RNA Interference and Gene Silencing: History and Overview
May 20, 2002
Post-transcriptional gene silencing (PTGS), which was
initially considered a bizarre phenomenon limited to petunias and a few
other plant species, is now one of the hottest topics in molecular biology
(1).
In the last few years, it has become clear that PTGS occurs in both plants
and animals and has roles in viral defense and transposon silencing
mechanisms. Perhaps most exciting, however, is the emerging use of PTGS
and, in particular, RNA interference (RNAi) — PTGS initiated by the
introduction of double-stranded RNA (dsRNA) — as a tool to knock out
expression of specific genes in a variety of organisms (reviewed in 1-3).
How was RNAi discovered? How does it work? Perhaps more
importantly, how can it be harnessed for functional genomics experiments?
This article will briefly answer these questions and provide you with
resources to find in depth information on PTGS and RNAi research.
A Bizarre Phenomenon is Discovered:
Cosuppression and PTGS in Plants
More
than a decade ago, a surprising observation was made in petunias. While
trying to deepen the purple color of these flowers, Rich Jorgensen and
colleagues introduced a pigment-producing gene under the control of a
powerful promoter. Instead of the expected deep purple color, many of the
flowers appeared variegated or even white. Jorgensen named the observed
phenomenon "cosuppression", since the expression of both the introduced
gene and the homologous endogenous gene was suppressed (1-5).
First thought to be a quirk of petunias, cosuppression
has since been found to occur in many species of plants. It has also been
observed in fungi, and has been particularly well characterized in
Neurospora crassa, where it is known as "quelling" (1-3).
But what causes this gene silencing effect? Although
transgene-induced silencing in some plants appears to involve
gene-specific methylation (transcriptional gene silencing, or TGS), in
others silencing occurs at the post-transcriptional level
(post-transcriptional gene silencing, or PTGS). Nuclear run-on experiments
in the latter case show that the homologous transcript is made, but that
it is rapidly degraded in the cytoplasm and does not accumulate (1,
3,
6).
Introduction of transgenes can trigger PTGS, however
silencing can also be induced by the introduction of certain viruses (2,
3).
Once triggered, PTGS is mediated by a diffusible, trans-acting molecule.
This was first demonstrated in Neurospora, when Cogoni and
colleagues showed that gene silencing could be transferred between nuclei
in heterokaryotic strains (1,
7).
It was later confirmed in plants when Palauqui and colleagues induced PTGS
in a host plant by grafting a silenced, transgene-containing source plant
to an unsilenced host (8).
From work done in nematodes and flies, we now know that the trans-acting
factor responsible for PTGS in plants is dsRNA (1-3).
Gene Silencing by dsRNA:
RNA Interference
RNAi Is Discovered in Nematodes
The first evidence that dsRNA could lead to gene silencing
came from work in the nematode Caenorhabditis elegans. Seven years
ago, researchers Guo and Kemphues were attempting to use antisense RNA to
shut down expression of the par-1 gene in order to assess its
function. As expected, injection of the antisense RNA disrupted expression
of par-1, but quizzically, injection of the sense-strand control
did too (9).
This result was a puzzle until three years later. It was
then that Fire and Mello first injected dsRNA — a mixture of both sense
and antisense strands — into C. elegans (10).
This injection resulted in much more efficient silencing than injection of
either the sense or the antisense strands alone. Indeed, injection of just
a few molecules of dsRNA per cell was sufficient to completely silence the
homologous gene's expression. Furthermore, injection of dsRNA into the gut
of the worm caused gene silencing not only throughout the worm, but also
in its first generation offspring (10).
The potency of RNAi inspired Fire and Timmons to try
feeding nematodes bacteria that had been engineered to express dsRNA
homologous to the C. elegans unc-22 gene. Surprisingly,
these worms developed an unc-22 null-like phenotype (11-13).
Further work showed that soaking worms in dsRNA was also able to induce
silencing (14).
These strategies, whereby large numbers of nematodes are exposed to dsRNA,
have enabled large-scale screens to select for RNAi-defective C.
elegans mutants and have led to large numbers of gene knockout studies
within this organism (15-18).
RNAi in Drosophila
RNAi has also been observed in Drosophila. Although a strategy
in which yeast were engineered to produce dsRNA and then fed to fruit
flies failed to work, microinjecting Drosophila embryos with dsRNA
does effect silencing (2).
Silencing can also be induced by "shooting" dsRNA into Drosophila
embryos with a "gene gun" or by engineering flies to carry DNA containing
an inverted repeat of the gene to be silenced. Over the last few years,
these RNAi strategies have been used as reverse genetics tools in Drosophila
organisms, embryo lysates, and cells to characterize various loss-of-function
phenotypes (2,
19-23).
GLOSSARY OF TERMS
Cosuppression - Silencing of
an endogenous gene caused by the introduction of a transgene or
infection by a virus. This term, which can refer to silencing at
the post-transcriptional (PTGS) or transcriptional (TGS) level,
has been primarily adopted by researchers working with plants.
Post-transcriptional Gene Silencing
(PTGS) - Silencing of an endogenous gene caused by the introduction
of a homologous dsRNA, transgene or virus. In PTGS, the transcript
of the silenced gene is synthesized but does not accumulate because
it is rapidly degraded. This is a more general term than RNAi, since
it can be triggered by several different means.
Quelling - PTGS
in Neurospora crassa induced by the introduction of a transgene.
RISC - RNA-induced
silencing complex. A nuclease complex, composed of proteins and
siRNA (see below), that targets and destroys endogenous mRNAs complementary
to the siRNA within the complex.
RNA interference (RNAi)
- Post-transcriptional gene silencing (PTGS) induced by the
direct introduction of dsRNA. The term "RNA interference" was first
used by researchers studying C. elegans.
siRNAs - Small
interfering RNAs. Current models of PTGS indicate that these 21-23
nucleotide dsRNAs mediate PTGS. Introduction of siRNAs can induce
PTGS in mammalian cells. siRNAs are apparently produced in vivo
by cleavage of dsRNA introduced directly or via a transgene or virus.
Amplification by an RNA-dependent RNA polymerase (RdRP) may occur
in some organisms. siRNAs are incorporated into the RNA-induced
silencing complex (RISC), guiding the complex to the homologous
endogenous mRNA where the complex cleaves the transcript.
The Biochemical Mechanism of RNAi
So how does injection of dsRNA lead to gene silencing?
Many research groups have diligently worked over the last few years to
answer this important question. A key finding by Baulcombe and Hamilton
provided the first clue. They identified RNAs of ~25 nucleotides in plants
undergoing cosuppression that were absent in non-silenced plants. These
RNAs were complementary to both the sense and antisense strands of the
gene being silenced (24).
Further work in Drosophila — using embryo lysates
and an in vitro system derived from S2 cells — shed more light on the
subject (3,
25,
26).
In one notable series of experiments, Zamore and colleagues found that
dsRNA added to Drosophila embryo lysates was processed to 21-23
nucleotide species. They also found that the homologous endogenous mRNA
was cleaved only in the region corresponding to the introduced dsRNA and
that cleavage occurred at 21-23 nucleotide intervals (26).
Rapidly, the mechanism of RNAi was becoming clear.
Current Models of the RNAi
Mechanism
Both biochemical and genetic approaches (see "The
Genes and Enzymes Involved in PTGS and RNAi" below for a discussion of
genetic approaches used to undersand RNAi) have led to the current models
of the RNAi mechanism. In these models, RNAi includes both initiation and
effector steps (27,
see also a Flash animation of "How
Does RNAi Work?", from reference 3).
In the initiation step, input dsRNA is digested into
21-23 nucleotide small interfering RNAs (siRNAs), which have also been
called "guide RNAs" (reviewed in 3,
18,
27).
Evidence indicates that siRNAs are produced when the enzyme Dicer, a
member of the RNase III family of dsRNA-specific ribonucleases,
processively cleaves dsRNA (introduced directly or via a transgene or
virus) in an ATP-dependent, processive manner. Successive cleavage events
degrade the RNA to 19-21 bp duplexes (siRNAs), each with 2-nucleotide 3'
overhangs (27,
28).
In the effector step, the siRNA duplexes bind to a
nuclease complex to form what is known as the RNA-induced silencing
complex, or RISC. An ATP-depending unwinding of the siRNA duplex is
required for activation of the RISC. The active RISC then targets the
homologous transcript by base pairing interactions and cleaves the mRNA
~12 nucleotides from the 3' terminus of the siRNA (3,
18,
27,
29).
Although the mechanism of cleavage is at this date unclear, research
indicates that each RISC contains a single siRNA and an RNase that appears
to be distinct from Dicer (27).
Because of the remarkable potency of RNAi in some organisms,
an amplification step within the RNAi pathway has also been proposed.
Amplification could occur by copying of the input dsRNAs, which would
generate more siRNAs, or by replication of the siRNAs themselves (see
"Possible Role for RNA-dependent RNA Polymerase" below). Alternatively
or in addition, amplification could be effected by multiple turnover events
of the RISC (3,
18,
27).
The Genes and Enzymes Involved in PTGS and RNAi
Possible Role for RNA-dependent RNA
Polymerase
Genetic screens in Neurospora, C.
elegans, and Arabidopsis have identified several genes that
appear to be crucial for PTGS and RNAi. Several of these, including
Neurospora qde-1, Arabidopsis SDE-1/SGS-2 and C. elegans
ego-1, appear to encode RNA-dependent RNA polymerases (RdRPs). At
first glance, it might be assumed that this is proof that an RdRP activity
is required for RNAi. Certainly the existence of an RdRP might explain the
remarkable efficiency of dsRNA-induced silencing if it amplifed either the
dsRNA prior to cleavage or the siRNAs directly. But mutants of these genes
have varying phenotypes, which makes the role of RdRP in RNAi difficult to
discern (1,
3,
17,
18).
In C. elegans ego-1 mutants ("ego" stands for
"enhancer of glp-1"), RNAi functions normally in somatic cells, but
is defective in germline cells where ego-1 is primarily expressed.
In Arabidopsis SDE-1/SGS-2 mutants ("SGS" stands for
suppressor of gene silencing), siRNAs are produced when dsRNA is
introduced via an endogenously replicating RNA virus, but not when
introduced by a transgene. It has been proposed that perhaps the viral
RdRP is substituting for the Arabidopsis enzyme in these mutants
(1,
3,
17,
18).
Although no homolog of an RdRP has been found in flies or humans, an RdRP
activity has recently been reported in Drosophila embryo lysates
(30).
One model of amplification, termed the "random degradative PCR" model,
suggests that an RdRP uses the guide strand of an siRNA as a primer for
the target mRNA, generating a dsRNA substrate for Dicer and thus more
siRNAs (27,
30).
Evidence supporting this model has been found in worms, whereas
experimental results refuting the model have been obtained from
Drosophila embryo lysates (26,
27).
RNAi Initiators
Two C.
elegans genes, rde-1 and rde-4 ("rde" stands for "RNAi
deficient"), are believed to be involved in the initiation step of RNAi.
Mutants of these genes produce animals that are resistant to silencing by
injection of dsRNA, but silencing can be effected in these animals by the
transmission of siRNA from heterozygous parents that are not silencing
deficient. The C. elegans rde-1 gene is a member of a large family
of genes and is homologous to the Neurospora qde-2 ("qde" stands
for "quelling deficient") and the Arabidopsis AGO1 genes ("AGO"
stands for "argonaute"; AGO1 was previously identified to be
involved in Arabidopsis development). Although the function of
these genes in PTGS is unclear, a mammalian member of the RDE-1 family has
been identified as a translation initiation factor. Interestingly,
Arabidopsis mutants of AGO1, which are defective for
cosuppression, also exhibit defects in leaf development. Thus some
processes or enzymes involved in PTGS may also be involved in development
(1,
3,
17,
18).
RNAi Effectors
Important
genes for the effector step of PTGS include the C. elegans rde-2
and mut-7 genes. These genes were initially identified from
heterozygous mutant worms that were unable to transmit RNAi to their
homozygous offspring (16).
Worms with mutated rde-2 or mut-7 genes exhibit defective
RNAi, but interestingly, they also demonstrate increased levels of
transposon activity. Thus, silencing of transposons appears to occur by a
mechanism related to RNAi and PTGS. Although the rde-2 gene product
has not yet been identified, the mut-7 gene encodes a protein with
homology to the nuclease domains of RNase D and a protein implicated in
Werner syndrome (a rapid aging disease) in humans (1,
3,
17,
18,
31).
Perhaps this protein is a candidate for the nuclease activity required for
target RNA degradation.
PTGS Has Ancient Roots
Discoveries from both genetic and biochemical approaches point
to the fact that PTGS has deep evolutionary roots. Proposals have been put
forth that PTGS evolved as a defense mechanism against transposons or RNA
viruses, perhaps before plants and animals diverged (1,
3,
17,
18).
Interestingly, it was noted by many researchers that
disruption of genes required for RNAi often causes severe developmental
defects. This observation suggested a link between RNAi and at least one
developmental pathway.
A group of small RNA molecules, known as small temporal
RNAs (stRNAs), regulates C. elegans developmental timing through
translational repression of target transcripts. Research indicates that
the C. elegans lin-4 and let-7 stRNAs are generated
from 70-nt transcripts following the folding of these longer transcripts
into a stem-loop structure. The folded RNA molecules are cleaved to
produce 22-nt stRNAs by the enzyme Dicer (called DCR-1 in C.
elegans). Thus Dicer generates both siRNAs and stRNAs, and represents
an intersection point for the RNAi and stRNA pathways (32-34).
Recently, nearly 100 additional ~22 nt RNA molecules,
termed microRNAs (miRNAs), were identified in Drosophila, C.
elegans, and HeLa cells (35-38).
Much like lin-4 and let-7, these miRNAs are formed from
precursor RNA molecules that fold into a stem-loop secondary structure.
The newly discovered ~22 nt miRNAs are believed to play a role in
regulation of gene expression, and at least two of them are known to
require Dicer for their production (37).
It appears that the use of small RNAs for both gene regulation and RNAi is
a common theme throughout evolution.
Inducing RNAi in Mammalian Cells — From Mechanism to
Application
Non-specific Gene Silencing by Long
dsRNAs
While the natural presence of RNAi had been observed in a
variety of organisms (plants, protozoa, insects, and nematodes), evidence
for the existence of RNAi in mammalian cells took longer to establish.
Transfection of long dsRNA molecules (>30 nt) into most mammalian cells
causes nonspecific suppression of gene expression, as opposed to the
gene-specific suppression seen in other organisms. This suppression has
been attributed to an antiviral response, which takes place through one of
two pathways.
In one pathway, long dsRNAs activate a protein kinase,
PKR. Activated PKR, in turn phoshorylates and inactivates the translation
initiation factor, eIF2a, leading to repression of translation. (39)
In the other pathway, long dsRNAs activate RNase L, which leads to
nonspecific RNA degradation (40).
A number of groups have shown that the dsRNA-induced
antiviral response is absent from mouse embryonic stem (ES) cells and at
least one cell line of embryonic origin. (41,
42)
It is therefore possible to use long dsRNAs to silence specific genes in
these specific mammalian cells. However, the antiviral response precludes
the use of long dsRNAs to induce RNAi in most other mammalian cell
types.
siRNAs Bypass the Antiviral
Response
Interestingly, dsRNAs less than 30 nt in length do not
activate the PKR kinase pathway. This observation, as well as knowledge
that long dsRNAs are cleaved to form siRNAs in worms and flies and that
siRNAs can induce RNAi in Drosophila embryo lysates, prompted
researchers to test whether introduction of siRNAs could induce
gene-specific silencing in mammalian cells (43).
Indeed, siRNAs introduced by transient transfection were found to
effectively induce RNAi in mammalian cultured cells in a sequence-specific
manner. The effectiveness of siRNAs varies — the most potent siRNAs result
in >90% reduction in target RNA and protein levels (44-46).
The most effective siRNAs turn out to be 21 nt dsRNAs with 2 nt 3'
overhangs. Sequence specificity of siRNA is very stringent, as single base
pair mismatches between the siRNA and its target mRNA dramatically reduce
silencing (44,
47).
Unfortunately, not all siRNAs with these characteristics are effective.
The reasons for this are unclear but may be a result of positional effects
(46,
48,
49).
For current recommendations on designing siRNAs, see "siRNA
Design".
RNAi as a Tool for Functional Genomics
Although the history and mechanism of RNAi and PTGS are
fascinating, many researchers are most excited about RNAi's potential use
as a functional genomics tool. Already RNAi has been used to ascertain the
function of many genes in Drosophila, C. elegans, and
several species of plants. With the knowledge that RNAi can be induced in
mammalian cells by the transfection of siRNAs, many more researchers are
beginning to use RNAi as a tool in human, mouse and other mammalian cell
culture systems.
In early experiments with mammalian cells, the siRNAs
were synthesized chemically (Ambion is one of several companies that offer
custom siRNA
synthesis). Recently, Ambion introduced a kit (the Silencer™
siRNA Construction Kit) to produce siRNAs by in vitro transcription,
which is a less expensive alternative to chemical synthesis, particularly
when multiple different siRNAs need to be synthesized. Once made, the
siRNAs are introduced into cells via transient transfection. Due to
differences in efficacy, most researchers will synthesize 3–4 siRNAs to a
target gene and perform pilot experiments to determine the most effective
one. Transient silencing of more than 90% has been observed with this type
of approach (44-46,
48,
49).
So far, injection and transfection of dsRNA into cells
and organisms have been the main method of delivery of siRNA. And while
the silencing effect lasts for several days and does appear to be
transferred to daughter cells, it does eventually diminish. Recently,
however, a number of groups have developed expression vectors to
continually express siRNAs in transiently and stably transfected mammalian
cells (50-56).
Some of these vectors have been engineered to express small hairpin RNAs
(shRNAs), which get processed in vivo into siRNAs-like molecules capable
of carrying out gene-specific silencing (50,
53,
54,
56).
The vectors contain the shRNA sequence between a polymerase III (pol III)
promoter and a 4-5 thymidine transcription termination site. The
transcript is terminated at position 2 of the termination site (pol III
transcripts naturally lack poly(A) tails) and then folds into a stem-loop
structure with 3' UU-overhangs. The ends of the shRNAs are processed in
vivo, converting the shRNAs into ~21 nt siRNA-like molecules, which in
turn initiate RNAi (50).
This latter finding correlates with recent experiments in C.
elegans, Drosophila, plants and Trypanosomes, where RNAi has
been induced by an RNA molecule that folds into a stem-loop structure
(reviewed in 3).
Another siRNA expression vector developed by a different
research group encodes the sense and antisense siRNA strands under control
of separate pol III promoters (52).
The siRNA strands from this vector, like the shRNAs of the other vectors,
have 5 thymidine termination signals. Silencing efficacy by both types of
expression vectors was comparable to that induced by transiently
transfecting siRNA.
The recent studies on RNAi have taken the research world
by storm. The ability to quickly and easily create loss-of-function
phenotypes has researchers rushing to learn as much as they can about RNAi
and the characteristics of effective siRNAs. In the future, RNAi may even
hold promise for development of gene-specific therapeutics. Much has been
learned about this powerful technique, but additional information becomes
available on an almost daily basis (see The RNA Interference
Resource to learn about the very latest RNAi research and tools). It
is not an understatement to say that the field of functional genomics is
being revolutionized by RNAi.
References
- Cogoni C, and Macino G. (2000) Post-transcriptional
gene silencing across kingdoms. Genes Dev 10: 638-643. (Abstract)
- Guru T. (2000). A silence that speaks volumes.
Nature 404, 804-808. (Article)
- Hammond SM, Caudy AA, Hannon GJ. (2001)
Post-transcriptional Gene Silencing by Double-stranded RNA. Nature
Rev Gen 2: 110-119. (Abstract)
- Napoli C, Lemieux C, and Jorgensen R. (1990)
Introduction of a chalcone synthase gene into Petunia results in
reversible co-suppression of homologous genes in trans. Plant
Cell 2: 279-289.
- Jorgensen RA, Cluster PD, English J, Que Q, and Napoli
CA. (1996) Chalcone synthase cosuppression phenotypes in petunia
flowers: comparison of sense vs. antisense constructs and single-copy
vs. complex T-DNA sequences. Plant Mol Biol 31: 957-973.
(Abstract)
- Ingelbrecht I, Van Houdt H, Van Montagu M, and
Depicker A. (1994) Posttranscriptional silencing of reporter transgenes
in tobacco correlates with DNA methylation. Proc Natl Acad Sci
USA 91: 10502-10506. (Abstract,
PDF of
Article)
- Cogoni C, Irelan JT, Schumache, M, Schmidhauser T,
Selker EU, and Macino G. (1996) Transgene silencing of the al-1
gene in vegetative cells of Neurospora is mediated by a
cytoplasmic effector and does not depend on DNA-DNA interactions or DNA
methylation. EMBO J 15: 3153-3163. (Abstract)
- Palauqui JC, Elmayan T, Pollien JM, and Vaucheret H.
(1998) Systemic acquired silencing: transgene-specific
post-transcriptional silencing is transmitted by grafting from silenced
stocks to non-silenced scions. EMBO J 16: 4738-4745. (Article)
- Guo S, and Kempheus KJ. (1995). Par-1, a gene
required for establishing polarity in C. elegans embryos, encodes
a putative Ser/Thr kinase that is asymmetrically distributed.
Cell 81: 611-620. (Abstract)
- Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE,
and Mello CC. (1998). Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature
391: 806-811. (Article)
- Timmons, L., and Fire, A. (1998) Specific
interference by ingested dsRNA. Nature 395: 854.
- Timmons L, Court D, and Fire A. (2001) Ingestion of
bacterially expressed dsRNAs can produce specific and potent genetic
interference in Caenorhabditis elegans. Gene
263:103-112. (Abstract)
- Hunter CP. (2000) Shrinking the Black Box of RNAi.
Current Biology 10: R137-R140. (Article)
- Tabara H, Grishok A, and Mello CC. (1998) RNAi in
C. elegans: soaking in the genome sequence. Science
282: 430-431.
- Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG,
and Ahringer J. (2000) Effectiveness of specific RNA-mediated
interference through ingested double-stranded RNA in Caenorhabditis
elegans. Genome Biology 2: 2.1-2.10. (Article)
- Grishok A, Tabar H, and Mello CC. (2000) Genetic
requirements for inheritance of RNAi in C. elegans.
Science 287: 2494-2497. (Abstract)
- Sharp PA, and Zamore PD. (2000) RNA Interference.
Science 287: 2431-2433.
- Sharp PA. RNA Interference-2001. (2001) Genes
Dev 15: 485-490.
- Kennerdell JR, and Carthew RW. (1998) Use of
dsRNA-mediated genetic interference to demonstrate that frizzled
and frizzled 2 act in the wingless pathway. Cell
95: 1017-1026. (Abstract)
- Kennerdell JR, and Carthew RW. (2000) Heritable gene
silencing in Drosophila using double-stranded RNA. Nature
Biotech 18: 896-898. (Abstract)
- Dzitoyeva S, Dimitrijevic N, Manev H. (2001)
Intra-abdominal injection of double-stranded RNA into anesthetized adult
Drosophila triggers RNA interference in the central nervous
system. Mol Psychiatry 6(6):665-670.
- Worby CA, Simonson-Leff N, Dixon JE. (2001) RNA
interference of gene expression (RNAi) in cultured Drosophila
cells. Sci STKE Aug 14, 2001(95):PL1.
- Schmid A, Schindelholz B, Zinn K. (2002)
Combinatorial RNAi: a method for evaluating the functions of gene
families in Drosophila. Trends Neurosci
25(2):71-74.)
- Hamilton AJ, Baulcombe DC. (1999) A species of small
antisense RNA in posttranscriptional gene silencing in plants.
Science 286: 950-952. (Abstract)
- Hammond S, Bernstein E, Beach D, and Hannon G.
(2000). An RNA-directed nuclease mediates post-transcriptional gene
silencing in Drosophila cells. Nature, 404:
293-298. (Abstract)
- Zamore PD, Tuschl T, Sharp PA, and Bartel DP. (2000).
RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at
21 to 23 nucleotide intervals. Cell 101: 25-33. (Abstract)
- Hutvagner G, and Zamore PD. (2002) RNAi: nature
abhors a double-strand. Curr Opin Genetics & Development
12:225-232.
- Bernstein E, Caudy AA, Hammond SA, and Hannon GJ.
(2001) Role for a bidentate ribonuclease in the initiation step of RNA
interference. Nature 409:363-366.
- Nykanen A, Haley B, and Zamore PD. (2001) ATP
requirements and small interfering RNA structure in the RNA interference
pathway. Cell 107:309-321.
- Lipardi C, Wei Q, and Paterson BM. (2001) RNAi as
random degradative PCR. siRNA primers convert mRNA into dsRNA that are
degraded to generate new siRNAs. Cell 107:297-307.
- Ketting RF, Haverkamp TH, van Luenen HG, and Plasterk
RH. (1999). Mut-7 of C. elegans, required for transposon
silencing and RNA interference, is a homolog of Werner syndrome helicase
and RNase D. Cell 99: 133-141. (Abstract)
- Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S,
Ha I, Baillie DL, Fire A, Ruvkun G, and Mello CC. (2001) Genes and
mechanisms related to RNA interference regulate expression of the small
temporal RNAs that control C. elegans developmental timing.
Cell 106:23À34.
- Hutvagner G, McLachlan J, Pasquinelli AE, Balint E,
Tuschl T, and Zamore PD. (2001) A cellular function for the
RNA-interference enzyme Dicer in the maturation of the let-7
small temporal RNA. Science 293(5531):834-838.
- Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon
GJ, and Plasterk RH. (2001) Dicer functions in RNA interference and in
synthesis of small RNA involved in developmental timing in C.
elegans. Genes Dev 15(20):2654-2659.
- Lagos-Quintana M, Rauhut R, Lendeckel W, and Tuschl
T. (2001) Identification of novel genes coding for small expressed RNAs.
Science 294:853-858.
- Lau NC, Lim LP, Weinstein EG, and Bartel DP. (2001)
An abundant class of tiny RNAs with probable regulatory roles in
Caenorhabditis elegans. Science 294:858-862.
- Lee RC, and Ambrose V. (2001) An extensive class of
small RNAs in Caenorhabditis elegans. Science
294:862-864.
- Ruvkun G. (2001) Glimpses of a tiny RNA world.
Science 294:797-799.
- Manche L, Green SR, Schmedt C, and Mathews MB.
(1992). Interactions between double-stranded RNA regulators and the
protein kinase DAI. Mol. Cell. Biol. 12:5238-5248.
- Minks MA, West DK, Benvin S, and Baglioni C. (1979).
Structural requirements of double-stranded RNA for the activation of
2'-5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa
cells. J. Biol. Chem. 254:10180-10183.
- Yang S, Tutton S, Pierce E, and Yoon K. (2001)
Specific double-stranded RNA interference in undifferentiated mouse
embryonic stem cells. Mol. Cell. Biol. 21(22):7807-7816.
- Paddison PJ, Caudy A, and Hannon GJ. (2002). Stable
suppression of gene expression by RNAi in mammalian cells. Proc.
Natl. Acad. Sci. USA 99(3):1443-1448.
- Elbashir SM, Lendeckel W, and Tuschl T. (2001) RNA
interference is mediated by 21- and 22-nucleotide RNAs Genes Dev
15(2):188-200.
- Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber
K, and Tuschl T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA
interference in cultured mammalian cells. Nature 411:
494-498. (Abstract)
- Caplen NJ, Parrish S, Imani F, Fire A, and Morgan RA.
(2001) Specific inhibition of gene expression by small double-stranded
RNAs in invertebrates and vertebrate systems. Proc. Natl. Acad. Sci.
USA 98: 9746-9747.
- Holen T, Amarzguioui M, Wiiger M, Babaie E, and Prydz
H. (2002) Positional effects of short interferning RNAs targeting the
human coagulation trigger Tissue Factor. Nucleic Acids Research
30(8):1757-1766.
- Elbashir SM, Martinez J, Patkaniowska A, Lendeckel W,
Tuschl T. (2001) Functional anatomy of siRNA for mediating efficient
RNAi in Drosophila melanogaster embryo lysate. EMBO J
20: 6877-6888.
- Jarvis RA, and Ford LP. (2001) The siRNA Target Site
Is an Important Parameter for Inducing RNAi in Human Cells.
TechNotes 8(5): 3-5. (Article)
- Brown D, Jarvis R, Pallotta V, Byrom M, and Ford L.
(2002) RNA Interference in Mammalian Cell Culture: Design, Execution and
Analysis of the siRNA Effect. TechNotes 9(1): 3-5. (Article)
- Brummelkamp TR, Bernards R, and Agami R. (2002). A
system for stable expression of short interfering RNAs in mammalian
cells. Science 296:550-553.
- Lee NS, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A,
Salvaterra P, and Rossi J. (2002). Expression of small interfering RNAs
targeted against HIV-1 rev transcripts in human cells. Nature
Biotechnol. 20:500-505.
- Miyagishi M, and Taira K. (2002). U6-promoter-driven
siRNAs with four uridine 3' overhangs efficiently suppress targeted gene
expression in mammalian cells. Nature Biotechnol.
20:497-500.
- Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, and
Conklin DS. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific
silencing in mammalian cells. Genes & Dev. 16:948-958.
- Paul CP, Good PD, Winer I, and Engelke DR. (2002).
Effective expression of small interfering RNA in human cells. Nature
Biotechnol. 20:505-508.
- Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester
WC, and Shi Y. (2002). A DNA vector-based RNAi technology to suppress
gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA
99(6):5515-5520.
- Yu J-Y, DeRuiter SL, and Turner DL. (2002). RNA
interference by expression of short-interfering RNAs and hairpin RNAs in
mammalian cells. Proc. Natl. Acad. Sci. USA
99(9):6047-6052.
Additional Resources
RNA interference
http://www.nature.com/nature/fow/000316.html
Flash Animation: How Does RNAi Work?
Hammond, S.M., Caudy, A.A., Hannon, G.J. (2001) Post-transcriptional
Gene Silencing by Double-stranded RNA. Nature Rev Gen 2:
110-119.
www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.html
back to top