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

Thank you