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BY
Kamlesh Kumar ChandelPh.D. Scholar
Department of Genetics and Plant Breeding
RNAiRNA interference
Content
I. Overview view /
History
II. Mechanism /
Process
III. Function
IV. Applications
V. RNAi Glossary
VI. References
Nobel Prize in 2006
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
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.
(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
RNAi is a fantastic discovery , all the RNAi idea will be describing in these video
Time -
Line
Discovery of RNAi or
PTGS(Post transcriptional gene
silencing)
•Also called Co-suppression
Suppression was mostly
due to increased
degradation of the mRNAs
(from the endogenous and
introduced genes)
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 genes became
suppressed!
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.
•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)
RNA interference also reported
in
Nobel Prize in 2006
( Field of Physiology &
Medicine)
-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
RNAi discovered in Nematode {Caenorhabditis elegans} 1998
(first animal) while attempting to use antisense RNA in vivo
Craig Mello & Andrew Fire
Control “sense” RNAs also produced suppression of target gene!
sense RNAs were contaminated with dsRNA.
dsRNA was the suppressing agent.
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.
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.
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.
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.
Mechanism of RNA
interference
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
effect.Conclusion: 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.
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
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
Responses to Mechanical Stimuli
17
HIV levels can
be reduced by
30-50 fold by
siRNA!!!
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.
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.
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
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
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
http://www.rnaiweb.com/RNAi/RNAi_Web/
http://www.rnainterference.org/Sequences.html
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
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