486 | JULY 2001 | VOLUME 2 www.nature.com/reviews/genetics

H I G H L I G H T S

Since its discovery in Caenorhabditiselegans, RNA interference (RNAi) hasrevolutionized gene-function analy-sis. The success of RNAi lies in itssimplicity when introduced intocells of certain organisms, double-stranded (ds) RNAs can cause asequence-specific mRNA degrada-tion that mimics a loss-of-functionphenotype. Elbashir and colleaguesnow show for the first time that RNAican work consistently and specificallyin mammalian cells in response toshort dsRNA molecules (calledsiRNAs). Their study also reveals theexistence of two pathways onegeneral and one sequence specific that are activated in mammalian cellsin response to dsRNA.

For those interested in genefunction, RNAi-mediated geneknock-down is a useful and rapidalternative to gene knockouts orconventional antisense technology,and it works efficiently in plantsand in many invertebrates.Although RNAi has previously beenshown to work in mammalianoocytes and embryos, in mam-malian cells dsRNAs longer than 30nucleotides cause a general,sequence non-specific, mRNAdegradation and translational shut-down. As very short dsRNAs medi-ate specific RNAi effects inDrosophila, Elbashir et al. testedwhether such siRNAs might alsowork in mammalian cells. Theysynthesized 21-mer dsRNA mole-cules with sequence homology tothree luciferase genes and co-trans-fected plasmids containing thesegenes with the correspondingsiRNAs into four different types ofmammalian cell lines. The authors

determined siRNA effectiveness bymeasuring luciferase luminescenceand found that, although the extentof suppression varies — possiblydepending on the level of targetgene expression — suppression iscompletely sequence specific.Furthermore, siRNA is effective atsignificantly lower concentrationsthan antisense or ribozymes ingene-targeting experiments. Similarresults were also obtained for sup-pressing endogenous genes.

Elbashir et al. investigated theeffects of longer, supposedly non-specific, dsRNAs and found thatthey also act in a sequence-specificmanner, but that this is normallymasked by the non-specific effects.Their observations indicate thatthere are two pathways in mam-malian cells that can be activated inresponse to dsRNAs.

RNAi provides unequalled flexi-bility for rapidly analysing gene function and can be used to

It’s been a long time coming, but now two papers report a clear-cutidentification by linkage mapping ofa gene involved in a common humandisorder — Crohn disease (CD).Importantly, they also indicate how the innate immune system might beinvolved in the aetiology of CD,because the identified gene — NOD2— encodes an intracellular receptor for bacterial lipopolysaccharides (LPS) that activates NFκB, a target of the innate immune signalling pathway and a transcriptional regulator of inflammatory genes.

CD is a chronic inflammatory gutdisorder, thought to be caused by an abnormal inflammatory response to enteric microbes. In 1996, a CDsusceptibility locus, IBD1, was identified on chromosome 16. Littleprogress has been made since, but it is this locus that the two research teams —one European, the other US-based —tackled in their studies, using positional-cloning and candidate-gene strategies,respectively.

Hugot et al. took a decisive step when theyidentified association of CD to an allele of achromosome-16 microsatellite marker.Despite the borderline significance of thisassociation, the authors went on to identifyputative transcripts in the region of thismarker, and identified over 30 singlenucleotide polymorphisms (SNPs) bysequencing the region from affected andunaffected individuals. Several turned outto be non-synonymous variants in achromosome-16 gene, NOD2. Three of theseSNPs — each independently associated withdisease susceptibility — altered the leucine-rich repeat (LRR) region of NOD2, which isrequired for LPS recognition.

Having previously identified NOD2, Oguraet al. considered it a candidate for CD becauseof its chromosome-16 location. Onsequencing the gene from CD individuals,they identified an insertion that caused twoframeshift mutations in the LRR region andthe premature truncation of NOD2. In invitro assays, this mutant NOD2 producedconsiderably diminished levels of NFκBactivation in response to bacterial LPScompared to wild-type NOD2.

So how could NOD2 contribute tosusceptibility to CD? The innate immunesystem regulates the immediate immuneresponse to bacterial pathogens,components of which are recognized inhost immune cells by specific receptors,such as NOD2. A defect in this recognitionmight lead to an exaggeratedinflammatory reaction being mediated by the adaptive immune system.Alternatively, NOD2 might act to triggercytokines that dampen inflammatoryresponses. Although NOD2 does notaccount for all susceptibility to CD, it doesprovide a first glimpse into the aetiology of the disease and should speed thediscovery of other CD loci and futuretherapies, and improve its diagnosis. AsJohn Todd comments in an accompanyingNews and Views, these papers arehopefully the first of many such successesin grappling with the genetic basis ofmultifactorial, common disease.

Jane Alfred

References and linksORIGINAL RESEARCH PAPERS Hugot, J.-P. et al.Association of NOD2 leucine-rich repeat variants withsusceptibility to Crohn’s disease. Nature 411, 599–603(2001) | Ogura, Y. et al. A frameshift mutation in NOD2associated with susceptibility to Crohn’s disease. Nature411, 603–606 (2001)FURTHER READING Todd, J. A. Tackling commondisease. Nature 411, 537–539 (2001)WEB SITE NIH Crohn disease web links

Opening the flood gates?

M U LT I FA C TO R I A L D I S E A S E

It’s a knock-down

T E C H N O LO G Y

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NATURE REVIEWS | GENETICS VOLUME 2 | JULY 2001 | 487

The identification of the Drosophila period (per)locus — published in 1971 by Ronald Konopkaand Seymour Benzer — was a singular landmarkin behavioural genetics research. They were thefirst to show that genetics could be used todissect behaviour, in this case the internal clock,or circadian rhythm, that underlies manyaspects of behaviour in diverse organisms. Sincethen, many more genes involved in circadianrhythms have been identified (at least nine inmammals); the basic molecular mechanism ofthe clock has been defined; and the generalfeatures of the clock are known to be conservedin most living organisms. But little is knownabout the connections between the cellularworkings of the clock and the patterns ofbehaviour that it influences. Two new studiesprovide valuable insights into how suchknowledge might be obtained.

The study by Zheng et al. begins with ananalysis of the mouse gene Per (mPer1) — one ofthree mammalian per homologues. By knockingout Per, the authors show that its clock functionsdiffer from those of Per2 (mPer2). This reinforcesthe idea that the core mechanism of themammalian clock is more complex than inDrosophila. To investigate further the differentroles of Per and Per2, the authors used microarraystudies to identify genes that might be regulatedby the clock in Per, Per2 and double-knockoutmutants. They found 16 genes with reproduciblecircadian expression that was dependent on Per,Per2, or both genes. Two genes in particularshowed circadian expression and were dependenton both Per and Per2: Alas1 and Alas2, whichencode rate-limiting enzymes involved in haem

biosynthesis. Because haem is an essential part ofmany proteins, such as metabolic and signallingproteins, the authors speculate that the circadianregulation of these genes might feed into a rangeof physiological processes.

In the second study, Shimomura et al. used aquantitative genetics approach to identify locithat underlie the difference in circadian rhythms(as measured by wheel-running behaviour)between two inbred mouse strains. Theirphenotypic analysis measured five aspects ofcircadian behaviour, such as the length of thecircadian cycle and the phase angle ofentrainment — how the onset of activity relatesto the light–dark cycles. In addition to searchingfor independent quantitative trait loci, theauthors also looked for epistatic interactionsbetween loci, by incorporating a genome-widesearch for loci that had an effect only incombination with another locus. They found 14 loci that had a significant effect on circadianbehaviour, including several that interactedepistatically. Importantly, most of these locimapped to regions outside those containingknown mammalian circadian genes.

In the 30 years since period was first identified,geneticists have built on the sound foundationslaid by the pioneers of this field, but before thevision of Benzer and his colleagues can berealized, a good deal more work needs to be done.Both of these new lines of research hold greatpromise for finding new genes that functionsomewhere between the cellular clock and itsoutput — circadian behaviour.

Mark Patterson

References and linksORIGINAL RESEARCH PAPERS Zheng, B. et al. Nonredundant rolesof the mPer1 and mPer2 genes in the mammalian circadian clock. Cell105, 683–694 (2001) | Shimomura, K. et al. Genome-wide epistaticinteraction analysis reveals complex genetic determinants of circadianbehavior in mice. Genome Res. 11, 959–980 (2001)FURTHER READING Weiner, J. Time, Love and Memory (Faber &Faber, New York, 1999)WEB SITES Cheng Chi Lee’s lab | Joe Takahashi’s lab

Watching the clock

B E H AV I O U R A L G E N E T I C S

determine gene function on agenome-wide scale, as recentlyshown in C. elegans. The findings ofElbashir et al. have important con-sequences for those investigatinggene function in mammalian cells.But before these applicationsbecome practical in mammaliansystems, it will be essential to estab-lish easy and efficient ways of deliv-ering siRNA into mammalian cellsand rapid phenotypic screens.Questions regarding the biologicalrole of endogenous dsRNAs in generegulation also remain to beanswered.

Magdalena Skipper

References and linksORIGINAL RESEARCH PAPER Elbashir, S. M.et al. Duplexes of 21-nucleotide RNAs mediateRNA interference in cultured mammalian cells.Nature 411, 494–498 (2001) FURTHER READING Bass, B. The shortanswer. Nature 411, 428–429 (2001) |Hammond, S. M. et al. Post-transcriptionalgene silencing by double-stranded RNA. Nature Rev. Genet. 2, 110–119 (2001)

The Thames flood barrier, UK.

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