RNA-interference

INTRODUCTION: silencing of homogenous gene expression triggered by dsRNA is

called as RNAi introduction of long dsRNA into the cell of plant, invertebrates as well as
mammals leads to the sequence spscific degradation of the homologous gene transcripts .
long dsRNA mol is cleaved by an RNAs3 enzyme called dicer this generates small 21-23
nt long dsRNA mol called as small interfering RNA (siRNA). siRNA binds to the
protein complex called RNA induced silencing complex which has helicase activity that
unwind two strands of RNA mol. The antisense RNA strads so generated pair with the
target RNA mol. & endonuclease activity then hydrolyse the target RNA at the site where
the antisense RNA strand is bound.& thus it cause the permanent inhibition of the gene.

TERMINOLOGIES RELATED TO RNAi

• 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
• RNAi - 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
• Argonaute - A family of proteins containing multiple domains and involved in
RNA interference (RNAi). Argonatue is the main component of RNAi effector
complex, the so called siRNA induced silencing complex (RISC) and aids in
target recognition and cleavage during RNAi.
• 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.
• 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.
• Ribozyme - Ribozymes are RNA molecules that act as enzymes in the absence
of proteins.
• 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. 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 (siRNA) - Small Interfering RNA (siRNA) is
21~23-nt double-stranded RNA molecules. It guides the cleavage and degradation
of its cognate RNA
HISTORICAL BACKGROUND

Guo S, and First noticed that sense RNA was as effective as antisense RNA
1995 C. elegans
Kemphues KJ. for suppressing gene expression in worm

First described RNAi phenomenon in C. elegans by injecting

1998 Fire et al. dsRNA into C. elegans which led to an efficient sequence- C. elegans
specific silencing and coined the term "RNA Interference".

Reported processing of long dsRNA by Rnase III (Dicer) into

2000 Zamore et al. Drosophila
shorter fragments of 21-23-nt intervals in Drosophila extracts

2001 Cloned Dicer, the RNase III enzyme that is evolutionarily

Bernstein et al. conserved and contains helicase and PAZ domains, as well as C. elegans
　 two dsRNA-binding domains.

Tuschl T and
2001 First described RNAi in mammalian cells Mammals
colleagues

Paddison et al.
Short hairpin RNAs (shRNAs) induce sequence-specific
2003 Sui et al. Mammals
silencing in mammalian cells.
Paul et al.

First reported that siRNAs can be used therapeutically in whole

2003 Song et al. Mammals
animals.

Kawasaki and Taira First observed that siRNA silences gene at transcriptional level
2004 Human
Morris et al. possibly through directing de novo DNA methylation.

Acuity First phase I clinical trial of siRNA drug for age-related macular
2004 Human
Pharmaceuticals degeneration (AMD)

Andrew Fire and Won Noble Prize in Physiology or Medicine for discovering
2006
Craig Mello RNAi mechanism.

First reported that small dsRNA induces gene expression

2006 Li et al Human
activation, a phenomenon termed as RNAa

Mechanism of RNA interference

A. On entering the cell, long dsRNAs act as a trigger of RNAi process.

B. It is first processed by the RNAse III enzyme Dicer in an ATP-dependent reaction.

C. Dicer processes dsRNAs into 21-23 nt short interfering RNA (siRNA) with 2-nt 3'
overhangs. siRNA can also be synthesized outside the cell and then be introduced into a
cell.

D. The siRNAs are incorporated into the RNA-inducing silencing complex (RISC) which
consists of an Argonaute (Ago) protein as one of its main components. Ago cleaves and
discards the passenger (sense) strand of the siRNA duplex leading to activation of the
RISC.

E and F. The remaining guide (antisense) strand of the siRNA guides RISC to its
homologous mRNA, resulting in the endonucleolytic cleavage of the target mRNA

ADVANTAGES:
1) Role in medicine :The dsRNAs that trigger RNAi may be usable as drugs
eg treatment of macular degeneration
2) antiviral defence mechanism
3)Analysis of gene function in invertebrates , plants & mammals
4)Used to produce transgenic plants
ex RNAi has been used to produce low caffeine coffee

LIMITATION
1)Short lived: RNAi based on exogenous siRNAs is short lived for 4-6 days.
2)Longer RNA molecule trigger antiviral response & leads to suppression of gene
expression.
3)Is not highly efficient.