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`1
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`
`RNA SILENCING
`AND NONCODING
`RNA
`
`1259 The Other RNA World
`
`VIEWPOINTS
`
`
`
`
`
`RNA silenc-
`ing of green
`fluorescent
`protein (GFP)
`(center) in leaves from Nicotiana benthami-
`anais suppressedby an animal(left; B2 pro-
`tein of flock house virus) or a plant (right)
`viral suppressor, leading to enhanced GFP
`:
`expression (lighter green/yellow areas). The
`role of RNA silencing in defending both
`plant and animal genomes from invading
`foreign nucleic acids, the mechanisms
`underlying RNAsilencing, and noncodin
`g
`g
`RNAsare considered in this special section.
`[Image: Shou-WeiDing]
`
`1263
`
`1260 An Expanding Universe of
`Noncoding RNAsG. Storz
`RNASilencing: The Genome’s
`Immune System
`R HEA! Plaster
`Pr ea
`1265 Ancient Pathways
`Programmedby SmallRNAs
`g'
`y,
`P. D. Zamore
`REVIEW
`1270 RNA-Dependent RNAPolymerases,Viruses, and RNASilencingP. Ahlquist
`
`See also Science's STKE on p. 1195 and Report onp. 1319
`
`1319
`
`1313 Vitamin D ReceptorAs anIntestinal Bile
`Acid Sensor M. Makishima,T. T. Lu, W.Xie, G.
`K. Whitfield, H. Domoto, R. M. Evans, M.R.
`Haussler, D.J. Mangelsdorf
`1316 Heterotopic Shift of Epithelial-Mesenchymal
`Interactions in Vertebrate Jaw Evolution
`Y. Shigetani, F. Sugahara,Y. Kawakami,
`Y. Murakami,S. Hirano,S. Kuratani
`Induction and Suppression of RNA
`Silencing by an AnimalVirus H.Li, W. X.Li,
`S.W.Ding
`Is Face Processing Species-Specific During
`theFirst Year of Life? O.Pascalis,
`M. de Haan,C. A. Nelson
`y1323 Direct Recognition of Cytomegalovirus by
`1248
`Activating and Inhibitory NK Cell Receptors
`H.Arase,E. S. Mocarski, A. E. Campbell, A. B.Hill,
`L. L. Lanier
`
`1321
`
`1305
`Tracking the rise of dinosaurs
`
`
`
`
`RESEARCH
`
`1275
`
`BREVIA
`Rebirth of Novae as Distance Indicators
`Dueto Efficient, Large TelescopesM. Della
`Valle and R. Gilmozzi
`
`1276
`1250
`
`1280
`
`1285
`
`1290
`
`1293
`
`1297
`
`1300
`
`1302
`
`¢ 1305
`1215
`
`RESEARCH ARTICLES
`
`Premature Aging in Mice Deficient in DNA
`Repair and Transcription J. de Boer, J. O.
`Andressoo,J. de Wit, J. Huijmans, R. B. Beems,
`H. van Steeg, G. Weeda, G.T.J. van der Horst,
`W. van Leeuwen,A. P. N. Themmen,
`M. Meradji, J. H. J. Hoeijmakers
`
`Structural Basis of Transcription
`Initiation: RNA Polymerase Holoenzyme
`at 4 A Resolution K. S. Murakami, S. Masuda,
`S.A. Darst
`
`Structural Basis of Transcription Initiation:
`An RNAPolymerase Holoenzyme-DNA
`Complex K.S. Murakami, S. Masuda,
`E.A. Campbell, O. Muzzin, S.A. Darst
`REPORTS
`Formationof a Matter-Wave Bright Soliton
`L. Khaykovich, F. Schreék, G.Ferrari, T. Bourdel,
`J. Cubizolles, L. D. Carr, ¥, Castin, C. Salomon
`Electrochemistry and|Electrogenerated
`Chemiluminescence from Silicon
`Nanocrystal QuantumBots Z. Ding,
`B. M. Quinn,S. K. Haram;’L.E.Pell,
`B.A. Korgel, A. J. Bard
`Global AzimuthalAnisotropyin the
`Transition ZoneJ. Trampert and
`H.Jan van Heijst
`Seismic Evidence for Olivine Phase
`Changesatthe 410- and 660-Kilometer
`Discontinuities S. Lebedev, S. Chevrot,
`R. D.van der Hilst
`Identity and Search in Social Networks
`D.J. Watts,P. S. Dodds, M.E. J. Newman
`Ascent of Dinosaurs Linked to an Iridium
`Anomaly at the Triassic-Jurassic Boundary
`P. E. Olsen, D. V. Kent, H.-D. Sues, C. Koeberl,
`H. Huber, A. Montanari, E. C. Rainforth,S.J.
`Fowell, M. J. Szajna, B. W. Hartline
`C-Cadherin Ectodomain Structure and
`Implications for Cell Adhesion Mechanisms
`T. J. Boggon,J. Murray, S. Chappuis-Flament,
`E. Wong, B. M. Gumbiner, L. Shapiro
`
`AY
`
`AMERICAN
`ASSOCIATION FOR THE
`ADVANCEMENTOF
`SCIENCE
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`www.sciencemag.org
`
`SCIENCE
`
`VOL 296
`2
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`17 MAY 2002
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`1193
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`RNA SILENCING AND NONCODING RNA] 22
`VIEWPOINT
`
`An Expanding Universe of Noncoding RNAs
`
`Gisela Storz
`
`Noncoding RNAs (ncRNAs) have been found to have roles in a great
`variety of processes,
`including transcriptional regulation, chromosome
`replication, RNA processing and modification, messenger RNA stability
`and translation, and even protein degradation and translocation. Recent
`studies indicate that ncRNAs are far more abundant and important than
`initially imagined. These findings raise several fundamental questions:
`How many ncRNAsare encoded by a genome? Given the absence of a
`diagnostic open reading frame, how can these genes be identified? How
`can all the functions of ncRNAsbe elucidated?
`
`Over the years, a number of RNAsthat do
`not function as messenger RNAs (mRNAs),
`transfer RNAs
`(tRNAs), or
`ribosomal
`RNAs
`(rRNAs) have been discovered,
`mostly fortuitously. The non-mRNAs have
`been given a variety of names (/, 2); the
`term small RNAs (sRNAs) has been pre-
`dominant
`in bacteria, whereas the term
`noncoding RNAs (ncRNAs) has been pre-
`dominant in eukaryotes and will be used
`here. ncRNAsrangein size from 21 to 25 nt
`for
`the
`large
`family of microRNAs
`(miRNAs) that modulate development
`in
`Caenorhabditis elegans, Drosophila, and
`mammals (3—8), up to ~100 to 200 nt for
`sRNAs commonly found as translational
`regulators in bacterial cells (9, 10) and to
`>10,000 nt for RNAs involved in gene
`
`
`
`Cell Biology and Metabolism Branch, National Insti-
`tute of Child Health and Human Development, Na-
`tional Institutes of Health, Bethesda, MD 20892, USA.
`E-mail: storz@helix.nih.gov
`
`silencing in higher eukaryotes (//—/3). The
`functions described for ncRNAs thus far
`are extremely varied (Table 1).
`Some ncRNAsaffect transcription and
`chromosomestructure. The Escherichia coli
`6S RNA binds to the bacterial «7° holoen-
`zyme and modulates promoter use (/4), and
`the human 7SK RNAbinds andinhibits the
`transcription elongation factor P-TEFb (J5,
`16). Another human ncRNA, SRA RNA,was
`identified as interacting with progestin ste-
`roid hormone receptor and may serve as a
`coactivator of transcription (17). Several ex-
`tremely long ncRNAsdetected in insect and
`mammalian cells have been implicated in
`silencing genes and changing chromatin
`structure across large chromosomal regions
`(/1-13). Examples include the human Xist
`RNArequired for X chromosomeinactiva-
`tion and mouse Air RNA required for auto-
`somal gene imprinting. The Xist RNA is pro-
`duced by the inactive X chromosome and
`spreads in cis along the chromosome (/3).
`The chromosome-associated RNA has been
`
`proposedto recruit proteins that affect chro-
`matin structure; however, much remainsto be
`learned about the mechanism by which Xist
`and other
`long ncRNAs establish and/or
`maintain gene silencing. Another eukaryote-
`specific RNAthatis required for proper chro-
`mosomereplication and structure is the te-
`lomerase RNA. This ncRNA is an integral
`part of the telomerase enzyme andserves as
`the template for the synthesis of the chromo-
`some ends (18).
`ncRNAs play roles in RNA processing
`and modification. The catalytic ribonuclease
`P (RNase P) RNA,found in organisms from
`all kingdoms,
`is responsible for processing
`the 5’ end of precursor tRNAs and some
`rRNAs (19).
`In eukaryotes, small nuclear
`RNAs (snRNAs) are central to splicing of
`pre-mRNAs(20), and small nucleolar RNAs
`(snoRNAs) direct
`the 2'-O-ribose methyl-
`ation (C/D-box type) and pseudouridylation
`(H/ACA-box type) of rRNA,
`tRNA, and
`ncRNAsbyforming basepairs with sequenc-
`es near the sites to be modified (2/). Ho-
`mologs of the two classes of snoRNAs have
`been found in archaea (22); however, coun-
`terparts have not yet been identified in bac-
`teria, even though the rRNAsare modified.
`The less ubiquitous guide RNAs (gRNAs)
`present in kinetoplasts direct the insertion or
`deletion of ‘uridine
`residues
`into mRNA
`(RNA editing)’ by mechanisms that involve
`base-pairing as\well (23, 24).
`ncRNAs also regulate mRNA stability
`
`Genesilencing
`Replication
`RNAprocessing
`RNA modification
`
`RNAstability
`mRNAtranslation
`
`Table 1. Processes affected by ncRNAs.
`
` Process Example Function Reference
`
`
`
`Transcription
`184-nt E. coli 6S
`Modulates promoter use
`(9, 14)
`331-nt human 7SK
`Inhibits transcription elongation factor P-TEFb
`(15, 16, 46)
`875-nt human SRA
`Steroid receptor coactivator
`(12, 17)
`16,500-nt human Xist
`Required for X-chromosomeinactivation
`(72, 13)
`~100,000-nt human Air
`Required for autosomal gene imprinting
`(77)
`451-nt human telomerase RNA
`Core of telomerase and telomere template
`(18, 46)
`377-nt E. coli RNase P
`Catalytic core of RNase P
`(9, 19)
`186-nt human U2 snRNA
`Core of spliceosome
`(20, 46)
`102-nt S. cerevisiae U18 C/D snoRNA
`Directs 2’-O-ribose methylation of target rRNA
`(21, 47)
`189-nt S. cerevisiae snR8 H/ACA snoRNA
`Directs pseudouridylation of target rRNA
`(21, 47)
`68-nt T. brucei gCYb gRNA
`Directs the insertion and excision of uridines
`(23, 24, 48)
`80-nt £. coli RyhB sRNA
`Targets mRNAsfor degradation?
`(27)
`Eukaryotic miRNA?
`Targets mRNAsfor degradation?
`(7, 8)
`109-nt E. coli OxyS
`Repressestranslation by occluding ribosome binding
`(9, 10)
`87-nt E. coli DsrA sRNA
`Activates translation by preventing formation of an
`(9, 10)
`inhibitory MRNA structure
`Represses translation by pairing with 3’ end of
`target MRNA
`Directs addition of tag to peptides onstalled
`Protein stability
`ribosomes
`Integral componentof signal recognition particle
`Protein
`translocation
`central to protein translocation across
`membranes
`
`
`22-nt C. elegans lin-4 miRNA
`363-nt E£. coli tmRNA
`114-nt E. coli 4.55 RNA
`
`(7, 8)
`(9, 28)
`(9, 29)
`
`1260
`17 MAY 2002 VOL 296 3SCIENCE www.sciencemag.org
`
`3
`
`
`
`————— RNA SILENCING AND NONCODING RNA
`
`
`
`ncRNAswhere base-pairing (often <10 base
`pairs and discontinuous) with another RNA
`or DNA molecule is central to function. The
`snoRNAsthat direct RNA modification, the
`bacterial RNAs that modulate translation by
`forming base pairs with specific
`target
`mRNAs, and probably most of the miRNAs
`are examplesofthis category. Some ncRNAs
`mimic the structures of other nucleic acids;
`the 6S RNA structure is reminiscent of an
`open bacterial promoter, and the tmRNAhas
`features of both tRNAs and mRNAs. Other
`ncRNAs, such as the RNase P RNA, have
`catalytic functions. Although synthetic RNAs
`have been selected to have a variety of bio-
`chemical functions,
`the number of natural
`ncRNAs shownto have catalytic function is
`limited. Most, if not all, ncRNAsare associ-
`ated with proteins that augment their func-
`tions; however, some ncRNAs, such as the
`snRNAsand the SRP RNA,serve key struc-
`tural roles in RNA-protein complexes. Sev-
`eral ncRNAsfit into more than one mecha-
`nistic category; the telomerase RNA provides
`the base-pairing template for telomere syn-
`thesis andis an integral part of the telomerase
`ribonucleoprotein complex. The mechanisms
`of action for a number of ncRNAs(such as
`the 7SK RNA) are not known, and it
`is
`probable that some ncRNAsact in ways that
`have not yet been established. Some investi-
`gators have suggested that many ncRNAsare
`vestiges of a world in which RNAcarried out
`all of the functions in a primitive cell. How-
`ever, given the versatility of RNA and the
`fact that the properties of RNA provide ad-
`vantages over peptides for some mechanisms,
`it is likely that a number of ncRNAs have
`evolved more recently (30, 31).
`
`How Many ncRNAsExist?
`The first ncRNAs were identified in the
`1960s on the basis of their high expression;
`these RNAs were detected by direct labeling
`and separation on polyacrylamide gels. Oth-
`ers were later found by subfractionation of
`nuclear extracts or by association with spe-
`cific proteins. A few were identified by mu-
`tations or phenotypes resulting from overex-
`pression. The serendipitous discoveries of
`many of these ncRNAswerethefirst glimps-
`es of their existence, but this work did not
`presage the vast numbers that appear to be
`encoded by a genome.
`Several systematic searches for ncRNA
`genes have been carried out in the past 4
`years. Among the computation-based search-
`es, there have been screens of the yeast Sac-
`charomyces cerevisiae and archaeal Pyrococ-
`cus genomes for the short conserved motifs
`present in snoRNAs(32, 33). In other search-
`es, the intergenic regions of S. cerevisiae, E.
`coli, Methanococcus jannaschii, and Pyro-
`coccus furiosus chromosomes have been
`scanned for properties
`indicative of an
`
`
`
`
`
`
`
`
`
`
`
`discovered
`first
`The
`translation.
`and
`miRNAs, C. elegans lin-4 and let-7, repress
`translation by forming base pairs with the 3’
`end of target mRNAs (7, 8). Many of the
`recently identified miRNAsare likely to act
`in a similar fashion. However, it is conceiv-
`able that some membersofthis large family
`target mRNAsfor degradation,as is the case
`for the similarly sized small interfering RNAs
`(siRNAs) that are processed and amplified
`from exogenously added, double-stranded
`RNAand lead to gene suppression in a pro-
`cess termed RNA interference (25, 26). As
`yet there is no evidence for miRNAsin bac-
`teria, archaea, or fungi, but it might be fruitful
`to search for RNAs of <25 nt in these organ-
`isms. Several ncRNAs have been found to
`regulate translation and possibly mRNAsta-
`bility in E. coli (9, 10, 27). These sRNAs
`formbasepairsat variouspositions with their
`target mRNAs, and they have been shown to
`repress translation by occluding the ribosome
`binding site and to activate translation by
`
`preventing the formation of inhibitory mRNA
`structures.
`
`Finally, ncRNAsaffect protein stability
`and transport. One unique bacterial sRNA is
`recognized as both a tRNA and an mRNA by
`stalled ribosomes (tmRNA) (28). Alanylated
`tmRNAis delivered to the A site of a stalled
`ribosome;
`the nascent polypeptide is trans-
`ferred to the alanine-charged tRNAportion of
`tmRNA. The problematic transcript then is
`replaced by the mRNAportion of tmRNA,
`which encodes a tag for degradation of the
`stalled peptide. It is not yet clear whether
`there is a counterpart to this coding RNA in
`archaeal and eukaryotic cells. In contrast, a
`small cytoplasmic RNA that forms the core
`of the signal recognition particle (SRP) re-
`quired for protein translocation across mem-
`branes is found in organisms from all king-
`doms(29).
`The mechanismsof action for the charac-
`terized ncRNAscan be groupedinto several
`general
`categories
`(Fig.
`1). There
`are
`
`
`
` 4.55 RNA
`
`
`
`RNA polymerase
`
`Ribosome
`
`Fig. 1. Different mechanisms of ncRNA(red) action. (A) Direct base-pairing with target RNA or
`DNA molecules is central to the function of some ncRNAs: Eukaryotic snoRNAsdirect nucleotide
`Modifications (green star) by forming basepairs with flanking sequences, and the E. coli OxyS RNA
`represses translation by forming base pairs with the Shine-Dalgarno sequence (green box) and
`occluding ribosomebinding. (B) Some ncRNAs mimic the structure of other nucleic acids: Bacterial
`RNA polymerase may recognize the 6S RNA as an open promoter, and bacterial ribosomes
`Fecognize tmRNAas both a tRNA and an mRNA.(C) ncRNAsalso can function as an integral part
`of a larser RNA-protein complex, such as the signal recognition particle, whose structure has been
`Partially determined (49).
`
`
`
`www.sciencemag.org SCIENCE, VOL 296 17 MAY 2002
`
`1261
`
`
`4
`
`
`
`
`
`
`recent discovery of hundreds of new ncRNAs
`illustrates that the “RNome”also will need to
`be characterized before a complete tally of
`the number of genes encoded by a genome
`can be achieved.
`
`studyingall aspects of biology should keep
`ncRNAsin mind. The phenotypesassociat-
`ed with specific mutations may be due to
`defects in a ncRNAinstead of being due to
`defects in a protein, as is usually expected.
`Investigators
`developing
`purification
`schemes for specific proteins or activities
`should be aware of the possible presence of
`an ncRNA component; many purification
`procedures are designed to remove nucleic
`acids. There may be ncRNAslurking be-
`hind many an unexplained phenomenon.
`
`WwONaW
`
`in
`
`ncRNA gene. Criteria for identifying candi-
`date intergenic regions have included large
`gaps between protein-coding genes (34), ex-
`tendedstretches of conservation between spe-
`cies with the same gene order (35, 36), or-
`phan promoter or terminator sequences (34,
`WhatAreAll the Functions of
`36, 37), presence of GC-rich regions in an
`ncRNAs?
`organisms with a high AT content (38), and
`Anastonishing variety of ncRNA functions
`conserved RNA secondary structures (39,
`have already been found, but
`there are
`40). Other searches for ncRNAs have in-
`many ncRNAsfor which the cellular roles
`volved large-scale cloning efforts that have
`are still unknown. For instance, Y RNAs,
`taken into account specific ncRNA proper-
`small cytoplasmic RNAsassociated with
`ties. In studies of mouse (4/, 42) and the
`the Ro autoantigen in several different or-
`References and Notes
`archaeon Archaeoglobus fulgidus (22), total
`ganisms, are still enigmatic even after
`1. S. R. Eddy, Nature Rev. Genet. 2, 919 (2001).
`RNAbetween 50 to 500 nt wasisolated, and
`2. Non-mRNAshave been denoted ncRNA = noncoding
`many years of study (45). With the more
`arrays of cDNA clones obtained from the
`RNA, snmRNA = small non-mRNA, sRNA = small
`systematic
`identification of
`increasing
`RNA were screened with oligonucleotides
`RNA, fRNA = functional RNA, and oRNA = other
`numbers of ncRNAs, the question of how to
`corresponding to the most abundant known
`RNA, andit is likely that the nomenclature of these
`RNAswill need to berevisited.
`elucidate the functions of all ncRNAs is
`RNAs.Clones showing the lowest hybridiza-
`3. M. Lagos-Quintana, R. Rauhut, W. Lendeckel, T.
`becoming more and more prominent.
`tion signal then were randomly sequenced.In
`Tuschl, Science 294, 853 (2001).
`Approachesthat have succeeded previously
`recent screens for C. elegans, Drosophila,
`-
`. N.C. Lau, L. P. Lim, E. G. Weinstein, D. P. Bartel,
`are an obviousplace to start in answering the
`and human miRNAs, RNA moleculesofless
`Science 294, 858 (2001).
`question of function, but it is likely that new
`. R. C. Lee, V. Ambros, Science 294, 862 (2001).
`than 30 nt were isolated, and cDNA clones
`. Z. Mourelatos et al., Genes Dev. 16, 720 (2002).
`approachesalso will need to be developed. For
`were generated upon the ligation of primers
`. G. Ruvkun, Science 294, 797 (2001).
`genetically tractable organisms, ncRNA knock-
`to the 5’ and 3’ ends of the RNA(3, 4) or
`. H. Grosshans,F. J. Slack, J. Cell Biol. 156, 17 (2002).
`out or overexpression strains can be screened
`upon RNAtailing (5). Other miRNAs were
`.
`_K. M. Wassarman, A. Zhang, G. Storz, Trends Micro-
`for differences in phenotypes(such asviability)
`isolated and cloned on the basis of their
`biol. 7, 37 (1999).
`10. S. Altuvia, E. G. H. Wagner, Proc. Natl. Acad. Sci.
`or whole- genome expression patterns. The
`association with a complex composed ofthe
`U.S.A. 97, 9824 (2000).
`functions of several ncRNAswereidentified by
`human Gemin3, Gemin4, and IF2C proteins
`11. F. Sleutels, R. Zwart, D. P. Barlow, Nature 415, 810
`the biochemical
`identification of associated
`(6). In most studies, Northern blots have been
`(2002).
`proteins, and the development of more system-
`carried out to confirm that the cloned genes
`12. V. A. Erdmann, M. Szymanski, A. Hochberg, N. de
`Groot, J. Barciszewski, Nucleic Acids Res. 28, 197
`atic methods for characterizing ncRNA-associ-
`are expressed as small
`transcripts. These
`(2000).
`ated proteins should be fruitful. As the knowl-
`blots also have provided information about
`13. P. Avner, E. Heard, Nature Rev. Genet. 2, 59 (2001).
`edge base of what sequencesare critical for the
`spatial and temporal expression patterns as
`14. K. M. Wassarman, G. Storz, Cell 101, 613 (2000).
`formation of specific structures or for base-
`well as potential precursor and degradation
`15. Z. Yang,,QsZhu, K. Luo, Q. Zhou, Nature 414, 317
`pairing expands,and as computer programs for
`(2001).¢ 3;
`products.
`16. V. T. Nguyet, T. Kiss, A. A. Michels, O. Bensaude,
`predicting structures improve, computational
`Despite the success of the recent system-
`Nature 414? 322 (2001).
`approachesshould become an increasingly im-
`atic efforts, it is certain that not all ncRNAs
`17. RB. Lanz #fal., Cell 97, 17 (1999).
`portant avenue for elucidating the functions of
`have been detected. Estimates for the number
`18. J.-L. Chen,’M:-A, Blasco, C. W. Greider, Cell 100, 503
`ncRNAs. The three-dimensionalstructures of
`of sRNAsin E. coli range from 50 to 200 (J,
`(2000)..
` #
`19. D. N. Frank, N. R. Pace, Annu. Rev. Biochem. 67, 153
`only a limited number of RNAs and RNA-
`35), and estimates for the number of miRNAs
`(1998). *
`protein complexes have been solved. An in-
`in C. elegans range from hundreds to thou-
`20. C. L. Will, R. Luhrmann, Curr. Opin. Cell Biol. 13, 290
`crease in the structural database may bring to
`sands (7). There also are many non-protein-
`(2001).
`,
`light recognizable RNA or RNA-protein do-
`coding regionsofthe bacterial and eukaryotic
`21. T. Kiss, EMBO j. 20, 3617 (2001).
`mains associated with specific functions.
`22. T.-H. Tang et al., Proc. Natl. Acad. Sci. U.S.A.,
`chromosomes for which transcription is de-
`press.
`Information about when ncRNAsare ex-
`tected (43, 44), but it is not known how many
`23. M. L. Kable, S. Heidmann, K. D. Stuart, Trends Bio-
`pressed and where ncRNAsare localized is
`of these regions encode defined, functional
`chem. Sci. 22, 162 (1997).
`useful for all experiments aimed at probing
`ncRNAs. Extensions of the various systemat-
`24. L. Simpson, O. H. Thiemann,N.J. Savill, J. D. Alfonzo,
`D. A. Maslov, Proc. Natl. Acad. Sci. U.S.A. 97, 6986
`function. Many of the C. elegans miRNAs
`ic searches should leadto the identification of
`(2000).
`are synthesized only at very specific times in
`more ncRNAs. However, limitations of the
`25. K. Nishikura, Cell 107, 415 (2001).
`development, and thus they have also been
`current approaches should be noted. Most of
`26. P. D. Zamore, Science 296, 1265 (2002).
`called small
`temporal RNAs
`(stRNAs).
`the computation methods have focusedon the
`27. E. Massé, S. Gottesman, Proc. Natl. Acad. Sci. U.S.A.
`Among the snoRNAs, some are expressed
`99, 4620 (2002).
`intergenic regions. It has already been shown
`28. R. Gillet, B. Felden, Mol. Microbiol. 42, 879 (2001).
`exclusively in the brain (4/), and one of the
`that some of the ncRNAsare processed from
`29. R. J. Keenan, D. M. Freymann,R. M. Stroud, P. Walter,
`bacterial sRNAsis only detected upon oxida-
`longer protein- or rRNA-encoding transcripts
`Annu. Rev. Biochem. 70, 755 (2001).
`tive stress (9, 10).
`It
`is likely that other
`(42). It also is quite possible that ncRNAsare
`30. V. Y. Kuryshev, B. V. Skryabin, J. Kremerskothen, J.
`ncRNAswill be found to have very defined
`Jurka, J. Brosius, J. Mol. Biol. 309, 1049 (2001).
`expressed from the opposite strand of pro-
`31. W. Wang, F. G. Brunet, E. Nevo, M. Long, Proc. Natl.
`expression and localization patterns and that
`tein-coding genes. Onthe other hand, expres-
`Acad. Sci, U.S.A. 99, 4448 (2002).
`these will be critical to function.
`sion-based methods may miss ncRNAsthat
`32. T. M. Lowe,S. R. Eddy, Science 283, 1168 (1999).
`There are many more ncRNAs than was
`are synthesized under very defined condi-
`33. C. Gaspin, J. Cavaillé, G. Erauso, J.-P. Bachellerie, J.
`Mol. Biol. 297, 895 (2000).
`;
`ever suspected. A big challenge for the future
`tions, such as in response toaspecific envi-
`34. W.M.Olivas, D. Muhlrad,R. Parker, Nucleic Acids Res.
`will be to identify the whole complement of
`ronmental signal, during a specific stage in
`25, 4619 (1997).
`ncRNAs andto elucidate their functions. This is
`development,or in a specific cell type. Much
`35. K. M. Wassarman,F. Repoila, C. Rosenow,G.Storz, S.
`an exciting time for investigators whose work
`Gottesman, Genes Dev. 15, 1637 (2001).
`attention has been focused on characterizing
`36. L. Argamanetal., Curr. Biol. 11, 941 (2001).
`has focused on ncRNAs. However, scientists
`the “proteome”of a sequenced organism. The
`
`
`
`
`
`RNA SILENCING AND NONCODING RNA
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`5
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`|ee
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`RNA SILENCING AND NONCODING
`
`RNA
`
`in press.
`37. S. Chenet al., Biosystems,
`38. R.J. Klein, Z. Misulovin, S. Eddy, Proc. Natl. Acad. Sci.
`U.S.A.,
`in press.
`39. E. Rivas,R. J. Klein, T. A. Jones, S. R. Eddy, Curr. Biol.
`11, 1369 (2001).
`40. R. J. Carter, |. Dubchak, S. R. Holbrook, Nucleic Acids
`Res, 29, 3928 (2001).
`1. J. Cavaillé et al., Proc. Natl. Acad. Sci. U.S.A. 97,
`14311 (2000).
`42. A. Hiittenhofer et al., EMBO J. 20, 2943 (2001).
`
`&
`
`43. D. W.Selinger et al., Nature Biotechnol. 18, 1262
`(2000).
`44. K. E. Plant, S. J. E. Routledge, N. J. Proudfoot, Mol.
`Cell. Biol. 21, 6507 (2001).
`45. X. Chen, A. M. Quinn, S. L. Wolin, Genes Dev. 14, 777
`(2000).
`46. J. Gu, Y. Chen, R. Reddy, Nucleic Acids Res. 26, 160
`(1998).
`47. D. A. Samarsky, M. J. Fournier, Nucleic Acids Res. 27,
`161 (1999).
`
`48. A. E. Souza, T. Hermann,H. U. Géringer, Nucleic Acids
`Res. 25, 104 (1997).
`49. R. T. Batey, R. P. Rambo, L. Lucast, B. Rha, J. A.
`Doudna,Science 287, 1232 (2000).
`| thank S. Altuvia, J. Brosius, S. Gottesman, K. M.
`Wassarman,and A. Zhangforhelpful discussions and
`comments on the manuscript. | also thank them and
`many other investigators for extensive discussion of
`the nomenclature.
`
`50.
`
`VIEWPOINT
`
`RNASilencing: The Genome’s
`Immune System
`
`Ronald H. A. Plasterk
`
`
`
`
`
`
`
`
`
`which allows the vertebrate immune system
`to raise a massive immuneresponse (//—/4).
`
`The Function of RNASilencing
`Neither nematodes nor flies normally en-
`counter highly concentrated double-stranded
`RNA (dsRNA) of identical sequence to one
`of their endogenous genes. Nevertheless, ge-
`netic analysis indicates that the number of
`genes required for genesilencing triggered by
`exogenous dsRNAis probably larger than 10
`(15-18). What is the natural function of this
`elaborate pathway?
`The clearest picture is seen in plants,
`where PTGSand virus-induced gene silenc-
`ing are recognized as mechanismsthat pro-
`tect against frequently occurring viral infec-
`tions (6, 19). An advantage of this defense
`system is that the defensive signal can spread,
`such that inoculation in one area of a leaf can
`
`confer immunity on surrounding cells. A
`study in this issue showsthat an animalvirus
`also encodes a suppressor of RNA interfer-
`ence (RNAi),
`supporting the notion that
`RNAi mayhave an antiviral function in ani-
`mals as well
`(20).
`In nematodes,
`loss of
`function of genes required for RNAiresults
`in the activation of multiple transposable el-
`ements in the germline (/5), indicating that
`they function to repress the spreading of
`transposons within the genome of subsequent
`generations of worms.
`Protection against viruses and transposons
`may bethe natural function of the core of the
`RNAi pathway, but it does not explain all
`aspects of what
`is now considered to be
`RNAi. One of the most striking features of
`RNAiin C. elegans is the systemic effect.
`Injection of naked dsRNAinto one region of
`the animal may affect gene expression else-
`where, and dsRNA present in the lumen of
`the gut as part of the food is apparently taken
`up and affects gene expression in progeny
`that arises in the gonads (2/). In plants, graft-
`ing experiments have shown immunity trav-
`eling over 30 cm of stem tissue (22);
`this
`
`
`
`
`
`Genomesare databasessensitive to invasion by viruses. In recent years, a
`defense mechanism. has been discovered, which turns out to be conserved
`amongeukaryotes. The system can be comparedto the immune system in
`several ways: It has specificity against foreign elements and the ability to
`amplify and raise a massive response against an invading nucleic acid. The
`latter property is beginning to be understood at the molecularlevel.
`‘
`SOc
`All genomes of complex orgatlismis are po-
`tential
`targets of invasion by),viruses and
`transposable elements. Forty-five'percent of
`the human genome consists oftvemnants of
`previous transposon/Virus invasions and ele-
`ments that are still active to date: 21% long
`interspersed nuclear elements, 13% short in-
`terspersed nuclear elements, 8% retroviruses,
`and 3% DNA-transposons, as compared with
`less than 2% that encodes (nontransposon)
`proteins. A priori, one would expect
`that
`organisms needto fight off such invasions to
`prevent the genome from being completely
`taken over by molecular invaders. The two '
`problems with which the organism is faced in
`protecting the integrity of the genome are
`similar to those faced by the vertebrate im-
`mune system: (i) how to recognize self from
`nonself, and (ii) how to amplify an initial
`response in a specific fashion.
`The vertebrate immune system fights off
`invaders using a two-step strategy: a large
`repertoire of antibody-encoding genesis gen-
`erated from a limited set of gene segments by
`combinatorial gene rearrangements, and this
`Tepertoire is stored in a distributed fashion
`over large numbersofcells. After infection,
`clonal selection and expansion of a few of
`these cells results in an immune response
`Specifically directed to the immunogen. The
`vertebrate immune system has solved the
`Specificity problem by initially generating a
`- More orless random repertoire, which, during
`a Phase of early development,is limited by a
`filtering process, called tolerance induction:
`
`
`cells raised against self antigens are excluded
`from the mature immune system.
`How does the genome recognize invaders
`and raise an overwhelming and specific “im-
`mune response” against them? One strategy
`to suppress transposons maybethe selective
`methylation of transposon sequences in the
`genome(/), although it has also been argued
`that this phenomenonis a secondary effect of
`suppression (2). This will not be discussed
`further, but see a recent review for more
`information (3). In recent years, an RNA-
`based silencing mechanism has emergedthat
`is ancient, conserved among species from
`different kingdoms
`(fungi,
`animals,
`and
`plants), and very likely acts as the “immune
`system” of the genome. This system was
`initially independently discovered and stud-
`ied in different organisms before it was rec-
`ognized that the underlying mechanisms are
`at some level
`identical. Posttranscriptional
`gene silencing (PTGS) and co-suppression in
`plants (4, 5), as well as RNA-mediated virus
`resistance in plants (6), RNA interference in
`animals [first discovered in Caenorhabditis
`elegans (7)], and silencing in fungi [“quell-
`ing” in Neurospora (8)] and algae (9) are all
`based on the same core mechanism. This
`
`conclusion is based on the discovery of com-
`mon mechanistic elements [such as the small
`interfering RNAs (siRNAs) (/0)] and of ho-
`mology between genes
`required for
`this
`mechanism in plants, animals, and fungi and
`algae.
`The precise mechanism of this group of
`phenomena, nowreferred to as RNA silenc-
`ing, is being rapidly unraveled. The aspect
`that I specifically address here is the equiva-
`lent in RNA silencing of “clonal selection,”
`
`Hubrecht Laboratory, Centre for Biomedical Genetics,
`Uppsalalaan 8, 3584 CT Utrecht, Netherlands. E-mail:
`Plasterk@niob.knaw.nl
`
`
`
`www.sciencemag.org SCIENCg VOL 296
`
`17 MAY 2002
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