RNAi is a powerful, conserved biological process through which the small, double-stranded RNAs specifically silence the expression of homologous genes, largely through degradation of their cognate mRNA.

1. RNA interference
RNAi
BY
Kamlesh Kumar Chandel
Ph.D. Scholar
Department of Genetics and Plant Breeding

2. Content
I. Overview view /
History
II. Mechanism /
Process
III. Function
IV. Applications
V. RNAi Glossary
VI. References
Nobel Prize in 2006

3. RNAi
 RNAi is a powerful, conserved biological process
through which the small, double-stranded RNAs
specifically silence the expression of homologous
genes, largely through degradation of their cognate
mRNA.
 its responsible for post-transcriptional gene silencing of the
gene from which it was derived
 Endogenous cellular mechanisms
 Effecter molecules for functional genomics
 Great potential as therapeutic agents for treatment
of human disease

4. RNA interference Technology
 RNAi is used to block the expression of genes and
create phenotypes that can potentially yield clues
about the function of these genes.
 In the post-genomic era, the elucidation of the
physiological function of genes has become the rate-limiting
step in the quest to develop ‘gene-based
drugs’ and RNAi could potentially play a pivotal role in
the validation of such novel drugs.
RNAi is a fantastic discovery , all the RNAi idea will be describing in these video
(1) http://www.youtube.com/watch?v=H5udFjWDM3E&feature=related
(2) http://www.youtube.com/watch?v=A-l8tqjm4Vg&feature=related
(3) http://www.youtube.com/watch?v=3kdhYCJFmZc&feature=related
(4) http://www.youtube.com/watch?v=kCxQdXX0Dbk
(5) http://www.youtube.com/watch?v=h1kayIVEfcY&feature=related

5. Time -
Line

6. Discovery of RNAi or
PTGS
(Post transcriptional gene
silencing)
First discovered in plants
(R. Jorgensen, 1990)
•When Jorgensen introduced
a re-engineered gene into
petunia that had a lot of
homology with an
endogenous petunia gene,
•both Also genes called became
Co-suppression
suppressed!
Suppression was mostly
due to increased
degradation of the mRNAs
(from the endogenous and
introduced genes)
Flowers from 3 different transgenic petunia
plants carrying copies of the chimeric DFR
gene above. The flowers had low DFR
mRNA levels in the non-pigmented areas,
but gene was still being transcribed.

7. RNA interference also reported
in
•Jorgensen 1990
•van der Krol 1990
Gene injection (pigmentation
Enzyme-petunias)
Expectation: more red color
Co-suppression of transgene
and endogenous gene.
Bill Douherty and
Lindbo 1993
•Gene injection with a
complete tobacco
etch virus particle.
•Expectation: virus
expression
Co-suppression of
transgene
and virus particles via
RNA.
Hamilton and Baulcombe 1998
•Identification of short antisense
RNA sequences
Fire and Mello 1998
Injection of dsRNA into C. elegans
RNA interference (RNAi) or gene
silencing
Ambros 1993 (2000)
Identification of small
RNA in C. elegans
(micro RNA)

8. RNAi discovered in Nematode {Caenorhabditis elegans} 1998
(first animal) while attempting to use antisense RNA in vivo
Nobel Prize in 2006
( Field of Physiology &
Medicine)
Craig Mello & Andrew Fire
Control “sense” RNAs also produced suppression of target gene!
sense RNAs were contaminated with dsRNA.
dsRNA was the suppressing agent.
-RNAi can be induced in C. elegans in
three simple ways:
-Injection of dsRNA into the worm
gonads
-Soaking the worms in dsRNA solution
-Feeding the worms engineered
bacteria producing dsRNA

9. Double-stranded RNA (dsRNA) induced interference
of the Mex-3 mRNA in the Nematode
{Caenorhabditis elegans}
Antisense RNA (c) or
dsRNA (d) for the mex-
3 (mRNA) was injected
into C. elegans
ovaries, and then mex-
3 mRNA was detected
in embryos by in situ
hybridization with a
mex-3 probe.
(a) control embryo
(b) control embryo hyb.
with mex-3 probe
Conclusions: (1) dsRNA reduced mex-3 mRNA better than antisense
mRNA. (2) the suppressing signal moved from cell to cell.

10. Mechanism/Process
 A cellular mechanism that degrades unwanted RNAs
in the cytoplasm but not in the nucleus
What happens ?
 dsRNA is processed into shorter interfering (siRNAs)
that guide the targeted cleavage of homologous RNA.

11. Mechanism of RNA interference
(RNAi)
 dsRNA are chopped into
short interfering RNAs
(siRNA) by Dicer.
 2. The siRNA-Dicer
complex recruits
additional components
to form an RNA-Induced
Silencing Complex
(RISC). The siRNA
unwinds.
 3. The unwound siRNA
base pairs with
complementary mRNA,
thus guiding the RNAi
machinery to the target
mRNA.
 4. The target mRNA is
effectively cleaved and
subsequently degraded
– resulting in gene
silencing.

12. A model for the mechanism of RNAi
- Silencing triggers in the form of double-stranded
RNA may be presented in the cell as
synthetic RNAs, replicating viruses or may be
transcribed from nuclear genes.
- These are recognized and processed into
small interfering RNAs by Dicer.
- The duplex siRNAs are passed to RISC
(RNA-induced silencing complex)
- The complex becomes activated by unwinding
of the duplex.
- Activated RISC complexes can regulate gene
expression at many levels:
•Promoting RNA degradation
•Translational inhibition
•Chromatin remodelling
- Amplification of the silencing signal in plants
may be accomplished by siRNAs priming RNA-directed
RNA polymerase (RdRP)-dependent
synthesis of new dsRNA.

13. Mechanism of RNA
interference

14. Mechanism of RNAi : Role of
Dicer
 Cells (plants and animals) undergoing RNAi
contained small fragments (~25 nt) of the RNA
being suppressed.
 A nuclease (Dicer) was purified from Drosophila
embryos that still had small RNA fragments
associated with it, both sense and antisense.
 The Dicer gene is found in all organisms that
exhibit RNAi, and mutating it inhibits the RNAi
Coenfcfleucsito.n: Dicer is the endonuclease that degrades dsRNA
into 21-24 nt fragments, and in higher eukaryotes also pulls
the strands apart via intrinsic helicase activity.

15. RNAi FUNCTIONS
- To regulates expression of protein coding
genes
- To mediates resistance to both exogenous
parasitic and exogenous pathogenic
nucleic acid
- To used experimentally to block gene
expression

16. RNAi applications
 Genome-wide RNAi screening
 Done in C. elegans
 19 757 protein coding genes (predicted)
 16 757 inactivated using RNAi
 New standard for systematic genome wide functional studies
 RNAi as a solution for mammalian genetics
 Defense against Infection by viruses
 Potential therapeutic use
 Prevents viral infection
 Inhibits the expression of viral antigens
 Suppresses the transcription of viral genome
 Blocks viral replication
 Silences viral accessory genes
 Hinders the assembly of viral particles & Displays roles in virus-host
interactions

17. Responses to Mechanical Stimuli
HIV levels can
be reduced by
30-50 fold by
17
siRNA!!!

19. Biotechnology & Agriculture
 RNA interference has been used for applications in
biotechnology, particularly in the engineering of food plants
that produce lower levels of natural plant toxins. Such
techniques take advantage of the stable and heritable
RNAi phenotype in plant stocks.
 For example, cotton seeds are rich in dietary protein but
naturally contain the toxic terpenoid product gossypol,
making them unsuitable for human consumption.
 RNAi has been used to produce cotton stocks whose
seeds contain reduced levels of delta-cadinene synthase,
a key enzyme in gossypol production, without affecting the
enzyme's production in other parts of the plant, where
gossypol is important in preventing damage from plant
pests.

20. Biotechnology & Agriculture
 Similar efforts have been directed toward the reduction of
the cyanogenic natural product linamarin in cassava
plants.
 Although no plant products that use RNAi-based genetic
engineering have yet passed the experimental stage,
development efforts have successfully reduced the levels
of allergens in tomato plants and decreased the
precursors of likely carcinogens in tobacco plants.
 Other plant traits that have been engineered in the
laboratory include the production of non-narcotic natural
products by the opium poppy, resistance to common plant
viruses, and fortification of plants such as tomatoes with
dietary antioxidants.

21. RNA interference
characteristics
 dsRNA needs to be directed against an exon,
not an intron in order to be effective
 Homology of the dsRNA and the target
gene/mRNA is required
 Targeted mRNA is lost (degraded) after RNAi
 The effect is non-stoichiometric; small
amounts of dsRNA can wipe out an excess of
mRNA (pointing to an enzymatic mechanism)
 ssRNA does not work as well as dsRNA

22. Advantage of RNAi
 Downregulation of gene expression simplifies
"knockout" analysis.
 Easier than use of antisense oligonucleotides.
siRNA more effective and sensitive at lower
concentration.
 Cost effective
 High Specifity
middle region 9-14 are most sensitive
 With siRNA, the researcher can simultaneously
perform experiments in any cell type of interest
 Can be labelled
 Ease of transfection by use of vector

23. Importance of RNAi
 Powerful for analyzing unknown genes in sequenced
genomes.
  efforts are being undertaken to target every human
gene via siRNAs
 Faster identification of gene function
 Gene therapy: down-regulation of certain genes/ mutated
alleles
 Cancer treatments
 knock-out of genes required for cell proliferation
 knock-out of genes encoding key structural proteins

24. http://www.rnaiweb.com/RNAi/RNAi_Web/

25. http://www.rnainterference.org/Sequences.html

26. RNAi Glossary
 Dicer – Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded
RNAs. Dicer processes long dsRNA into siRNA of 21-23 nt.
 Interferon – A small and highly potent molecule that functions in an autocrine and paracrine manner,
and that induces cells to resist viral replication. This term is related to RNAi because in mammals
introduction of dsRNA longer than 30 nt induces a sequence-nonspecific interferon response.
 Micro-RNA – Micro-RNAs (miRNA) are single-stranded RNAs of 22-nt that are processed from ~70-nt
hairpin RNA precursors by Rnase III nuclease Dicer. Similar to siRNAs, miRNAs can silence gene
activity via destruction of homologous mRNA in plants or blocking its translation in plants and animals.
 Post-Transcriptional Gene Silencing – Post-transcriptional gene silencing (PTGS) is a sequence-specific
RNA degradation system designed to act as an anti-viral defense mechanism. A form of PTGS
triggered by transgenic DNA, called co-suppression, was initially described in plants and a related
phenomenon, termed quelling, was later observed in the filamentous fungus Neurospora crassa
 Ribozyme – Ribozymes are RNA molecules that act as enzymes in the absence of proteins.
 RNA Interference – RNA Interference (RNAi), a term coined by Fire et al in 1998, is a phenomenon that
small double-stranded RNA (referred as small interference RNA or siRNA) can induce efficient
sequence-specific silence of gene expression.
 RNA-Directed DNA Methylation – RNA-directed DNA methylation (RdDM) is an RNA directed
silencing mechanism found in plants. Similar to RNA interference (RNAi), RdDM requires a double-strand
RNA that is cut into short 21-26-nt fragments. DNA sequences homologous to these short RNAs
are then methylated and silenced.
 RNA-Induced Silencing Complex – RNA-induced silencing complex (RISC) is an siRNA-directed
endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target.
 RNAi Trigger – RNAi triggers are double-stranded RNAs containing 21-23 nt sense and antisens
strands hybridized to have 2 nt overhangs at both 3' ends.
 Small Interfering RNA – Small Interfering RNA (siRNA) is 21-23-nt double-strand RNA. It guides the
cleavage and degradation of its cognate RNA.
Helicase – Enzyme responsible for unwinding double stranded molecule

27. References
http://www.rna.com/
http://www.cambridge.org/catalogue/catalogue.asp?isbn=0511081316
http://www.youtube.com/watch?v=H5udFjWDM3E&feature=related
http://www.youtube.com/watch?v=kCxQdXX0Dbk
http://arabidopsis.info/students/rohan/mechanismrnai.html
http://www.youtube.com/watch?v=h1kayIVEfcY&feature=related
* Simple, Efficient Production of Short Double-Stranded RNA Using RNase III (Judith E. Meis, EPICENTRE).website :
http://www.epibio.com/pdfforum/9_3dsrnarnaseiii.pdfmicroRNA formation and function
http://www.youtube.com/watch?v=_-9pROnSD-A
http://www.rnaiweb.com/RNAi/RNAi_Web_Resources/RNAi_Companies/RNAi_Therapeutics/index.html
http://www.alnylam.com/Programs-and-Pipeline/Programs/Liver-Cancer.php
(1)* Meister ,G., Tuschl ,T.. (2004). Mechanisms of gene silencing by double-stranded RNA. Natural. 431(7006). 343-9.
(2)* Kedde ,M., Strasser ,M.J., Boldajipour ,B., Oude Vrielink ,J.A., Slanchev ,K., le Sage ,C., Nagel ,R., Voorhoeve ,P.M., van
Duijse ,J., Ørom ,U.A., Lund ,A.H., Perrakis ,A., Raz ,E., Agami ,R.. (2007). RNA-binding protein Dnd1 inhibits microRNA
access to target mRNA. cell. 131(7). 1273-86.
(3)*Klionov ,M.S., Stoliarenko ,A.D., Riazanskiĭ ,S.S., Sokolova ,O.A., Konstantinov ,I.N., Gvozdev ,V.A.. (2007). Role of short RNAs in
regulating the expression of genes and mobile elements in germ cells. ONTOGENES. 38(3). 213-27.
(4)*Aalto ,A.P., Sarin ,L.P., van Dijk ,A.A., Saarma ,M., Poranen ,M.M., Arumäe ,U., Bamford ,D.H.. (2007). Large-scale
production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase.
RNA(New York .N.Y.). 13(3), 422-9.
http://books.google.jo/books?id=bjAm2mTbnPoC&pg=PA56&lpg=PA56&dq=repeat+-
associated+short+interfering+RNAs+(rasiRNAs)&source=bl&ots=ii34nFhrYx&sig=ABKvNRISLOdkGX0zwC4sW0i-qbU&hl=en&ei=SMXxSanVBcLm-
Ab1wLidDw&sa=X&oi=book_result&ct=result&resnum=1#PPP5,M1

28. Thank you