letters to nature
`
`and 10 Na3HP2O7. FV solution also contained 0.2 NaF and 0.1 Na3VO4. Rarely,
`irreversible current rundown still occurred with FVPP. The total Na+ concen-
`tration of all cytoplasmic solutions was adjusted to 30 mM with NaOH, and pH
`was adjusted to 7.0 with N-methylglucamine (NMG) or HCl. PIP2 liposomes
`(20–200 nm) were prepared by sonicating 1 mM PIP2 (Boehringer Mannheim)
`in distilled water. Reconstituted monoclonal PIP2 antibody (Perspective
`Biosystems, Framingham, MA) was diluted 40-fold into experimental solution.
`Current–voltage relations of all currents reversed at EK and showed charac-
`teristic rectification, mostly owing to the presence of Na+ in FVPP and possibly
`also residual polyamines. Current records presented (measured at 30 ⬚C,−30 mV
`holding potential) are digitized strip-chart recordings. Purified bovine
`brain Gbg29 was diluted just before application such that the final detergent
`(CHAPS) concentration was 5 ␮M. Detergent-containing solution was washed
`away thoroughly before application of PIP2, because application of phos-
`pholipid vesicles in the presence of detergent usually reversed the effects of
`Gbg; presumably, Gbg can be extracted from membranes by detergent plus
`phospholipids.
`Molecular biology. R188Q mutation was constructed by insertion of the
`mutant oligonucleotides between the Bsm1 and BglII sites of pSPORT–
`ROMK1 (ref. 11). A polymerase chain reaction (PCR) fragment (amino acids
`180–391) from pSPORT–ROMK1 R188Q mutant was subcloned into pGEX-
`2T vector (Pharmacia) for expression of R188Q mutant protein of GST–RKC.
`The construction, expression and purification of GST–IKC (amino acids 182–
`428 of IRK1), GST–GKC (180–462 of GIRK1), GST–IKN (1–86 of IRK1) have
`been described21,22.
`In vitro PIP2 binding assay. 3H-PIP2 in chloroform-methanol (1:1) (American
`−1 specific activity) was dried under N2
`Radiolabeled Chemicals; 0.4 ␮Ci nM
`and sonicated in 100 ␮l phosphate buffered saline (PBS) to form pure 3H-PIP2
`liposomes. Purified GST fusion protein (100 nM) was incubated with 3H-PIP2
`(0.2–1 ␮M) and precipitated by glutathione 4B-Sepharose beads. After 1 wash
`with PBS, the precipitates were dissolved in SDS gel loading buffer and counted
`in a beta-scintillation counter using a window for 3H. The bound 3H
`radioactivity was typically in the range ⬃2–8% of the total added. For co-
`immunoprecipitation, 25% PIP2 or PIP in 75% phosphatidylcholine (PC)
`background (30 ␮g PIP2 or PIP (Boehringer Mannheim) and 90 ␮g
`phosphatidylcholine (Sigma)), both in chloroform, were dried down together
`and sonicated in 300 ␮l PBS to form mixed liposome. GST fusion proteins were
`first incubated with 25% PIP2 or PIP liposome (100 ␮M) and PIP2 antibodies
`(1:100 dilution) for 2 h and with protein A–Sepharose for a further 30 min.
`After one wash with PBS, the immunoprecipitates were separated by 10% SDS–
`PAGE, probed with specific antibodies21,22, and visualized by ECL (Amersham).
`Each experiment was performed at least twice with similar results. The relative
`amount of immunoreactivity in each lane was quantified by serial dilutions of
`sample21.
`
`Received 6 June; accepted 13 October 1997.
`
`1. McNicholas, C. M., Wang, W., Ho, K., Hebert, S. C. & Giebisch, G. Regulation of ROMK1 K+ channel
`activity involves phosphorylation processes. Proc. Natl Acad. Sci. USA 91, 8077–8081 (1994).
`2. Fakler, B., Brandle, U., Glowatzki, E., Zenner, H.-P. & Ruppersberg, J. P. Kir2.1 inward rectifier K+
`channels are regulated independently by protein kinases and ATP hydrolysis. Neuron 13, 1413–1420
`(1994).
`3. Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N. & Jan, L. Y. Primary structure and functional
`expression of a rat G-protein-coupled muscarinic potassium channel. Nature 364, 802–806 (1993).
`4. Dascal, N. et al. Atrial G protein-activated K+ channel: expression cloning and molecular properties.
`Proc. Natl Acad. Sci. USA 90, 10235–10239 (1993).
`5. Krapivinsky, G. et al. The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly
`rectifying K+-channel proteins. Nature 374, 135–141 (1995).
`6. Lesage, F. et al. Molecular properties of neuronal G protein-activated inwardly rectifying K+ channels.
`J. Biol. Chem. 270, 28660–28667 (1995).
`7. Furukawa, T., Yamane, T., Terai, T., Katayama, Y. & Hiraoka, M. Functional linkage of the cardiac ATP-
`sensitive K+ channel to actin cytoskeleton. Pflugers Arch. 431, 504–512 (1996).
`8. Hilgemann, D. W. & Ball, R. Regulationof cardiac Na+, Ca2+ exchange and KATP potassium channels by
`PIP2. Science 273, 956–959 (1996).
`9. Fukami, K. et al. Antibody to phosphatidylinositol 4,5-bisphosphate inhibits oncogene-induced
`mitogenesis. Proc. Natl Acad. Sci. USA 85, 9057–9061 (1988).
`10. Kubo, Y., Baldwin, T. J., Jan, Y. N. & Jan, L. Y. Primary structure and functional expression of a mouse
`inward rectifier potassium channel. Nature 362, 127–133 (1993).
`11. Ho, K. et al. Cloning and expression of an inwardly rectifying ATP-regulated potassium channel.
`Nature 362, 31–38 (1993).
`12. Sui, J. L., Chan, K. W. & Logothetis, D. E. Na+ activation of the muscarinic K+ channel by a G-protein-
`independent mechanism. J. Gen. Physiol. 109, 381–390 (1996).
`13. Chan, K. W. et al. A recombinant inwardly rectifying potassium channel coupled to GTP-binding
`proteins. J. Gen. Physiol. 107, 381–397 (1996).
`14. Zhang, X., Jefferson, A. B., Auethavekiat, V. & Majerus, P. W. The protein deficient in Lowe syndrome
`is a phosphatidylinositol-4,5-bisphosphate 5-phosphatase. Proc. Natl Acad. Sci. USA 92, 4853–4856
`(1995).
`
`8
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`15. Fukami, K., Endo, T., Imamura, M. & Takenawa, T. a-Actinin and vinculin are PIP2-binding proteins
`involved in signaling by tyrosine kinase. J. Biol. Chem. 269, 1518–1522 (1994).
`16. Fan, Z. & Makielski, J. C. Anionic phospholipids activate ATP-sensitive potassium channels. J. Biol.
`Chem. 272, 5388–5395 (1997).
`17. Schacht, J. Inhibition by neomycin of polyphosphoinositide turnover in subcellular fractions of
`guinea-pig cerebral cortex in vitro. J. Neurochem. 27, 1119–1124 (1976).
`18. Kim, J., Mosior, M., Chung, L. A., Wu, H. & McLaughlin, S. Binding of peptides with basic residues to
`membrane containing acidic phospholipids. Biophys. J. 60, 135–148 (1991).
`19. Harlan, J. E., Yoon, H. S., Hajduk, P. J. & Fesik, S. W. Structural characterization of the interaction
`between a pleckstrin homology domain and phosphatidylinositol 4,5-bisphosphate. Biochemistry 34,
`9859–9864 (1995).
`20. Reuveny, E. et al. Activation of the cloned muscarinic potassium channel by G protein bg subunits.
`Nature 370, 143–146 (1994).
`21. Huang, C.-L., Slesinger, P. A., Casey, P. J., Jan, Y. N. & Jan, L. Y. Evidence that direct binding of Gbg to
`the GIRK1 protein-gated inwardly rectifying K+ channel is important for channel activation. Neuron
`15, 1133–1143 (1995).
`22. Huang, C.-L., Jan, Y. N. & Jan, L. Y. Binding of Gbg to multiple regions of G protein-gated inward
`rectifier K+ channels. FEBS Lett. 405, 291–298 (1997).
`23. Krapivinsky, G., Krapivinsky, L., Wickman, K. & Clapham, D. E. Gbg binds directly to the G protein-
`gated K+ channel, IKACh. J. Biol. Chem. 270, 29059–29062 (1995).
`24. Janmey, P. A. Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly.
`Annu. Rev. Physiol. 56, 169–191 (1994).
`25. Penniston, J. T. Plasma membrane Ca2+-pumping ATPases. Ann. NY Acad. Sci. 402, 291–303 (1982).
`26. Pitcher, J. A., Touhara, K., Payne, E. S. & Lefkowitz, R. J. Pleckstrin homology domain-mediated
`membrane association and activation of the b-adrenergic receptor kinase requires coordinate
`interaction with Gbg and lipid. J. Biol. Chem. 270, 11707–11710 (1995).
`27. Tagliaalatela, M., Wible, B. A., Caporaso, R. & Brown, A. M. Specification of the pore properties by the
`carboxyl terminus of inward rectifying K+ channels. Science 264, 844–847 (1994).
`28. Clapham, D. E. & Neer, E. J. New roles for G protein bg-dimers in transmembrane signaling. Nature
`365, 403–406 (1993).
`29. Casey, P. J., Graziano, M. P. & Gilman, A. G. G protein bg subunits from bovine brain and retina:
`equivalent catalytic support of ADP-ribosylation of a subunit by pertussis toxin but differential
`interactions with Gsa. Biochemistry 28, 611–616 (1989).
`
`Acknowledgements. We thank E. Phan for technical assistance; I. Bezprozvanny, C. Dessauer, D. Logo-
`thetis, C.-C. Lu, O. Moe, S. Muallem and H. Yin for discussions and advice; L. Jan for GIRK1 and ROMK1
`antibodies; C. Dessauer and A. Gilman for Gai1; P. Casey for Gbg; and R. Alpern for support and
`encouragement. This work was supported by grants from the NKF of Texas (C.L.H.) and from the AHA
`and NIH (D.W.H.).
`
`Correspondence and requests for materials should be addressed to C.L.H. (e-mail: chuan1@mednet.
`swmed.edu).
`
`Potent and specific
`genetic interference by
`double-stranded RNA in
`Caenorhabditis elegans
`
`Andrew Fire*, SiQun Xu*, Mary K. Montgomery*,
`Steven A. Kostas*†, Samuel E. Driver‡ & Craig C. Mello‡
`
`* Carnegie Institution of Washington, Department of Embryology,
`115 West University Parkway, Baltimore, Maryland 21210, USA
`† Biology Graduate Program, Johns Hopkins University,
`3400 North Charles Street, Baltimore, Maryland 21218, USA
`‡ Program in Molecular Medicine, Department of Cell Biology,
`University of Massachusetts Cancer Center, Two Biotech Suite 213,
`373 Plantation Street, Worcester, Massachusetts 01605, USA
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Experimental introduction of RNA into cells can be used in
`certain biological systems to interfere with the function of an
`endogenous gene1,2. Such effects have been proposed to result
`from a simple antisense mechanism that depends on hybridiza-
`tion between the injected RNA and endogenous messenger RNA
`transcripts. RNA interference has been used in the nematode
`Caenorhabditis elegans to manipulate gene expression3,4. Here we
`investigate the requirements for structure and delivery of the
`interfering RNA. To our surprise, we found that double-stranded
`RNA was substantially more effective at producing interference
`than was either strand individually. After injection into adult
`animals, purified single strands had at most a modest effect,
`whereas double-stranded mixtures caused potent and specific
`interference. The effects of this interference were evident in
`both the injected animals and their progeny. Only a few molecules
`of injected double-stranded RNA were required per affected cell,
`arguing against stochiometric interference with endogenous
`
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`letters to nature
`
`mRNA and suggesting that there could be a catalytic or amplifica-
`tion component in the interference process.
`Despite the usefulness of RNA interference in C. elegans, two
`features of the process have been difficult to explain. First, sense and
`antisense RNA preparations
`are
`each sufficient
`to cause
`interference3,4. Second, interference effects can persist well into the
`next generation, even though many endogenous RNA transcripts
`are rapidly degraded in the early embryo5. These results indicate a
`fundamental difference in behaviour between native RNAs (for
`example, mRNAs) and the molecules responsible for interference.
`We sought to test the possibility that this contrast reflects an underlying
`difference in RNA structure. RNA populations to be injected are
`
`generally prepared using bacteriophage RNA polymerases6. These
`polymerases, although highly specific, produce some random or
`ectopic transcripts. DNA transgene arrays also produce a fraction of
`aberrant RNA products3. From these facts, we surmised that the
`interfering RNA populations might include some molecules with
`double-stranded character. To test whether double-stranded character
`might contribute to interference, we further purified single-stranded
`RNAs and compared interference activities of individual strands with
`the activity of a deliberately prepared double-stranded hybrid.
`The unc-22 gene was chosen for initial comparisons of activity.
`unc22 encodes an abundant but nonessential myofilament pro-
`tein7–9. Several thousand copies of unc-22 mRNA are present in each
`
`8
`
`Table 1 Effects of sense, antisense and mixed RNAs on progeny of injected animals
`
`Gene
`
`segment
`
`Size
`(kilobases)
`...................................................................................................................................................................................................................................................................................................................................................................
`unc-22-null mutants: strong twitchers7,8
`unc-22
`
`Injected RNA
`
`F1 phenotype
`
`unc22A* Exon 21–22
`
`unc22B Exon 27
`
`742
`
`1,033
`
`Wild type
`Wild type
`Strong twitchers (100%)
`
`Wild type
`Wild type
`Strong twitchers (100%)
`
`Sense
`Antisense
`Sense þ antisense
`Sense
`Antisense
`Sense þ antisense
`Sense þ antisense
`unc22C Exon 21–22†
`Strong twitchers (100%)
`785
`...................................................................................................................................................................................................................................................................................................................................................................
`fem-1-null mutants: femal (no sperm)13
`fem-1
`fem1A Exon 10‡
`
`Hermaphrodite (98%)
`Sense
`Hermaphrodite (⬎98%)
`Antisense
`Sense þ antisense
`Female (72%)
`Sense þ antisense
`Hermaphrodite (⬎98%)
`556
`Intron 8
`fem1B
`...................................................................................................................................................................................................................................................................................................................................................................
`unc-54-null mutants: paralysed7,11
`unc-54
`
`531
`
`unc54A Exon 6
`
`unc54B Exon 6
`
`unc54C Exon 1–5
`
`unc54D Promoter
`
`unc54E Intron 1
`
`hlh1A
`
`Exons 1–6
`
`hlh1B
`
`Exons 1–2
`
`hlh1C
`
`Exons 4–6
`
`576
`
`651
`
`1,015
`
`567
`
`369
`
`1,033
`
`438
`
`299
`
`Sense
`Antisense
`Sense þ antisense
`Sense
`Antisense
`Sense þ antisense
`Sense þ antisense
`Sense þ antisense
`Sense þ antisense
`Sense þ antisense
`Wild type (100%)
`386
`unc54F Intron 3
`...................................................................................................................................................................................................................................................................................................................................................................
`hlh-1-null mutants: lumpy-dumpy larvae16
`hlh-1
`Wild type (⬍2% lpy-dpy)
`Sense
`Wild type (⬍2% lpy-dpy)
`Antisense
`Lpy-dpy larvae (⬎90%)k
`Sense þ antisense
`Lpy-dpy larvae (⬎80%)k
`Sense þ antisense
`Lpy-dpy larvae (⬎80%)k
`Sense þ antisense
`Sense þ antisense
`Wild type (⬍2% lpy-dpy)
`697
`Intron 1
`hlh1D
`...................................................................................................................................................................................................................................................................................................................................................................
`myo-3-driven GFP transgenes¶
`
`Wild type (100%)
`Wild type (100%)
`Paralysed (100%)§
`
`Wild type (100%)
`Wild type (100%)
`Paralysed (100%)§
`
`Arrested embryos and larvae (100%)
`
`Wild type (100%)
`
`Wild type (100%)
`
`myo-3::NLS::gfp::lacZ
`
`gfpG
`
`Exons 2–5
`
`lacZL
`
`Exon 12–14
`
`myo-3::MtLS::gfp
`
`730
`
`830
`
`Sense
`Antisense
`Sense þ antisense
`Sense þ antisense
`
`Makes nuclear GFP in body muscle
`
`Nuclear GFP–LacZ pattern of parent strain
`Nuclear GFP–LacZ pattern of parent strain
`Nuclear GFP–LacZ absent in 98% of cells
`Nuclear GFP–LacZ absent in ⬎95% of cells
`
`Makes mitochondrial GFP in body muscle
`
`Mitochondrial-GFP pattern of parent strain
`Mitochondrial-GFP pattern of parent strain
`Mitochondrial-GFP absent in 98% of cells
`
`gfpG
`
`Exons 2–5
`
`730
`
`Sense
`Antisense
`Sense þ antisense
`Sense þ antisense
`Mitochondrial-GFP pattern of parent strain
`830
`Exon 12–14
`lacZL
`...................................................................................................................................................................................................................................................................................................................................................................
`Each RNA was injected into 6–10 adult hermaphrodites (0:5 ⫻ 106 –1 ⫻ 106 molecules into each gonad arm). After 4–6 h (to clear prefertilized eggs from the uterus), injected animals were
`transferred and eggs collected for 20–22 h. Progeny phenotypes were scored upon hatching and subsequently at 12-24-h intervals.
`* to obtain a semiquantitative assessment of the relationship between RNA dose and phenotypic response, we injected each unc22A RNA preparation at a series of different concentrations
`(see figure in Supplementary information for details). At the highest dose tested (3:6 ⫻ 106 molecules per gonad), the individual sense and antisense unc22A preparations produced some
`visible twitching (1% and 11% of progeny, respectively). Comparable doses of double-stranded unc22A RNA produced visible twitching in all progeny, whereas a 120-fold lower dose of
`double-stranded unc22A RNA produced visible twitching in 30% of progeny. † unc22C also carries the 43-nucleotide intron between exons 21 and 22. ‡ fem1A carries a portion (131
`nucleotides) of intron 10. § Animals in the first affected broods (layed 4–24 h after injection) showed movement defects indistinguishable from those of unc-54-null mutants. A variable fraction
`of these animals (25%–75%) failed to lay eggs (another phenotype of unc-54-null mutants), whereas the remainder of the paralysed animals did lay eggs. This may indicate incomplete
`interference with unc-54 activity in vulval muscles. Animals from later broods frequently show a distinct partial loss-of-function phenotype, with contractility in a subset of body-wall muscles.
`k Phenotypes produced by RNA-mediated interference with hlh-1 included arrested embryos and partially elongated L1 larvae (the hlh-1-null phenotype). These phenotypes were seen in
`virtually all progeny after injection of double-stranded hlh1A and in about half of the affected animals produced after injection of double-stranded hlh1B and double-stranded hlhlC. A set of
`less severe defects was seen in the remainder of the animals produced after injection of double-stranded hlh1B and double-stranded hlh1C). The less severe phenotypes are characteristic
`of partial loss of function of hlh-1 (B. Harfe and A.F., unpublished observations). ¶ the host for these injections, strain PD4251, expresses both mitochondrial GFP and nuclear GFP–LacZ (see
`Methods). This allows simultaneous assay for interference with gfp (seen as loss of all fluorescence) and with lacZ (loss of nuclear fluorescence). The table describes scoring of animals as
`L1 larvae. Double-stranded gfpG caused a loss of GFP in all but 0–3 of the 85 body muscles in these larvae. As these animals mature to adults, GFP activity was seen in 0–5 additional body-
`wall muscles and in the 8 vulval muscles. Lpy-dpy, lumpy-dumpy.
`
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`letters to nature
`
`striated muscle cell3. Semiquantitative correlations between unc-22
`activity and phenotype of the organism have been described8:
`decreases in unc-22 activity produce an increasingly severe twitch-
`ing phenotype, whereas complete loss of function results in the
`additional appearance of muscle structural defects and impaired
`motility.
`Purified antisense and sense RNAs covering a 742-nucleotide
`segment of unc-22 had only marginal interference activity, requiring
`a very high dose of injected RNA to produce any observable effect
`(Table 1). In contrast, a sense–antisense mixture produced highly
`effective interference with endogenous gene activity. The mixture
`was at least two orders of magnitude more effective than either
`single strand alone in producing genetic interference. The lowest
`dose of the sense–antisense mixture that was tested, ⬃60,000
`molecules of each strand per adult, led to twitching phenotypes in
`an average of 100 progeny. Expression of unc-22 begins in embryos
`
`C
`
`A
`
`B
`
`5.0kb
`
`1.0kb
`
`A
`
`B
`
`unc-22
`
`fem-1
`
`unc-54
`
`hlh-1
`
`B
`
`F
`
`D
`
`E
`
`D
`
`B
`
`A
`
`C
`
`C
`A
`
`gfp fusions
`
`Nuclear
`
`Mitochondrial
`
`myo-3 5'
`
`NLS
`
`gfp
`
`lacZ
`
`myo-3 5'
`
`MtLS
`
`gfp
`
`G
`
`G
`
`1.0kb
`
`L
`
`Figure 1 Genes used to study RNA-mediated genetic interference in C. elegans.
`
`Intron–exon structure for genes used to test RNA-mediated inhibition are shown
`
`(grey and filled boxes, exons; open boxes, introns; patterned and striped boxes,
`5⬘ and 3⬘ untranslated regions. unc-22. ref. 9, unc-54, ref.12, fem-1, ref.14, and hlh-1,
`ref.15). Each segment of a gene tested for RNA interference is designated with the
`
`name of the gene followed by a single letter (for example, unc22C). These
`
`segments are indicated by bars and upper-case letters above and below each
`
`gene. Segments derived from genomic DNA are shown above the gene; seg-
`
`ments derived from cDNA are shown below the gene. NLS, nuclear-localization
`
`sequence; MtLS, mitochondrial localization sequence.
`
`8
`
`containing ⬃500 cells. At this point, the original injected material
`would be diluted to at most a few molecules per cell.
`The potent interfering activity of the sense–antisense mixture
`could reflect the formation of double-stranded RNA (dsRNA) or,
`conceivably, some other synergy between the strands. Electrophoretic
`analysis indicated that the injected material was predominantly
`double-stranded. The dsRNA was gel-purified from the annealed
`mixture and found to retain potent interfering activity. Although
`annealing before injection was compatible with interference, it was
`not necessary. Mixing of sense and antisense RNAs in low-salt
`concentrations (under conditions of minimal dsRNA formation) or
`rapid sequential injection of sense and antisense strands were
`sufficient to allow complete interference. A long interval (⬎1 h)
`between sequential injections of sense and antisense RNA resulted
`in a dramatic decrease in interfering activity. This suggests that
`injected single strands may be degraded or otherwise rendered
`inaccessible in the absence of the opposite strand.
`A question of specificity arises when considering known cellular
`responses to dsRNA. Some organisms have a dsRNA-dependent
`protein kinase that activates a panic-response mechanism10. Con-
`ceivably, our sense–antisense synergy might have reflected a non-
`specific potentiation of antisense effects by such a panic mechanism.
`This is not the case: co-injection of dsRNA segments unrelated to
`unc-22 did not potentiate the ability of single unc-22-RNA strands
`to mediate inhibition (data not shown). We also investigated
`whether double-stranded structure could potentiate interference
`activity when placed in cis to a single-stranded segment. No such
`potentiation was seen: unrelated double-stranded sequences located
`5⬘ or 3⬘ of a single-stranded unc-22 segment did not stimulate
`interference. Thus, we have only observed potentiation of inter-
`ference when dsRNA sequences exist within the region of homology
`with the target gene.
`The phenotype produced by interference using unc-22 dsRNA
`was extremely specific. Progeny of injected animals exhibited
`behaviour that precisely mimics loss-of-function mutations in
`unc-22. We assessed target specificity of dsRNA effects using three
`additional genes with well characterized phenotypes (Fig. 1, Table
`1). unc-54 encodes a body-wall-muscle heavy-chain isoform of
`myosin that is required for full muscle contraction7,11,12; fem-1
`encodes an ankyrin-repeat-containing protein that is required in
`hermaphrodites for sperm production13,14; and hlh-1 encodes a C.
`elegans homologue of myoD-family proteins that is required for
`proper body shape and motility15,16. For each of these genes,
`injection of related dsRNA produced progeny broods exhibiting
`
`Control RNA (ds-unc22A)
`
`ds-gfpG RNA
`
`ds-lacZL RNA
`
`a
`
`b
`
`c
`
`d
`
`e
`
`f
`
`g
`
`h
`
`i
`
`L1
`
`Adult
`
`Adult
`
`Figure 2 Analysis of RNA-interference effects in individual cells.Fluorescence
`
`musculature expresses active GFP in the adult animal in e. f, Two rare GFP-
`
`micrographs show progeny of injected animals from GFP-reporter strain PD4251.
`
`positive cells in an adult: both cells express both nuclear-targeted GFP–LacZ and
`
`a–c, Progeny of animals injected with a control RNA (double-stranded
`
`mitochondrial GFP. g–i, Progeny of animals injected with ds-lacZL RNA:
`
`(ds)-unc22A). a, Young larva, b, adult, c, adult body wall at high magnification.
`
`mitochondrial-targeted GFP seems unaffected, while the nuclear-targeted GFP–
`
`These GFP patterns appear identical to patterns in the parent strain, with
`
`LacZ is absent from almost all cells (for example, see larva in g). h, A typical adult,
`
`prominent fluorescence in nuclei (nuclear-localized GFP–LacZ) and mitochondria
`
`(mitochondrially targeted GFP). d–f, Progeny of animals injected with ds-gfpG.
`
`Only a single active cell is seen in the larva in d, whereas the entire vulval
`
`with nuclear GFP–LacZ lacking in almost all body-wall muscles but retained in
`vulval muscles. Scale bars represent 20 ␮m.
`
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`the known null-mutant phenotype, whereas the purified single RNA
`strands produced no significant interference. With one exception,
`all of the phenotypic consequences of dsRNA injection were those
`expected from interference with the corresponding gene. The
`exception (segment unc54C which led to an embryonic- and
`larval-arrest phenotype not seen with unc-54-null mutants) was
`illustrative. This segment covers the highly conserved myosin-
`motor domain, and might have been expected to interfere with
`activity of other highly related myosin heavy-chain genes17. The
`unc54C segment has been unique in our overall experience to date:
`effects of 18 other dsRNA segments (Table 1; and our unpublished
`observations) have all been limited to those expected from pre-
`viously characterized null mutants.
`The pronounced phenotypes seen following dsRNA injection
`indicate that interference effects are occurring in a high fraction of
`
`a
`
`c
`
`b
`
`d
`
`Figure 3 Effects of mex-3 RNA interference on levels of the endogenous mRNA.
`
`Interference contrast micrographs show in situ hybridization in embryos. The
`1,262-nt mex-3 cDNA clone20 was divided into two segments, mex-3A and mex-
`
`3B, with a short (325-nt) overlap (similar results were obtained in experiments with
`
`no overlap between interfering and probe segments). mex-3B antisense or
`
`dsRNA was injected into the gonads of adult animals, which were fed for 24 h
`
`before fixation and in situ hybridization (ref. 5; B. Harfe and A.F., unpublished
`
`observations). The mex-3B dsRNA produced 100% embryonic arrest, whereas
`⬎90% of embryos produced after the antisense injections hatched. Antisense
`probes for the mex-3A portion of mex-3 were used to assay distribution of the
`
`endogenous mex-3 mRNA (dark stain). four-cell-stage embryos are shown;
`
`similar results were observed from the one to eight cell stage and in the germ
`
`line of injected adults. a, Negative control showing lack of staining in the absence
`
`of the hybridization probe. b, Embryo from uninjected parent (showing normal
`pattern of endogenous mex-3 RNA20). c, Embryo from a parent injected with
`
`purified mex-3B antisense RNA. These embryos (and the parent animals) retain
`
`the mex-3 mRNA, although levels may be somewhat less than wild type. d,
`
`Embryo from a parent injected with dsRNA corresponding to mex-3B; no mex-3
`RNA is detected. Each embryo is approximately 50 ␮m in length.
`
`letters to nature
`
`8
`
`cells. The phenotypes seen in unc-54 and hlh-1 null mutants, in
`particular, are known to result from many defective muscle cells11,16.
`To examine interference effects of dsRNA at a cellular level, we used
`a transgenic line expressing two different green fluorescent protein
`(GFP)-derived fluorescent-reporter proteins in body muscle. Injec-
`tion of dsRNA directed to gfp produced marked decreases in the
`fraction of fluorescent cells (Fig. 2). Both reporter proteins were
`absent from the affected cells, whereas the few cells that were
`fluorescent generally expressed both GFP proteins.
`The mosaic pattern observed in the gfp-interference experiments
`was nonrandom. At low doses of dsRNA, we saw frequent inter-
`ference in the embryonically derived muscle cells that are present
`when the animal hatches. The interference effect in these differ-
`entiated cells persisted throughout larval growth: these cells pro-
`duced little or no additional GFP as the affected animals grew. The
`14 postembryonically derived striated muscles are born during early
`larval stages and these were more resistant to interference. These
`cells have come through additional divisions (13–14 divisions
`versus 8–9 divisions for embryonic muscles18,19). At high concen-
`trations of gfp dsRNA, we saw interference in virtually all striated
`body-wall muscles, with occasional lone escaping cells, including
`cells born during both embryonic and postembryonic development.
`The non-striated vulval muscles, which are born during late larval
`development, appeared to be resistant to interference at all tested
`concentrations of injected dsRNA.
`We do not yet know the mechanism of RNA-mediated inter-
`ference in C. elegans. Some observations, however, add to the debate
`about possible targets and mechanisms.
`First, dsRNA segments corresponding to various intron and
`promoter sequences did not produce detectable interference
`(Table 1). Although consistent with interference at a post-transcrip-
`tional level, these experiments do not rule out interference at the
`level of the gene.
`Second, we found that injection of dsRNA produces a pro-
`nounced decrease or elimination of the endogenous mRNA tran-
`script (Fig. 3). For this experiment, we used a target transcript (mex-
`3) that is abundant in the gonad and early embryos20, in which
`straightforward in situ hybridization can be performed5. No endo-
`genous mex-3 mRNA was observed in animals injected with a
`dsRNA segment derived from mex-3. In contrast, animals into
`which purified mex-3 antisense RNA was injected retained sub-
`stantial endogenous mRNA levels (Fig. 3d).
`Third, dsRNA-mediated interference showed a surprising ability
`to cross cellular boundaries. Injection of dsRNA (for unc-22, gfp or
`lacZ) into the body cavity of the head or tail produced a specific and
`robust interference with gene expression in the progeny brood
`(Table 2). Interference was seen in the progeny of both gonad
`arms, ruling out the occurrence of a transient ‘nicking’ of the gonad
`
`Table 2 Effect of site of injection on interference in injected animals and their progeny
`
`Progeny phenotype
`Injected-animal phenotype
`Site of injection
`dsRNA
`...................................................................................................................................................................................................................................................................................................................................................................
`None
`Gonad or body cavity
`No twitching
`No twitching
`None
`Gonad or body cavity
`Strong nuclear and mitochondrial GFP expression
`Strong nuclear and mitochondrial GFP expression
`
`unc22B
`unc22B
`unc22B
`
`gfpG
`gfpG
`
`Gonad
`Body-cavity head
`Body-cavity tail
`
`Gonad
`Body-cavity tail
`
`Weak twitchers
`Weak twitchers
`Weak twitchers
`
`Strong twitchers
`Strong twitchers
`Strong twitchers
`
`Lower nuclear and mitochondiral GFP expression
`Lower nuclear and mitochondrial GFP expression
`
`Rare or absent nuclear and mitochondiral GFP expression
`Rare or absent nuclear and mitochondrial GFP expression
`
`Rare or absent nuclear-GFP expression
`Lower nuclear GFP expression
`Gonad
`lacZL
`Rare or absent nuclear-GFP expression
`Lower nuclear GFP expresison
`Body-cavity tail
`lacZL
`...................................................................................................................................................................................................................................................................................................................................................................
`The GFP-reporter strain PD4251, which expresses both mitochondrial GFP and nuclear GFP–LacZ, was used for injections. The use of this strain allowed simultaneous