, and silences
endogenous genes carrying homologousendogenous genes carrying homologous
sequences by their degradation at both thesequences by their degradation at both the
transcriptional and posttranscriptional levels.transcriptional and posttranscriptional levels.
This process is widely used to ‘knock-down’This process is widely used to ‘knock-down’
the expression of genes of interest tothe expression of genes of interest to
explore phenotypes.explore phenotypes.
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Transcriptional Vs Post-transcriptionalTranscriptional Vs Post-transcriptional
gene silencinggene silencing
TGSTGS
Promoters silenced
Genes hypermethylated
in promoter region
Purpose - Viral
immunity?
PTGSPTGS
Promoters active
Gene hypermethylated
in coding region
Purpose - Viral
immunity?
This has recently been termed as “RNAi”
S. Grant (1999)
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RNA interference was first observed in petunias,
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.
The phenomenon they called “co-suppression”.
05/25/1505/25/15 S. S. JenaS. S. Jena

Flower pigmentation in PetuniaFlower pigmentation in Petunia
Attempted to over-
express
chalcone synthase
(anthrocyanin
pigment) gene
in petunia.
(trying to darken
flower color)
Caused the loss
of pigment.
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History cont.History cont.
1995- Guo and Kemphues:1995- Guo and Kemphues:
Injection of either antisense or sense RNAsInjection of either antisense or sense RNAs
in the germline ofin the germline of C. elegansC. elegans was equallywas equally
effective at silencing homologous targeteffective at silencing homologous target
genesgenes
1998- Mello and Fire:1998- Mello and Fire:
Extension of above experimentsExtension of above experiments,,
combination of sense and antisense RNAcombination of sense and antisense RNA
(= dsRNA) was 10 times more effective(= dsRNA) was 10 times more effective
than single strand RNAthan single strand RNA
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RNAi was first discovered in 1998 by Andrew
Fire and Craig Mello in the nematode worm
Caenorhabditis elegans.
Awarded Nobel Prize 2006 for this Path-
breaking concept.
This technique has proven effective in
Drosophila, plants, and recently, in
mammalian cell culture. (Sharp 1999;
Hammond et al. 2001).
RNAi discoveryRNAi discovery
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In 2006,In 2006, Andrew Z. FireAndrew Z. Fire andand Craig C. MelloCraig C. Mello shared the Nobelshared the Nobel
Prize in Physiology or Medicine for their work on RNAPrize in Physiology or Medicine for their work on RNA
interference in the nematode worminterference in the nematode worm Caenorhabditis elegansCaenorhabditis elegans..
Andrew Z. Fire Craig Mello at the 2006 Nobel Prize
lecture.
Craig C.Mello
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Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis
elegans.
Nature 391: 806-811(1998)
Since this paper, the new technology
irreversibly revolutionized genetics and
led to the publication of many papers.
Science crowned RNAi as its
"Breakthrough of the
Year" in 2002.05/25/1505/25/15 S. S. JenaS. S. Jena

RNAi is triggered by either endogenous or
exogenous dsRNA.
dsRNA needs to be directed against an exon,
not an intron in order to be effective
RNAi is a self-defence mechanism of eukaryotic
cells to combat infection by RNA viruses and
transposons.
The effect is non-stoichiometric; small amounts
of dsRNA can wipe out an excess of mRNA
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Double-stranded RNAsDouble-stranded RNAs
are produced by:are produced by:
Transcription of inverted repeats
Viral replication
Transcription of RNA by RNA-
dependent RNA-polymerases (RdRP)
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RNAi can be induced byRNAi can be induced by
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Involves Two steps
Initiator step:
Long dsRNA is cleaved in to siRNA
fragments (21-23 nt).
Effecter step:
Fragments are incorporated in to a protein
complex, dissociated & used as a guiding
sequence to recognize homologous mRNA
that is subsequently cleaved.
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StepsSteps
Sense strand/
Passenger strand
Antisense strand/
Guide strand
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GeneGene
silencing bysilencing by
RNAiRNAi
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1.1. Dicer/ DroshaDicer/ Drosha
2.2. Small RNAs (siRNA/ miRNA)Small RNAs (siRNA/ miRNA)
3.3. RNA-Induced Silencing Complex (RISC)RNA-Induced Silencing Complex (RISC)
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1.DICER1.DICER
Enzyme involved in the initiation of RNAi.
Rnase-III-like dsRNA-specific ribonuclease
(200 KDa).
ATP-dependent nucleases.
Acts as a dimer.
Dicer homologs exist in many organisms
including C. elegans, Drosophila, yeast and
humans.
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Dicer’s domains
a. Helicase domain (amino-
terminal end)
b. PAZ (Piwi-Argonaut-Zwille)
domain. (for Protein-protein
interaction)
c. Dual Rnase III motifs in the
carboxy-terminal end
d. dsRNA binding domain
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Cleavage by DicerCleavage by Dicer
Dicer cleaves dsRNA, leaves 3’ OH overhangs
and 5’ phosphate groups.
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Dicer trims the exogenous ds RNA to form
siRNA (small interfering RNA).
Efficiently silencing siRNA duplexes consists
of 21 nt sense and 21 nt antisense siRNAs
which form a 19 bp double helix with 2 nt
3′-overhanging ends.
2. siRNA (small interfering RNA)2. siRNA (small interfering RNA)
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RNAi byRNAi by
siRNAsiRNA
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Knockdown of a gene by artificialKnockdown of a gene by artificial
introduction of siRNAintroduction of siRNA
Transfection of an exogenous siRNA isTransfection of an exogenous siRNA is
problematic as dsRNA structure cannot beproblematic as dsRNA structure cannot be
permanently maintained.permanently maintained.
Modify the siRNA by the introduction of a loopModify the siRNA by the introduction of a loop
between the two strands & allow it to bebetween the two strands & allow it to be
expressed by an appropriate vector, e.g. aexpressed by an appropriate vector, e.g. a
plasmid.plasmid.
This can be processed into a functional siRNA.This can be processed into a functional siRNA.
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RNAi byRNAi by
In-VivoIn-Vivo
TranscribedTranscribed
siRNA (shRNA)siRNA (shRNA)
Transcription
cassette usually uses
an RNA polymerase
III promoter, such as
U6 or H1, which
direct the
transcription of small
nuclear RNA's.
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Vectors for shRNA expressionVectors for shRNA expression

How long does RNAi last?How long does RNAi last?
Silencing caused by injection (synthetic
siRNA) lasts for several days and appear
to be transferred to daughter cells, but
eventually diminish
Cells transfected with siRNA expression
vector experience steady, long-term
mRNA inhibition
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Micro RNA are single stranded RNA moleculesMicro RNA are single stranded RNA molecules
of about 21-23 nucleotide in length, whichof about 21-23 nucleotide in length, which
regulate gene expression.regulate gene expression.
MicroRNA are encoded by genes that areMicroRNA are encoded by genes that are
transcribed from DNA but not translated in totranscribed from DNA but not translated in to
protein; instead they are processed fromprotein; instead they are processed from
primary transcripts known asprimary transcripts known as pri-miRNApri-miRNA toto
stem-loop structures calledstem-loop structures called pre miRNApre miRNA andand
finally to functionalfinally to functional miRNAmiRNA..
miRNA (micro RNA)miRNA (micro RNA)
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miRNA ProcessingmiRNA Processing
Transcribed from endogenous gene as pri-miRNA
 Primary miRNA: long with multiple hairpins
Cleaved by Drosha into pre-miRNA
 Precursor miRNA: ~70nt imperfect hairpins
 Exported from nucleus
Cleaved by Dicer into mature miRNA
 21-25nt

miRNA ProcessingmiRNA Processing
Pri-miRNAPri-miRNA
pre-miRNApre-miRNA
MatureMature
miRNAmiRNA
Drosha
Dicer
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miRNA Vs siRNAmiRNA Vs siRNA
Similarity:Similarity:
1.1. They originate from double stranded structureThey originate from double stranded structure
2.2. More or less of the same size consisting ofMore or less of the same size consisting of
approximately 20-30 nt.approximately 20-30 nt.
3.3. Both are processed by Dicer or Dicer-like enzyme (DCL)Both are processed by Dicer or Dicer-like enzyme (DCL)
4.4. RISC uses both as targeting sequencesRISC uses both as targeting sequences
5.5. They play a vital role in the RNAi process by directingThey play a vital role in the RNAi process by directing
PTGSPTGS
Differences:Differences:
miRNA is derived frommiRNA is derived from endogenousendogenous DNA, while siRNADNA, while siRNA
is derived from exogenous dsDNA.is derived from exogenous dsDNA.
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Comparison of miRNA & siRNA actionComparison of miRNA & siRNA action
Transcriptional silencing
Inhibition of translation mRNA degradation

hpRNA (hairpin RNA)hpRNA (hairpin RNA)
Another form ofAnother form of
dsRNA deriveddsRNA derived
from a long piecefrom a long piece
of single strandedof single stranded
RNA containingRNA containing
inverted repeatsinverted repeats
and a hairpin loopand a hairpin loop
connecting them.connecting them.
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Overview of small RNA moleculesOverview of small RNA molecules

Multi-component ribonuclease (500 KDa) which
contains a member of the argonaute family,
having
PAZ & PIWI domains
RNA binding proteins
RNA helicase proteins
Assembling the RISC complex requires ATP,
while RNA cleavage does not.
3. RISC complex3. RISC complex
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RNAi AssayRNAi Assay
RT-PCR
Western blot
Histological, physiological, structural,
biochemical assay of transgenic
cells or animals.
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RNAi evolved to protect the genome from
viruses (or other invading DNAs or RNAs).
Secures genome stability by keeping mobile
elements silent.
Keep heterochromatic regions condensed and
transcriptionally suppressed.
Prevent over expression of gene
RNAi regulate the development of organisms.
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Applications of RNAiApplications of RNAi
Used to determine the function of a specific
gene by silencing its expression i.e.
Knocking-out a gene’s normal function.
Silencing of over-expressed or aberrantly
expressed disease causing genes.
e.g. cancer treatmente.g. cancer treatment
Generating improved crop varieties in terms of
disease & insect resistance, enhancing
nutritional qualities, and much more.
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Examples of RNAi ApplicationsExamples of RNAi Applications
Potent and specific inhibition of HIV type 1
replication by RNA interference.
Used to silence the gene(s) responsible forUsed to silence the gene(s) responsible for
production of the neurotoxin ‘production of the neurotoxin ‘BOAABOAA’ in’ in
Lathyrus sativusLathyrus sativus..
Monsanto is exploring RNAi to maximize theMonsanto is exploring RNAi to maximize the
production of beneficialproduction of beneficial mono-unsaturatedmono-unsaturated
fats in soybeanfats in soybean, while minimizing saturated, while minimizing saturated
fats and eliminating trans-fats.fats and eliminating trans-fats.
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Case StudiesCase Studies
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05/25/1505/25/15 S. S. JenaS. S. Jena

AbstractAbstract
Development of RNAi by microinjection of dsRNA inDevelopment of RNAi by microinjection of dsRNA in
the brown plant-hopper for gene function study.the brown plant-hopper for gene function study.
The conjunctive between prothorax and mesothoraxThe conjunctive between prothorax and mesothorax
was selected as the injection site and 50 nl aswas selected as the injection site and 50 nl as
injection volume.injection volume.
Three genes with different expression patterns wereThree genes with different expression patterns were
selected to evaluate the RNAi efficiency.selected to evaluate the RNAi efficiency.
CalreticulinCalreticulin - ubiquitously expressed gene- ubiquitously expressed gene
Cathepsin B-like proteaseCathepsin B-like protease - gut specific gene- gut specific gene
NlNlββ22 - central nervous system (CNS) specific- central nervous system (CNS) specific
genegene05/25/1505/25/15

Abstract cont.Abstract cont.
A comparableA comparable 40%40% decrease of genedecrease of gene
expression was observed at the 4th dayexpression was observed at the 4th day
after injection for the ubiquitouslyafter injection for the ubiquitously
expressedexpressed calreticulincalreticulin and the gut specificand the gut specific
cathepsin-B genescathepsin-B genes,,
But only 25% decrease was observed at theBut only 25% decrease was observed at the
5th day for the central nervous system5th day for the central nervous system
specificspecific NlNlββ2 gene2 gene..
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Result & discussionResult & discussion
Two volumes (25 nl &Two volumes (25 nl &
50 nl) of dsRNA-Cal50 nl) of dsRNA-Cal
were injected into L3were injected into L3
larvae and the RNAilarvae and the RNAi
efficiency onefficiency on
calreticulin expressioncalreticulin expression
was investigated bywas investigated by
qRT-PCR over sevenqRT-PCR over seven
days after the injection.days after the injection.
Knockdown of calreticulin expression
Calreticulin expression was reduced by 16.5 ± 4.7 % in one
day after injection with 50 nl dsRNA-Cal. The maximum
reduction of 43.8 ± 10.3 % occurred at the fourth day and
no effect was observed from the eighth day onward.
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Result & discussion cont.Result & discussion cont.
A similar experimentA similar experiment
was performed to inhibitwas performed to inhibit
nicotinic acetylcholinenicotinic acetylcholine
receptor (nAChR)receptor (nAChR) ββ22
subunit (Nlsubunit (Nlββ2)2)
At the first day afterAt the first day after
injection, the injection ofinjection, the injection of
dsRNA-NldsRNA-Nlββ2 only2 only
caused the negligiblecaused the negligible
RNAi.RNAi.
Knockdown of Nlβ2 expression by double
injection
The maximum reduction of 25.3 ± 5.2% of the transcript
level occurred at the fifth day, but not at the fourth day.
05/25/1505/25/15 S. S. JenaS. S. Jena

ConclusionConclusion
Different from calreticulin and cathepsin-B, RNAiDifferent from calreticulin and cathepsin-B, RNAi
efficiency on Nlefficiency on Nlββ2 was lower and the maximum2 was lower and the maximum
inhibition of Nlinhibition of Nlββ2 transcript occurred later.2 transcript occurred later.
These results demonstrate that different tissues orThese results demonstrate that different tissues or
genes seem to be unequally sensitive to genegenes seem to be unequally sensitive to gene
knockdown by dsRNA injection.knockdown by dsRNA injection.
Various parameters can be modified to increaseVarious parameters can be modified to increase
RNAi efficiency. e.g. a suitable injection site,RNAi efficiency. e.g. a suitable injection site,
injection volume, multi-injection.injection volume, multi-injection.
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AbstractAbstract
A serious shortcoming of many insecticides is thatA serious shortcoming of many insecticides is that
they can kill non-target species.they can kill non-target species.
To address this issue, the sequence specificity ofTo address this issue, the sequence specificity of
RNAi is harnessed to design orally-deliveredRNAi is harnessed to design orally-delivered
dsRNAs that selectively kill target species.dsRNAs that selectively kill target species.
Even closely related species can be selectivelyEven closely related species can be selectively
killed by feeding on dsRNAs that target the morekilled by feeding on dsRNAs that target the more
variable regions of genes, such as the 3’ UTRs.variable regions of genes, such as the 3’ UTRs.
05/25/1505/25/15 S. S. JenaS. S. Jena

Result & discussionResult & discussion
Designed species-specific dsRNAs to silence theDesigned species-specific dsRNAs to silence the
gene encoding the E-subunit of V-ATPase ingene encoding the E-subunit of V-ATPase in
four insect species: the fruit flyfour insect species: the fruit fly D. melanogasterD. melanogaster,,
the flour beetlethe flour beetle T. castaneumT. castaneum, the tobacco, the tobacco
hornwormhornworm M. sextaM. sexta, and the pea aphid, and the pea aphid A. pisumA. pisum..
V-ATPase is a membrane-bound protein that actsV-ATPase is a membrane-bound protein that acts
as a proton pump to establish the pH gradientas a proton pump to establish the pH gradient
within the gut lumen of many insects. Silencingwithin the gut lumen of many insects. Silencing
of the gene prevent growth and maturation ofof the gene prevent growth and maturation of
the larvae.the larvae.
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Cont.Cont.
All insects were first fed a series ofAll insects were first fed a series of
concentrations of conspecific dsRNA toconcentrations of conspecific dsRNA to
determine the LCdetermine the LC5050 values after a 1 weekvalues after a 1 week
period of feeding.period of feeding.
To examine the specificity of the dsRNA toTo examine the specificity of the dsRNA to
selectively kill the target species, the fourselectively kill the target species, the four
species of insects were fed each dsRNA atspecies of insects were fed each dsRNA at
the estimated LCthe estimated LC7575 dose for the targeteddose for the targeted
species.species.
05/25/1505/25/15 S. S. JenaS. S. Jena

Cont.Cont.
When insects were fed on a diet laced withWhen insects were fed on a diet laced with D.D.
melanogastermelanogaster-specific vATPase dsRNA only-specific vATPase dsRNA only
D. melanogasterD. melanogaster showed any evidence ofshowed any evidence of
reduced growth and development, while thereduced growth and development, while the
other three species were unaffected by theother three species were unaffected by the
Drosophila dsRNA.Drosophila dsRNA.
Similarly, by feeding each of the other threeSimilarly, by feeding each of the other three
species conspecific dsRNA, we selectivelyspecies conspecific dsRNA, we selectively
killed each species, without adverselykilled each species, without adversely
affecting any of the others.affecting any of the others.
05/25/1505/25/15 S. S. JenaS. S. Jena

Cont.Cont.
Each panel shows theEach panel shows the
mortalities resulting frommortalities resulting from
feeding on one species-feeding on one species-
specific dsRNA.specific dsRNA.
A)A) D. melanogasterD. melanogaster dsRNA;dsRNA;
B)B) M.M. sextasexta dsRNA;dsRNA;
C)C) T. castaneumT. castaneum dsRNA;dsRNA;
D)D) A. pisumA. pisum dsRNA.dsRNA.
Black bars representBlack bars represent
treatments with gus-dsRNAtreatments with gus-dsRNA
and white bars representand white bars represent
treatments with thetreatments with the
species-specific dsRNA.species-specific dsRNA.
05/25/1505/25/15
dsRNA-induced mortality in
four insect species

Conclusion of the studyConclusion of the study
The level of sequence divergence of the vATPaseThe level of sequence divergence of the vATPase
genes among the species tested was sufficientgenes among the species tested was sufficient
enough to identify portions of the gene thatenough to identify portions of the gene that
showed no 19–21 nt overlap, and therebyshowed no 19–21 nt overlap, and thereby
prevented RNAi induced silencing in the non-prevented RNAi induced silencing in the non-
target species.target species.
The appealing aspect of using dsRNA as aThe appealing aspect of using dsRNA as a
pesticide is that it is possible to design thepesticide is that it is possible to design the
pesticide to target only a single species or apesticide to target only a single species or a
group of related species, with minimal threat togroup of related species, with minimal threat to
other organisms.other organisms.
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Concluding RNAiConcluding RNAi
Remarkably, the RNAi machinery can handle
double-stranded RNA entering the cell as well as
double-stranded RNA generated within the cell.
The development of an organism and proper
function of its cells and tissues are dependent on
an intact RNAi machinery.
The discovery of RNAi has not only provided us with
a powerful new experimental tool to study the
function of genes but also raises expectations
about future applications of RNAi in medicine.
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med_res_avi.avi
05/25/1505/25/15 S. S. JenaS. S. Jena

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