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Genome-wide RNAi
Robert Barstead
In many species, double-stranded RNA can specifically and
effectively silence genes. This newly discovered biological
phenomenon, called RNA interference (RNAi), has practical
implications for functional genomics. As shown by two recent
reports, RNAi provides a rapid method to test the function of
genes in the nematode Caenorhabditis elegans; most of the
genes on C. elegans chromosome I and III have now been
tested for RNAi phenotypes. The results validate RNAi as a
powerful functional genomics tool for C. elegans, and point the
way for similar large-scale studies in other species.
Addresses
Oklahoma Medical Research Foundation, 825 NE 13th Street,
Oklahoma City, OK 73104, USA;
e-mail: [email protected]
Current Opinion in Chemical Biology 2001, 5:63–66
1367-5931/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
dsRNA double-stranded RNA
nt
nucleotide
PTGS
post-transcriptional gene silencing
rde
RNAi defective
RNAi
dsRNA interference
Introduction
The nematode Caenorhabditis elegans is susceptible to a full
range of genetic methods, and so it has contributed greatly to our understanding of many biological phenomena.
These methods include phenotypic analysis of either lossor gain-of-function mutants, the study of epistatic genetic
interactions to reveal genetic pathways, and suppressor
genetics to identify interacting pairs of genes. Recently,
geneticists working on C. elegans have been challenged to
apply genetic strategies to the study of those genes discovered by genome sequencing, which, within some small
margin of error, shows us that the number of protein-coding genes in C. elegans is about 19,000 [1]. New methods are
being developed to meet this challenge, including the epigenetic inactivation of genes through the introduction of
double-stranded RNA (dsRNA) homologous to the target
locus, a process known as dsRNA interference (RNAi). In
this review, I discuss recent efforts to use RNAi for largescale functional analysis of genes in C. elegans and other
model systems.
What is RNAi?
RNAi is a simple and rapid method to specifically inactivate gene function, first applied in the nematode C. elegans
[2–4]. The technique grew out of an observation that when
either the sense or antisense transcript from the C. elegans
gene par-1 was injected into the germline, the resulting
progeny showed par-1 phenotypes. Fire, Mello and coworkers [5–8] showed that this epigenetic gene
inactivation worked with other genes, and that the phenomenon is strongest with dsRNA. Recent reports show
that the technique also works in a wide variety of other
species including Arabidopsis thaliana [9], Xenopus laevis
[10], Drosophila melanogaster [11,12], Trypanosoma brucei
[13], Hydra magnipapillata [14] and zebrafish [15].
The phenomenon of RNAi points to the existence of an
exciting unexplored biological process. The mechanisms
that underlie this process are being addressed genetically
and biochemically. Some examples of this work are
described below.
Firstly, several RNAi defective (rde) mutants have been
identified in C. elegans [16•]. One of these, rde-1, was
cloned and found to be homologous to the piwi/sting/argonaute family of proteins [16•]. In other species, rde-1
homologues are required for the maintenance of stem-cell
populations. Secondly, C. elegans strains with mutations in
the mut-7 gene show high levels of spontaneous mutation
because of transposon hopping; mut-7 mutants are also
resistant to RNAi [17]. The authors of [17] suggest that
RNAi might be a means to control transposon activity. The
mut-7 gene encodes a protein with homology to RNaseD
and the Werner syndrome helicase and to the Neurospora
gene qde-3 [17]. Mutants of Qde-3 are defective for a phenomenon known as quelling (cosuppression), a particular
example of post-transcriptional gene silencing (PTGS)
whereby the introduction of a DNA transgene leads to the
epigenetic inactivation of itself and the chromosomal
homologue [18]. Thirdly, the C. elegans gene ego-1 was first
identified as necessary for germline development [19]. It
was found to be homologous to the qde-1 gene from
Neurospora that encodes an RNA-dependent RNA polymerase which is necessary for quelling [20]. On the basis of
homology data and the reasonable supposition that
quelling and RNAi might involve similar processes [21,22],
ego-1 mutants were tested and found to be defective for
RNAi [19], giving further experimental support for the
idea that PTGS/quelling in Neurospora and RNAi in C. elegans are related phenomena.
In Arabidopsis, also, an RNA-dependent RNA polymerase
gene, SDE1, is necessary for post-transcriptional gene
silencing mediated by transgenes [23]. SDE-1 mutants
lack the 25 nt (nucleotide) long sense and antisense RNAs
that are diagnostic of PTGS in Arabidopsis, suggesting that
the role of SDE-1 is to synthesize a dsRNA initiator of
silencing [23]. The involvement of small RNAs in gene
silencing is supported by results from experiments done
with a cell-free in vitro assay derived from Drosophila
embryo extracts in which dsRNA directs the sequencespecific degradation of mRNA [24•]. In the in vitro RNAi
reaction, the initiating 500 nt dsRNA is first processed to
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Proteomics and genomics
small segments 21–23 nt long [25•]. The target mRNA is
then cleaved within the region of identity of the dsRNA.
Cleavage occurs at sites 21–23 nt apart, the same interval
observed for the dsRNA itself, leading the authors to suggest that 21–23 nt fragments from the dsRNA guide
mRNA cleavage.
RNAi is now a routine method to study in vivo gene function in C. elegans [13,26–33]. In a typical experiment,
dsRNA from a selected target gene is synthesized in vitro
and injected into the germline of an adult nematode. The
progeny are then examined for phenotypes. Reports in the
literature indicate that for many genes the RNAi phenotypes correlate well with the phenotypes of genuine
loss-of-function mutants. Major exceptions, however, are
genes that act in the nervous system; such genes seem to be
refractory to RNAi injected into the germline. Driscoll and
co-workers [34] devised a remedy for this limitation in
which they constructed heritable transgenic animals that
express hairpin dsRNA in the nervous system. As reported,
however, though adequate for single-gene studies, the procedure was too inconsistent and complicated to apply on a
genome-wide scale [34]. A second exception to the general
reliability of RNAi comes from studies of the lir-1 gene
[35]. This gene exists in an operon with a second gene lin26; these two gene products are derived through the
processing of a single transcript. Mutants of lin-26 are inviable, whereas lir-1 mutants are viable. Labouesse and
co-workers [35] showed that RNAi directed at lir-1 causes a
lin-26-like phenotype. This may be an exceptional case,
however, as genes in two other operons behaved independently in RNAi experiments [35].
Genome-wide RNAi in C. elegans
Genomic studies are designed to rapidly provide sufficient information to encourage more detailed studies by
other investigators and to provide a comprehensive data
set that can be mined for correlations which otherwise
might escape detection if the data were collected ad hoc
by many individual investigators. These goals do not
depend on absolute reliability across the whole data set
and so the caveats regarding RNAi in C. elegans described
above, which may compromise reliability, are by themselves not sufficient to determine whether the system is
suitable for a genome-wide RNAi screen. Rather, the
decision to undertake such a genomics project is based
on an evaluation of the balance between data reliability,
cost, and logistical feasibility. In considering RNAi, one
of the most important logistical issues is the way in
which one applies the dsRNA. For C. elegans, microinjection is an effective method to introduce the dsRNA; this
method, however, is cumbersome on a large scale.
Nevertheless, as described below, one group of investigators has done a large-scale RNAi study in C. elegans
using microinjection [36•]. Other, more easily scaled
methods for the introduction of dsRNA include soaking
worms in solutions of dsRNA [37] and feeding worms
Escherichia coli expressing dsRNA [8].
On balance, therefore, RNAi in C. elegans is sufficiently
effective, robust and logistically feasible to support efforts
to examine systematically all C. elegans genes for RNAi
phenotypes. Two groups recently have begun such efforts:
Hyman and co-workers [36•] used RNAi to identify those
genes on C. elegans chromosome III that affect cell division.
Ahringer and co-workers [38•] used RNAi to examine the
function of about 90% of the genes on C. elegans chromosome I. Their results, described below, are a spectacular
validation of the value of genome-wide RNAi screens.
The Hyman group synthesized dsRNA for 2232 genes on
C. elegans chromosome III, representing about 96% of the
total number of protein-coding genes on this chromosome,
and then injected the dsRNA into the C. elegans germline
to test for RNAi phenotypes; in this case they used timelapse DIC (differential interference contrast) microscopy
to score for those dsRNAs that caused abnormalities in the
first two embryonic cell divisions [36•]. The abnormalities
included defects in meiotic division, nuclear appearance,
pace of development, and general embryonic appearance.
They found that 133 genes (6.1% of the tested genes)
showed such RNAi-induced phenotypes. They concluded
by extrapolation that the entire C. elegans genome might
have about 1000 genes essential for the first two cleavage
divisions. They drew the following additional conclusions
from their data. Firstly, the results compare favorably to
those obtained by classical genetics. Of the seven known
C. elegans chromosome III genes with early cell division
phenotypes, all showed RNAi phenotypes that matched
the genuine genetic mutant phenotype. This result validates the reliability of RNAi for this class of genes in
C. elegans. Secondly, only 11 of the 133 genes identified by
this work were associated with a function experimentally
verified in C. elegans. Another 104 genes, however, had no
previously identified function in C. elegans, but were conserved in other species. In cases where a function is known
in these other species, hypotheses regarding the function
in C. elegans of the genes in this set arise naturally. These
hypotheses now can be evaluated in light of the RNAi
phenotypes. Finally, 18 of the 133 genes could not be
ascribed a function on the basis of sequence homology
alone. At present, therefore, the RNAi phenotypes provide
the best functional data available for this set of genes.
The Ahringer group [38•] constructed a library of bacteria
expressing dsRNA for 2416 of the genes on C. elegans chromosome I; this represented about 87% of the total number
of predicted genes on this chromosome. They then fed
the bacterial strains to worms and looked for RNAiinduced phenotypes. Using a dissecting microscope,
animals were examined for anatomical abnormalities,
abnormal motility, altered sex ratios, and sterility. About
70 sequenced genes, identified using standard forwardgenetic methods, were already known to be on C. elegans
chromosome I. The Ahringer group saw RNAi-induced
phenotypes for 13.9% of the genes tested, thereby
increasing the number of sequenced genes on C. elegans
Genome-wide RNAi Barstead
chromosome I with known phenotypes from 70 to 378.
They were able to draw the following additional conclusions from their data. Firstly, they detected appropriate
phenotypes for 90% of the known embryonic lethals on
chromosome I. This validates the general reliability of
genome-wide RNAi on this class of genes from C. elegans.
Secondly, only one of eight neuronal genes gave phenotypes, confirming on a large scale the previous data
showing that this class of genes is refractory to RNAi from
C. elegans. Thirdly, evolutionarily conserved genes were
more likely to have an RNAi phenotype than genes that
were not conserved: 26% of the conserved genes showed
phenotypes compared with only 5% of the non-conserved
genes. Fourthly, extrapolating to the entire genome, the
data suggested that about 5400 of C. elegans genes might
have dissecting microscope phenotypes like that scored in
this study. Finally. the library of bacterial clones can
be replicated, thereby giving other investigators access to
a low-cost, simple means to do screens for other
RNAi phenotypes.
Conclusions
These reports show that RNAi is a powerful approach to
large-scale functional genomic studies of C. elegans.
Compared with typical classical genetic methods, RNAi
has the advantage that the sequence of the target is known,
thereby connecting mutant phenotypes with known genes.
Though more detailed studies depend on the recovery of
stable genetic mutants, RNAi is not simply a middling
substitute for proper classic genetic analysis; rather, one
could argue that RNAi may provide a simpler way to study
loss-of-function phenotypes of genes that are essential for
viability or genes that are expressed in the maternal
germline, so called maternal effect genes. Then again,
though RNAi does provide an entrées to the functional
analysis of many genes, it does not supersede classical
genetics in C. elegans because detailed genetic studies
depend on an examination of many types of mutant alleles
that allow for the application of the full range of genetic
methods including screens for suppressor mutants to identify important genetic interactions.
RNAi works in other species. Whether it can or should be
applied on a large scale to studies of these other species
depends, in part, on whether it is found to be sufficiently
reliable in these systems. Reliability is best judged by
comparing RNAi phenotypes of particular genes with the
phenotypes seen for genuine genetic mutants. This criterion cannot be applied to model organisms that lack an
extensive genetic foundation. Nevertheless, it is in just
such systems where RNAi may be most important.
Perhaps the lessons learned from the large-scale application of RNAi to C. elegans will serve as necessary guideposts
for future studies of such model systems.
Acknowledgements
I wish to thank Julie Ahringer and Andrew Fire for discussions and the
communication of unpublished results.
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References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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