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`ModernaTX, Inc. v. CureVac AG
`IPR2017-02194
`
`1
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`

`

`
`RESEARCH
`
`BREVIA
`Rebirth of Novae as Distance Indicators
`
`1275
`
`Due to Efficient, Large TeleScopes M. Della
`Valle and R. Gilmozzi
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`$1276
`1250
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`1280
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`1285
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`1290
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`i293
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`1297
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`1300
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`1302
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`$1305
`1215
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`1308
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`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 RNA Polymerase Holoenzyme-DNA
`Complex K. S. Murakami, S. Masuda,
`E. A. Campbell, 0. Muzzin, S.A. Darst
`
`REPORTS
`
`Formation of a Mattei'rWave Bright Soliton
`L. Khaykovich, F. Schre‘lfik, C.‘Ferrari,T. Bourdel,
`J. Cubizolles, L. D. Carr, Y, Castin, C. Salomon
`Electrochemistry and‘Electrogenerated
`Chemiluminescence fi’orn Silicon
`Nanocrystal Quantum‘pots Z. Ding,
`B. M. Quinn, 5. K. Haramf'L. E. Péll,
`B. A. Korgel,A. J. Bard
`
`Global AzimuthalAnisotropy in the
`Transition Zone J. Trampert and
`H. Jan van Heijst
`Seismic Evidence for Olivine Phase
`Changes at the 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
`
`RNA SILENCING
`
`AND NONCODING
`
`RNA
`
`1259 The Other RNA World
`
`VIEWPOINTS
`
`1260 An Expanding Universe of
`Noncoding RNAs G. Storz
`
`1263
`
`RNA Silencing:The Genome's
`Immune System
`R H A Plasterk
`'
`'
`'
`1265 Ancient Pathways
`Programmed by Small RNAs
`P. D. Zamore
`
`REVIEW
`
`
`
`RNA silenc-
`ing of green
`fluorescent
`
`
`
`protein (GFP)
`(center) in leaves from Nicotiana benthami—
`ana is suppressed by an animal (left; B2 pro-
`tein of flock house virus) or a plant (right)
`viral suppressor, leading to enhanced GFP
`_
`,
`expressnon (lighter green/yellow areas). The
`role of RNA Silencmg in defending both
`plant and animal genomes from invading
`foreign nucleic acids, the mechanisms
`underlying RNA silencing, and noncoding
`RNAs are considered in this special section.
`[Imagez Shou-Wei Ding]
`
`1270 RNA-Dependent RNA Polymerases, Viruses, and RNA Silencing P. Ahlquist
`
`See also Science’s STKE on p. 1 195 and Report on p. 1319
`
`1313 Vitamin D Receptor As an Intestinal Bile
`Acid Sensor M. Makishima,T. T. Lu,W. Xie, G.
`K.Whitfield, H. Domoto, R. M. Evans, M. R.
`Haussler, D. J. Mangelsdorf
`
`131 6 Heterotopic Shift of Epithelial-Mesenchymal
`Interactions in Vertebrate Jaw Evolution
`Y. Shigetani, F. Sugahara, Y. Kawakami,
`Y. Murakami, S. Hirano, S. Kuratani
`
`1319
`
`1321
`
`Induction and Suppression of RNA
`Silencing by an AnimalVirus H. Li,W. X. Li,
`S.W. Ding
`
`Is Face Processing Species-Specific During
`the First Year of Life? 0. Pascalis,
`M. de Haan, C.A. Nelson
`
`v1323
`1248
`
`Direct Recognition of Cytomegalovirus by
`Activating and Inhibitory NK Cell Receptors
`H.Arase, E. S. Mocarski, A. E. Campbell,A. B. Hill,
`L. L. Lanier
`
`1305
`
`Tracking the rise of dinosaurs
`
`i
`
`
`.
`77 7.. W
`"W
`Catchingrlymrphocytesw
`New on Science Expseas
`in the act
`
`
`
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`
`SCIENCE (ISSN 0036-8075) is published weekly on Friday, except the last week in December, by the American Association for
`the Advancement of Science, 1200 New York Avenue, NW, Washington, DC 20005. Periodicals Mail postage (publication No.
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`MERICAN
`ASSOCIATION FOR THE
`ADVANCEMENTCW
`&HENCE
`
`www.5ciencemag.org
`
`SCIENCE
`
`VOL 296
`2
`
`17 MAY 2002
`
`1193
`
`2
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`

`

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`
`RNA SILENCING AND NONCODING RNA -——-———-——‘~~
`a: l w a c: a a; ‘23
`
`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 ncRNAs are 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 ncRNAs be elucidated?
`
`Over the years, a number of RNAs that 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 (1, 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. ncRNAs range in size from 21 to 25 nt
`for
`the
`large
`family of microRNAs
`(miRNAs) that modulate development
`in
`Caenorhabditis elegans, Drosophz'la, and
`mammals (3—8), up to ~100 to 200 nt for
`sRNAs commonly found as translational
`regulators in bacterial cells (9, 10) and to
`>l0,000 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 (1143). The
`functions described for ncRNAs thus far
`are extremely varied (Table 1).
`Some ncRNAs affect
`transcription and
`chromosome structure. The Escherichia coli
`6S RNA binds to the bacterial (r70 holoen—
`zyme and modulates promoter use (14), and
`the human 7SK RNA binds and inhibits the
`transcription elongation factor P-TEFb (15,
`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 ncRNAs detected in insect and
`mammalian cells have been implicated in
`silencing genes and changing chromatin
`structure across large chromosomal regions
`(11713). Examples include the human Xist
`RNA required for X chromosome inactiva-
`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 (13).
`The chromosome-associated RNA has been
`
`proposed to recruit proteins that affect chro-
`matin structure; however, much remains to be
`learned about the mechanism by which Xist
`and other
`long ncRNAs establish and/or
`maintain gene silencing. Another eukaryote-
`specific RNA that is required for proper chro-
`mosome replication and structure is the te~
`lomerase RNA. This ncRNA is an integral
`part of the telomerase enzyme and serves 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’-0-ribose methyl—
`ation (C/D-box type) and pseudouridylation
`(H/ACA-box type) of rRNA,
`tRNA, and
`ncRNAs by forming base pairs with sequenc-t
`es near the sites to be modified (21). 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 rRNAs are modified.
`The less ubiquitous guide RNAs (gRNAs)
`present in ldfietoplasts direct the insertion or
`deletion of uridine residues
`into mRNA
`(RNA editing)" by mechanisms that involve
`base-pairing asxwell (23, 24).
`ncRNAs also regulate mRNA stability
`
`Table 1. Processes affected by ncRNAs.
`
`
`
`
` Process Example Function Reference
`
`
`
`
`
`
`
`
`
`
`
`Transcription
`
`Gene silencing
`
`Replication
`RNA processing
`
`RNA modification
`
`RNA stability
`
`mRNA translation
`
`184-nt E. coli 65
`331—nt human 7SK
`875—nt human SRA
`16,500-nt human Xist
`~100,000—nt human Air
`451-nt human telomerase RNA
`377—nt E. coli RNase P
`186—nt human U2 snRNA
`102—nt S. cerevisiae U18 C/D snoRNA
`189-nt S. cerevisiae snR8 H/ACA snoRNA
`68—nt T. brucei gCYb gRNA
`80—nt E. coli RyhB sRNA
`Eukaryotic miRNA?
`109-nt E. coli OxyS
`87-nt E. coli DsrA sRNA
`
`22—nt C. elegans [in—4 miRNA
`
`Protein stability
`
`363-nt E. coli tmRNA
`
`Modulates promoter use
`lnhibits transcription elongation factor P—TEFb
`Steroid receptor coactivator
`Required for X—chromosome inactivation
`Required for autosomal gene imprinting
`Core of telomerase and telomere template
`Catalytic core of RNase P
`Core of spliceosome
`Directs 2’-O—ribose methylation of target rRNA
`Directs pseudouridylation of target rRNA
`Directs the insertion and excision of uridines
`Targets mRNAs for degradation?
`Targets mRNAs for degradation?
`Represses translation by occluding ribosome binding
`Activates translation by preventing formation of an
`inhibitory mRNA structure
`Represses translation by pairing with 3’ end of
`target mRNA
`Directs addition of tag to peptides on stalled
`ribosomes
`
`(9, 74)
`(75, 76, 46)
`(72, 77)
`(72, 73)
`(77)
`(78, 46)
`(9, 79)
`(20, 46)
`(27, 47)
`(27, 47)
`(23, 24, 48)
`(27)
`(7, 8)
`(9, 70)
`(9, 70)
`
`(7, 8)
`
`(9, 28)
`
`Integral component of signal recognition particle
`Protein
`translocation
`central to protein translocation across
`membranes
`
`
`114—nt E. coli 4.55 RNA
`
`(9, 29)
`
`1 260
`17 MAY 2002 VOL 296 3SC|ENCE www.5ciencemag.org
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`3
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`

`

`—’——— RNA SILENCING AND NONCODING RNA
`
`
`
`
`
`
`
`
`
`
`
`
`
`discovered
`first
`The
`translation.
`and
`miRNAs, C. elegans [in-4 and let—7, repress
`translation by forming base pairs with the 3’
`end of target mRNAs (7, 8). Many of the
`recently identified miRNAs are likely to act
`in a similar fashion. However, it is conceiv—
`able that some members of this large family
`target mRNAs for degradation, as is the case
`for the similarly sized small interfering RNAs
`(siRNAS) that are processed and amplified
`from exogenously added, double-stranded
`RNA and lead to gene suppression in a pro-
`cess termed RNA interference (25, 26). As
`yet there is no evidence for miRNAs in 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 mRNA sta-
`bility in E. coli (9, 10, 27). These sRNAs
`fomi base pairs at various positions 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, ncRNAs affect protein stability
`and transport. One unique bacterial sRNA is
`recognized as both a tRNA and an mRNA by
`stalled ribosomes (tmRNA) (28). Alanylated
`tmRNA is delivered to the A site of a stalled
`
`the nascent polypeptide is trans-
`ribosome;
`ferred to the alanine-charged tRNA portion of
`tmRNA. The problematic transcript then is
`replaced by the mRNA portion 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 mechanisms of action for the charac—
`
`terized ncRNAs can be grouped into several
`general
`categories
`(Fig.
`1). There
`are
`
`
`
`
`
`
`
`
`RNA polymerase T
`
`
`
`tmRNA
`
`Ribosome
`
`
`
`4.58 RNA
`
`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 snoRNAs direct nucleotide
`modifications (green star) by forming base pairs with flanking sequences, and the E. coli OxyS RNA
`represses translation by forming base pairs with the Shine-Dalgarno sequence (green box) and
`Occluding ribosome binding. (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
`reCognize tmRNA as both a tRNA and an mRNA. (C) ncRNAs also can function as an integral part
`Of a larger RNA-protein complex, such as the signal recognition particle, whose structure has been
`partially determined (49)-
`
`ncRNAs where base-pairing (often <10 base
`pairs and discontinuous) with another RNA
`or DNA molecule is central to function. The
`snoRNAs that direct RNA modification, the
`bacterial RNAs that modulate translation by
`forming base pairs with specific
`target
`mRNAs, and probably most of the miRNAs
`are examples of this category. Some ncRNAs
`mimic the structures of other nucleic acids;
`the 6S RNA structure is reminiscent of an
`
`open bacterial promoter, and the tmRNA has
`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 shown to have catalytic function is
`limited. Most, if not all, ncRNAs are associ-
`ated with proteins that augment their func-
`tions; however, some ncRNAs, such as the
`snRNAs and the SRP RNA, serve key struc—
`tural roles in RNA-protein complexes. Sev-
`eral ncRNAs fit into more than one mecha-
`
`nistic category; the telomerase RNA provides
`the base-pairing template for telomere syn-
`thesis and is an integral part of the telomerase
`ribonucleoprotein complex. The mechanisms
`of action for a number of ncRNAs (such as
`the 78K RNA) are not known, and it
`is
`probable that some ncRNAs act in ways that
`have not yet been established. Some investi-
`gators have suggested that many ncRNAs are
`vestiges of a world in which RNA carried 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 ncRNAs Exist?
`The first ncRNAs were identified in the
`
`19605 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 ncRNAs were the first 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
`
`17 MAY 2002
`1261
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`

`
`
`
`
`RNA SILENCING AND NONCODING RNA
`
`
`
`ncRNA gene. Criteria for identifying candi-
`date intergenic regions have included large
`gaps between protein-coding genes (34), ex-
`tended stretches of conservation between spe—
`cies with the same gene order (35, 36), or-
`phan promoter or terminator sequences (34,
`36, 37), presence of GC-rich regions in an
`organisms with a high AT content (38), and
`conserved RNA secondary structures (39,
`40). Other searches for ncRNAs have iri—
`volved large-scale cloning efforts that have
`taken into account specific ncRNA proper—
`ties.
`].n studies of mouse (41, 42) and the
`archaeon Archaeoglobus fulgidus (22), total
`RNA between 50 to 500 nt was isolated, and
`
`arrays of cDNA clones obtained from the
`RNA were screened with oligonucleotides
`corresponding to the most abundant known
`RNAs. Clones showing the lowest hybridiza—
`tion signal then were randomly sequenced. In
`recent screens for C. elegans, Drosophila,
`and human miRNAs, RNA molecules of less
`than 30 nt were isolated, and cDNA clones
`were generated upon the ligation of primers
`to the 5’ and 3’ ends of the RNA (3, 4) or
`upon RNA tailing (5). Other miRNAs were
`isolated and cloned on the basis of their
`association with a complex composed of the
`human Gemin3, Gemin4, and IF2C proteins
`(6). In most studies, Northern blots have been
`carried out to confirm that the cloned genes
`are expressed as small
`transcripts. These
`blots also have provided information about
`spatial and temporal expression patterns as
`well as potential precursor and degradation
`products.
`Despite the success of the recent system—
`atic efforts, it is certain that not all ncRNAs
`have been detected. Estimates for the number
`of sRNAs in E. coli range from 50 to 200 (I,
`35), and estimates for the number of miRNAs
`in C. elegans range from hundreds to thou-
`sands (7). There also are many non—protein-
`coding regions of the bacterial and eukaryotic
`chromosomes for which transcription is de-
`tected (43, 44), but it is not known how many
`of these regions encode defined, functional
`ncRNAs. Extensions of the various systemat-
`ic searches should lead to the identification of
`more ncRNAs. However, limitations of the
`current approaches should be noted. Most of
`the computation methods have focused on the
`intergenic regions. It has already been shown
`that some of the ncRNAs are processed from
`longer protein- or rRNA—encoding transcripts
`(42). It also is quite possible that ncRNAs are
`expressed from the opposite strand of pro-
`tein-coding genes. On the other hand, expres-
`sion-based methods may miss ncRNAs that
`are synthesized under very defined condi—
`tions, such as in response to a specific envi—
`ronmental signal, during a specific stage in
`development, or in a specific cell type. Much
`attention has been focused on characterizing
`the “proteome” of a sequenced organism. The
`
`
`
`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.
`
`What Are All the Functions of
`ncRNAs?
`
`An astonishing variety of ncRNA functions
`have already been found, but
`there are
`many ncRNAs for which the cellular roles
`are still unknown. For instance, Y RNAs,
`small cytoplasmic RNAS associated with
`the R0 autoantigen in several different or-
`ganisms, are still enigmatic even after
`many years of study (45). With the more
`systematic
`identification of
`increasing
`numbers of ncRNAs, the question of how to
`elucidate the functions of all ncRNAs is
`becoming more and more prominent.
`Approaches that have succeeded previously
`are an obvious place to start in answering the
`question of function, but it is likely that new
`approaches also will need to be developed. For
`genetically tractable organisms, ncRNA knock-
`out or overexpression strains can be screened
`for differences in phenotypes (such as viability)
`or whole- genome expression patterns. The
`functions of several ncRNAs were identified by
`the biochemical
`identification of associated
`proteins, and the development of more system-
`atic methods for characterizing ncRNA-associ-
`ated proteins should be fi'uitfiil. As the knowl-
`edge base of what sequences are critical for the
`formation of specific structures or for base-
`pairing expands, and as computer programs for
`predicting structures improve, computational
`approaches should become an increasingly irn—
`portant avenue for elucidating the functions of
`ncRNAs. The three-dimensional structures of
`only a limited number of RNAs and RNA-
`protein complexes have been solved. An iri-
`crease in the structural database may bring to
`light recognizable RNA or RNA-protein do-
`mains associated With specific fimctions.
`Information about when ncRNAs are ex-
`
`pressed and where ncRNAs are localized is
`useful for all experiments aimed at probing
`function. Many of the C.’ elegans miRNAs
`are synthesized only at very specific times in
`development, and thus they have also been
`called small
`temporal RNAs
`(stRNAs).
`Among the snoRNAs, some are expressed
`exclusively in the brain (41), and one of the
`bacterial sRNAs is only detected upon oxida—
`tive stress (9, 10).
`It
`is likely that other
`ncRNAs will be found to have very defined
`expression and localization patterns and that
`these will be critical to function.
`There are many more ncRNAs than was
`ever suspected. A big challenge for the future
`will be to identify the whole complement of
`ncRNAs and to elucidate their fimctions. This is
`an exciting time for investigators whose work
`has focused on ncRNAs. However, scientists
`
`studying all aspects of biology should keep
`ncRNAs in mind. The phenotypes associat-
`ed with specific mutations may be due to
`defects in a ncRNA instead 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 ncRNAs lurking be-
`hind many an unexplained phenomenon.
`
`PWNQ‘F‘
`
`References and Notes
`1. S. R. Eddy, Nature Rev. Genet. 2, 919 (2001).
`2. Non—mRNAs have been denoted ncRNA = noncoding
`RNA. snmRNA 2 small non—mRNA, sRNA = small
`RNA, fRNA = functional RNA, and oRNA = other
`RNA, and it is likely that the nomenclature of these
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`in
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`rfi—
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`
`RNA SILENCING AND NONCODING
`
`RNA
`
`
`
`in press.
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`the nomenclature.
`
`SO.
`
`VIEWS’OINT
`
`RNA Silencing: The Genome's
`Immune System
`
`Ronald H. A. Plasterk
`
`Genomes are databases sensitive to invasion by viruses. In recent years, a
`defense mechanism. has been discovered, which turns out to be conserved
`among eukaryotes. The system can be compared to 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
`I
`latter property is beginning to be understood at the molecular level.
`;-
`l'i g
`'.
`
`All genomes of complex organisms are po—
`tential
`targets of invasion byiviruses and
`transposable elements. Forty—fiVe3percent of
`the human genome consists ofwremnants of
`previous transposon/virusinvasions 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 need to 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 genes is gen-
`erated from a limited set of gene segments by
`combinatorial gene rearrangements, and this
`repertoire is stored in a distributed fashion
`
`over large numbers of cells. 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 or less random repertoire, which, during
`a phase of early development, is limited by a
`filteiing process, called tolerance induction.
`
`
`Hubrecht Laboratory, Centre for Biomedical Genetics,
`Uppsalalaan 8. 3584 CT Utrecht, Netherlands E- mall
`Plasterk@niob.knaw.nl
`
`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 may be the selective
`methylation of transposon sequences in the
`genome (1), although it has also been argued
`that this phenomenon is 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 emerged that
`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) (10)] and of ho-
`mology between genes
`required for
`this
`mechanism in plants, animals, and fiingi and
`algae.
`The precise mechanism of this group of
`phenomena, now referred 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,”
`
`
`
`which allows the vertebrate immune system
`to raise a massive immune response (11—14).
`
`The Function of RNA Silencing
`Neither nematodes nor flies normally en-
`counter highly concentrated double-stranded
`RNA (dsRNA) of identical sequence to one
`of their endogenous genes. Neveitheless, ge-
`netic analysis indicates that the number of
`genes required for gene silencing triggered by
`exogenous dsRNA is probably larger than 10
`(15—18). What is the natural function of this
`elaborate pathway?
`The clearest picture is seen in plants,
`where PTGS and virus-induced gene silenc—
`ing are recognized as mechanisms that 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 shows that an animal virus
`also encodes a suppressor of RNA interfer-
`ence (RNAi),
`supporting the notion that
`RNAi may have an antiviral function in ani—
`mals as well
`(20).
`In nematodes,
`loss of
`function of genes required for RNAi results
`in the activation of multiple transposable el-
`ements in the gerrnline (15), indicating that
`they function to repress the spreading of
`transposons within the genome of subsequent
`generations of worms.
`Protection against viruses and transposons
`may be the 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
`RNAi in C. elegans is the systemic effect.
`Injection of naked dsRNA into 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 (21). In plants, graft-
`ing experiments have shown immunity trav-
`eling over 30 cm of stem tissue (22);
`this
`
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