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Ingrid Remy
Université de Montréal, Dépt de Biochimie,
C.P. 6128, Succursale centre-ville, Montréal,
Québec, H3C 3J7, Canada.
e-mail: ingrid.remy@umontreal.ca
TRENDS in Biotechnology
Vol.20 No.2 February 2002
http://tibtech.trends.com 0167-7799/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(01)01900-X
49
Research Update
A new approach to the inhibition of gene expression
Natasha J. Caplen
The induction of a specific down-regulation
in gene expression is an important research
tool.Recently,several related processes
including post-transcriptional gene
silencing (PTGS) and RNA interference
(RNAi) have been identified that generate
sequence-specific inhibition of gene
expression.Although the physiological
function of PTGS and RNAi is still being
elucidated,these pathways have been used
to rapidly determine gene function.The
recent observation of RNAi in mammalian
cells extends the possible applications of
these mechanisms.
Although PTGS (first identified in plants
and fungi) and RNAi (first observed in
Caenorhabditis elegans and Drosophila)
were identified independently, genetic
and biochemical analyses suggest a
strong evolutionary link between these
multi-step pathways [1–3]. These
pathways share some common elements,
including that they are triggered by
double stranded RNA (dsRNA) and that
the trigger dsRNA is enzymatically
processed into small fragments of 20 to
25 nucleotides (nts) in length – the
fragments appear to mediate the
sequence-specific recognition of the target
single stranded RNA (ssRNA) [4,5]. The
enzyme responsible for the cleavage of the
triggering dsRNA has been identified as a
dsRNA-specific RNase, called Dicer, that
generates dsRNA breakdown products
resembling those generated by RNase III
cleavage with a 5′phosphate group,
a3′hydroxyl group and two or three nt 3′
overhangs [6]. Evidence that these small
dsRNAs or short interfering RNAs
(siRNAs) act as guides for the enzymatic
complex required for the degradation of
the target ssRNA includes the cleavage
of the target mRNAat regular intervals
of ~21–23 nts in the region corresponding
to the input dsRNA [5]. Dicer, which also
has a potential helicase activity and an
additional domain of undetermined
function, has been shown to be essential
for RNAi in Drosophila and C. elegans
[6,7]. In addition, Dicer has a role in the
processing of transcripts required for
normal development [8,9]. So far, only one
protein component of the multi-subunit
complex or RNA-induced silencing
complex (RISC) required for the cleavage
of the target RNA has been identified.
This protein, Argonaute2, is a member of
a large family of proteins involved in germ
cell and stem cell production that contain
conserved domains known as PAZ and
PIWI domains [10]. Several genes with
PAZ or PIWI domains have been linked
with RNAi, including Dicer but, as yet, it
is unclear how most of these proteins are
involved in PTGS and RNAi [11]. Figure 1
shows a model of some aspects of dsRNA
mediated gene silencing. In some species,
there is evidence for the role of a RNA-
dependent RNA polymerase in PTGS and
RNAi [12,13], processes that induce
systemic spread of the gene silencing
TRENDS in Biotechnology
dsRNA
Invertebrate
systems
Mammalian
somatic
cells
mRNA target
Degraded transcript
RISC
Dicer
siRNA
PAZ/PIWI domain
containing proteins
plus other proteins
and factors
Synthetic
small
dsRNA
or
siRNA
Recycling of
RISC as a whole
or re-assembly
of components
following
mediation of
transcript
degradation
Fig. 1.A model of parts of the proposed RNAi pathway in
invertebrate and mammalian somatic cells. Processed or
synthesized dsRNAs of ∼20–23 nucleotides interact with
several proteins and factors to generate a complex that
mediates sequence-specific degradation of the target
transcript. It is currently unclear as to what extent the final
structure of the dsRNAs that mediate optimal inhibition
in somatic mammalian cells will resemble the naturally
processed siRNAs (short interfering RNAs) identified in
plants and invertebrates and so these structures have
been referred to as both small dsRNAs and siRNAs.
Abbreviation: RISC, RNA interfering silencing complex.