Long noncoding RNAs: functional
surprises from the RNA world
Jeremy E. Wilusz,1Hongjae Sunwoo,2and David L. Spector1,3
1Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA;2Graduate
Program in Molecular and Cellular Biology, Stony Brook University, Stony Brook, New York 11794
Most of the eukaryotic genome is transcribed, yielding
a complex network of transcripts that includes tens of
thousands of long noncoding RNAs with little or no
protein-coding capacity. Although the vast majority of
long noncoding RNAs have yet to be characterized thor-
transcriptional ‘‘noise’’ as a significant number have been
shown to exhibit cell type-specific expression, localiza-
tion to subcellular compartments, and association with
human diseases. Here, we highlight recent efforts that
have identified a myriad of molecular functions for long
act of noncoding RNA transcription is sufficient to posi-
tively or negatively affect the expression of nearby genes.
However, in many cases, the long noncoding RNAs
themselves serve key regulatory roles that were assumed
previously to be reserved for proteins, such as regulating
the activity or localization of proteins and serving as
organizational frameworks of subcellular structures. In
addition, many long noncoding RNAs are processed to
yield small RNAs or, conversely, modulate how other
that long noncoding RNAs can function via numerous
paradigms and are key regulatory molecules in the cell.
The sequencing of the human genome provided quite
a surprise to many when it was determined that there are
only ;20,000 protein-coding genes, representing <2% of
the total genomic sequence (International Human Ge-
nome Sequencing Consortium 2004). Since other less
complex eukaryotes like the nematode Caenorhabditis
elegans have a very similar number of protein-coding
genes, it quickly became clear that the developmental
and physiological complexity of humans probably cannot
be explained solely by the number of protein-coding
genes. Alternative pre-mRNA splicing of protein-coding
transcripts as well as post-translational modifications
of proteins increase the diversity and functionality of
the proteome, likely explaining part of this increased
complexity. In addition, there has been an explosion of
research addressing possible functional roles for the
other 98% of the human genome that does not encode
proteins. Rather unexpectedly, transcription is not lim-
ited to protein-coding regions, but is instead pervasive
throughout the mammalian genome as demonstrated by
large-scale cDNA cloning projects (Carninci et al. 2005;
Katayama et al. 2005) and genomic tiling arrays (Bertone
et al. 2004; Cheng et al. 2005; Birney et al. 2007; Kapranov
et al. 2007a). In fact, >90% of the human genome is likely
to be transcribed (Birney et al. 2007), yielding a complex
network of overlapping transcripts that includes tens of
thousands of long RNAs with little or no protein-coding
capacity (for review, see Kapranov et al. 2007b).
There is still some debate as to whether this pervasive
transcription represents largely useless transcription
(transcriptional ‘‘noise’’) (Wang et al. 2004; Struhl 2007;
Ebisuya et al. 2008) or if these noncoding RNAs (ncRNAs)
have functions that simply have not yet been identified
(for review, see Mattick 2004). Considering that it has
long been known that numerous noncoding transcripts—
such as transfer RNAs, ribosomal RNAs, and spliceoso-
mal RNAs—are critical components of many cellular
machines, it seems highly likely that additional ncRNAs
play key regulatory and functional roles. Supporting the
biological relevance of these transcripts, multiple studies
have shown that significant numbers of long ncRNAs are
regulated during development (Blackshaw et al. 2004;
Rinn et al. 2007; Dinger et al. 2008), exhibit cell type-
specific expression (Ravasi et al. 2006; Mercer et al. 2008),
localize to specific subcellular compartments (Hutchinson
et al. 2007; Sone et al. 2007; Clemson et al. 2009; Sasaki
et al. 2009; Sunwoo et al. 2009), and are associated with
human diseases (for review, see Costa 2005; Szymanski
et al. 2005; Prasanth and Spector 2007). In addition, evi-
has been found (Pollard et al. 2006; Pheasant and Mattick
2007; Ponjavic et al. 2007; Guttman et al. 2009).
In this review, we highlight recent studies that have
revealed how long ncRNAs can function on the molecu-
lar level (Fig. 1). Although the functions of only a limited
number of long noncoding transcripts have been identi-
fied, numerous paradigms are beginning to emerge. We
first highlight examples in which simply the act of ncRNA
transcription is sufficient to regulate the expression of
[Keywords: ncRNA; transcriptome; gene expression; transcriptional reg-
ulation; small RNAs; structural RNAs]
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nearby protein-coding genes. We then highlight how long
ncRNAs themselves serve key regulatory roles, with
a specific focus on functional paradigms. ncRNAs in-
volved in dosage compensation in mammals or Drosoph-
ila will be mentioned only briefly, as these have been
reviewed thoroughly elsewhere (Deng and Meller 2006;
Masui and Heard 2006; Payer and Lee 2008).
When the act of ncRNA transcription alone
may be enough
The act of transcribing ncRNAs can have profound con-
sequences on the ability of nearby genes to be expressed
(Katayama et al. 2005). For example, transcription of a
ncRNA across the promoter region of a downstream
protein-coding gene can directly interfere with transcrip-
tion factor binding, and thus prevent the protein-coding
gene from being expressed (Martens et al. 2004). In fact,
transcriptional interference mechanisms have been shown
to regulate key developmental decisions, such as where
homeotic (Hox) genes are expressed (Petruk et al. 2006)
and whether Saccharomyces cerevisiae enters into mei-
osis (Hongay et al. 2006). Even if not directly interfering
with a nearby promoter, transcription of ncRNAs can
induce histone modifications that repress transcription
initiation of overlapping protein-coding genes, as demon-
strated at the yeast PHO84 (Camblong et al. 2007) and
GAL1-10 gene clusters (Houseley et al. 2008). This is
because transcriptional elongation causes histone marks
to be added that prevent ‘‘spurious’’ transcription initia-
tion from sites within the body of the transcript (Carrozza
et al. 2005; Joshi and Struhl 2005; Keogh et al. 2005).
Noncoding transcription has even been shown to induce
the formation of heterochromatin at the p15 tumor
suppressor gene locus that persisted after noncoding
transcription was turned off, suggesting that the transient
expression of ncRNAs can have long-lasting heritable
effects on gene expression (Yu et al. 2008).
Of course, not every gene becomes silenced due to the
transcription of nearby ncRNAs. Continuous ncRNA
transcription has, for example, been suggested to prevent
the silencing of certain Hox genes by Polycomb group
(PcG) proteins (Bender and Fitzgerald 2002; Hogga and
Karch 2002; Rank et al. 2002; Schmitt et al. 2005).
Additionally, transcription of long ncRNAs upstream of
the Schizosaccharomyces pombe fbp1+locus induces
chromatin remodeling that is critical for transcriptional
activation of the downstream protein-coding gene (Hirota
et al. 2008). Interestingly, ncRNA transcription was found
to initiate in a stepwise manner from multiple sites up-
stream of the fbp1+promoter, causing chromatin opening
to proceed progressively toward the mRNA transcription
start site. The insertion of a transcriptional terminator
within these ncRNAs prevents downstream chromatin
ncRNAs function, many of which are highlighted here. Transcription from an upstream noncoding promoter (orange) can negatively (1)
or positively (2) affect expression of the downstream gene (blue) by inhibiting RNA polymerase II recruitment or inducing chromatin
remodeling, respectively. (3) An antisense transcript (purple) is able to hybridize to the overlapping sense transcript (blue) and block
recognition of the splice sites by the spliceosome, thus resulting in an alternatively spliced transcript. (4) Alternatively, hybridization of
the sense and antisense transcripts can allow Dicer to generate endogenous siRNAs. By binding to specific protein partners, a noncoding
transcript (green) can modulate the activity of the protein (5), serve as a structural component that allows a larger RNA–protein
complex to form (6), or alter where the protein localizes in the cell (7). (8) Long ncRNAs (pink) can be processed to yield small RNAs,
such as miRNAs, piRNAs, and other less well-characterized classes of small transcripts.
Paradigms for how long ncRNAs function. Recent studies have identified a variety of regulatory paradigms for how long
Long noncoding RNAs
GENES & DEVELOPMENT1495
remodeling, resulting in reduced recruitment of tran-
scription factors to the fbp1+promoter, and thus minimal
induction of the mRNA. Similar stepwise remodeling
of chromatin by ncRNAs has also been shown at the
S. pombe ade6-M26 locus (Hirota and Ohta 2009).
A critical remaining question is whether chromatin
remodeling occurs due to the act of ncRNA transcription,
implying that the ncRNAs are simply nonfunctional by-
products, or whether the ncRNAs themselves actively
play a role; e.g., by recruiting chromatin remodeling or
histone-modifying enzymes. At least at the yeast PHO5
locus, it appears to be the act of noncoding transcription
rather than the ncRNA itself that contributes to chroma-
tin plasticity and the ability of the protein-coding gene to
be rapidly induced (Uhler et al. 2007). Originating from
near the 39 end of the PHO5 gene is an ;2.4-kb antisense
ncRNA that is rapidly degraded by the exosome, a phe-
nomenon that has made detecting such unstable tran-
scripts often difficult, except when RNA decay activities
are depleted (Wyers et al. 2005; Davis and Ares 2006;
Preker et al. 2008). Expression of the PHO5 ncRNA in
trans had no effect on chromatin remodeling (Uhler et al.
2007). Therefore, rather than the unstable ncRNA itself
playing a functional role at the PHO5 locus, it was argued
that the actual act of ncRNA transcription affects the
local rate of nucleosome exchange and/or turnover,
allowing nucleosome eviction, and thus PHO5 transcrip-
tion, to occur much more rapidly in response to phos-
phate starvation (Uhler et al. 2007). It should be noted
that just because a transcript is degraded rapidly does not
mean that it is nonfunctional, as unstable transcripts
have, for example, been shown to repress transcription of
the yeast Ty1 retrotransposon in trans (Berretta et al.
At the human DHFR locus, a long noncoding transcript
originating from a region upstream of the major DHFR
promoter acts to repress expression of the downstream
protein-coding gene (Blume et al. 2003; Martianov et al.
2007). Interestingly, this ncRNA inhibits expression of
DHFR both in cis and in trans by forming an RNA–DNA
triplex structure with the DHFR promoter and directly
interacting with TFIIB, which results in the disruption
of the preinitiation complex at the DHFR promoter
(Martianov et al. 2007). Therefore, depending on the gene
locus, ncRNA transcription can have profound effects,
both negatively and positively, on the ability of neigh-
boring protein-coding genes to be expressed. In some
cases, the act of transcription is sufficient to have func-
tional consequences, but it is likely that many of the
ncRNAs produced may play yet-to-be-identified active
Long ncRNAs target proteins to specific genomic loci
to affect transcription patterns
PcG proteins are known to bind and silence the expres-
sion of more than a thousand mammalian genes (Boyer
et al. 2006; Bracken et al. 2006), yet how PcG proteins are
recruited to these specific target sites in mammalian cells
has been largely unclear. Several reports suggest that it
likely may be long ncRNAs that target PcG proteins to
specific genomic locations (Plath et al. 2003; Silva et al.
2003; Kohlmaier et al. 2004). Ezh2 (Enhancer of zeste
homolog 2), a histone methyltransferase and member of
Polycomb-repressive complex 2 (PRC2), was found to
directly bind a 1.6-kb-long ncRNA known as RepA (Zhao
et al. 2008). Interestingly, when transcription of a stably
integrated ectopic RepA gene locus was induced, PcG
proteins were recruited specifically to the gene locus,
indicating that RepA is sufficient to recruit PRC2 to
chromatin in vivo. The endogenous RepA ncRNA is
transcribed from the Repeat A region of the Xist gene
and has been proposed to play a key role in the early
stages of mammalian X-chromosome inactivation (Zhao
et al. 2008). Via a likely related mechanism, the ncRNA
HOTAIR, derived from the HOXC locus, has been shown
to interact with PcG proteins to regulate the HOXD locus
in trans (Rinn et al. 2007).
Trithorax group (TrxG) proteins counteract the actions
of PcG proteins to maintain active transcription states
and, interestingly, also may be recruited to their target
loci by long ncRNAs (Sanchez-Elsner et al. 2006). Certain
ncRNAs from the Hox loci were shown to interact
directly with the histone methyltransferase Ash1 in vitro
and were proposed to target TrxG proteins to chromatin
(Sanchez-Elsner et al. 2006). In fact, ectopic expression of
these ncRNAs in trans was found to activate Hox gene
expression in Drosophila S2 cells and wing imaginal discs
(Sanchez-Elsner et al. 2006). However, another report did
not observe a similar association between ectopic expres-
sion of these ncRNAs and transcriptional activation,
and instead suggested that ncRNA transcription inhib-
its expression of the nearby Hox genes (Petruk et al.
2006, 2007; for review, see Lempradl and Ringrose 2008).
Although there are still some discrepancies in the liter-
ature, it appears that certain ncRNAs play key roles in
maintaining the active or inactive state of gene expres-
sion by modulating where PcG and TrxG proteins are
Numerous long ncRNAs, including Airn and Kcnq1ot1,
are expressed from imprinted loci and have been sug-
gested to function as key players in assuring that only one
of the two parental alleles are expressed (for review, see
Prasanth and Spector 2007; Peters and Robson 2008; Royo
and Cavaille 2008). Recent work suggests that imprinted
genes within a single cluster can unexpectedly be si-
lenced by different mechanisms. In the mouse placenta,
the ;108-kb Airn ncRNA is required for the paternal-
specific silencing in cis of a 400-kb region that includes
the Slc22a3, Slc22a2, and Igf2r genes (Sleutels et al. 2002).
Analogous to Xist, Airn is retained in the nucleus (Seidl
et al. 2006) and appears to ‘‘coat’’ the imprinted locus on
the paternal chromosome (Nagano et al. 2008). However,
rather than uniformly localizing to the entire imprinted
domain, Airn preferentially accumulates at the Slc22a3
promoter (Nagano et al. 2008). Air then interacts with
the histone H3 Lys 9 methyltransferase G9a, leading
to methylation and silencing of the paternal Slc22a3
promoter. Deleting G9a results in the loss of Slc22a3
imprinting, yet has no effect on Igf2r, which remains
Wilusz et al.
1496GENES & DEVELOPMENT
monoallelically expressed. Therefore, even though Airn
is required for silencing both Slc22a3 and Igf2r on the
paternal chromosome, it must do so by different mech-
anisms. It will be of great interest to tease apart the
mechanisms by which ncRNAs are able to obtain such
high functional specificity and recruit different proteins
to different gene loci.
The imprinted ncRNA Kcnq1ot1 likewise accumulates
nonuniformly along the Kcnq1 domain and interacts
with G9a and PcG proteins (Pandey et al. 2008), as well
as functions by transcriptional interference (Kanduri etal.
2006; Mohammad et al. 2008). Interestingly, certaingenes
in the Kcnq1 domain are only imprinted in the placenta,
likely because the Kcnq1ot1 ncRNA interacts with G9a
and PcG proteins in a lineage-specific manner, resulting
in the establishment of repressive histone modifications
on these genes only in certain cell types (Pandey et al.
2008). Specifically, an interaction between Kcnq1ot1
with G9a and PcG proteins was detected in the placenta,
while no interaction was detected in the fetal liver.
Long ncRNAs modulate the activity of protein-binding
Many proteins bind to RNAs through a variety of RNA-
binding motifs to modulate the processing, localization,
and stability of the bound RNAs (for review, see Dreyfuss
et al. 2002). Naturally, the converse is also true—RNAs
can influence the activity and localization of the proteins
they bind. For example, long ncRNAs can serve as key
coactivators of proteins involved in transcriptional regu-
lation. The ;3.8-kb Evf-2 ncRNA, which is transcribed
from an ultraconserved region, forms a complex with the
homeodomain-containing protein Dlx2 (Feng et al. 2006).
Using reporter-based assays, it was shown that Dlx2 acts
a transcriptional enhancer only when the Evf-2 ncRNA
is also present. Similarly, the ncRNA HSR1 (heat-shock
RNA-1) forms a complex with HSF1 (heat-shock tran-
scription factor 1), enabling the transcription factor to
induce expression of heat-shock proteins during the
cellular heat-shock response (Shamovsky et al. 2006),
and an isoform of the ncRNA SRA (steroid receptor
RNA activator) functions as a transcriptional coactivator
of steroid receptors (Lanz et al. 1999). Conversely, non-
coding transcripts derived from SINEs (short interspersed
elements) bind to RNA polymerase II during heat shock
to inhibit transcription of other mRNAs, such as actin
(Allen et al. 2004; Espinoza et al. 2004; Mariner et al.
ncRNAs produced from the cyclin D1 (CCND1) pro-
moter region have been shown recently to function as
allosteric effectors of an RNA-binding protein known as
TLS (Translocated in Liposarcoma) (Wang et al. 2008).
These ncRNAs are variable in their lengths and of low
abundance (generally less than two copies per cell), but
are induced in response to DNA damage and remain
bound to the chromatin in the CCND1 promoter region.
Upon binding these ncRNAs, the TLS protein changes
from an inactive to an active conformation, such that it
binds and inhibits the enzymatic activities of the histone
acetyltransferases CBP and p300, thus silencing CCND1
Long ncRNAs also have been shown to modulate the
activity of proteins by regulating their subcellular local-
ization. The transcription factor NFAT (nuclear factor of
activated T cells) localizes to the cytoplasm until cal-
cium-dependent signals cause it to be imported into the
nucleus, where it activates transcription of target genes
(for review, see Hogan et al. 2003). One of the key regu-
lators of NFAT trafficking happens to be a ncRNA known
as NRON (noncoding repressor of NFAT) that is alterna-
tively spliced (0.8–3.7 kb) (Willingham et al. 2005). By
binding to members of the nucleocytoplasmic trafficking
machinery, NRON specifically inhibits the nuclear accu-
mulation of NFAT, but not that of other transcription
factors such as p53 and NFkB that also translocate from
the cytoplasm to nucleus.
Long ncRNAs as precursors for small RNAs
Recent genome-wide studies suggest that the function of
a significant fraction of long unannotated transcripts may
be to serve as precursors for small RNAs <200 nucleotides
(nt) in length (Kapranov et al. 2007a; Fejes-Toth et al.
2009). In addition to microRNAs (miRNAs) that are
usually generated via the sequential cleavage of long
transcripts by Drosha and Dicer (Cai et al. 2004; Lee
et al. 2004) and Piwi-interacting RNAs (piRNAs) that are
likely generated via processing of a single long transcript
(for review, see Aravin et al. 2007), there are many more
small RNAs whose functions and mechanisms of bio-
genesis are less clear. For example, small RNAs have been
found to cluster near the 59 and 39 ends of genes (Han et al.
2007; Kapranov et al. 2007a; Core et al. 2008; He et al.
2008; Preker et al. 2008; Seila et al. 2008; Fejes-Toth
et al.2009; Neil et al.2009; Taft etal. 2009; Xu etal. 2009).
Transfection of RNA mimetics to promoter-associated
small RNAs (PASRs) were found to reduce expression of
the overlapping mRNA promoter, indicating that these
newly identified small RNAs impact gene expression
(Fejes-Toth et al. 2009).
It now appears that many protein-coding mRNAs and
long ncRNAs may be post-transcriptionally processed to
yield many small RNAs that, curiously, have a 59 cap
structure (Fejes-Toth et al. 2009). Numerous small RNAs
identified using next-generation sequencing technology
were found to significantly overlap CAGE (cap analysis of
gene expression) tags, which are thought to mark the
59 ends of capped, long RNA transcripts (Fejes-Toth et al.
2009). Although many CAGE tags do mark transcription
start sites, significant numbers were found in exonic
regions and, in some cases, to even cross splice junctions,
meaning they must have arisen from at least partially
processed mRNAs. Therefore, it has been proposed that
mature long transcripts (both protein-coding mRNAs and
long ncRNAs) can be processed post-transcriptionally to
yield small RNAs, which are then modified by the
addition of a cap structure (Fig. 2A; Fejes-Toth et al. 2009).
By processing a single long transcript into multiple
smaller RNAs, each mature transcript can display a distinct
Long noncoding RNAs
GENES & DEVELOPMENT1497
subcellular localization and have a unique function. Re-
cent work from our group provides a clear example of
how a nascent transcript can be processed to yield two
ncRNAs that localize to distinct subcellular compart-
ments (Wilusz et al.2008). MALAT1 (Metastasis-associated
lung adenocarcinoma transcript 1), also known as NEAT2
(Hutchinson et al. 2007), is a long (;7-kb) ncRNA that
is misregulated in many human cancers (Ji et al. 2003; Lin
et al. 2007) and was shown previously to be retained spe-
cifically in the nucleus in nuclear speckles (Hutchinson
et al. 2007), domains that are thought to be involved
in the assembly, modification, and/or storage of the pre-
mRNA processing machinery (for review, see Lamond
and Spector2003). Upon probing for small RNAsmapping
to the MALAT1 locus, we identified a highly conserved
61-nt tRNA-like small RNA (Wilusz et al. 2008). In stark
contrast to the mature long MALAT1 transcript, the small
RNA is localized exclusively to the cytoplasm, and thus
we named it mascRNA, MALAT1-associated small cyto-
Unlike tRNAs that are transcribed by RNA polymerase
III, mascRNA is generated via processing of the MALAT1
nascent transcript (Fig. 2B; Wilusz et al. 2008). RNase P
recognizes the tRNA-like structure in the nascent RNA
polymerase II transcript and then cleaves to simulta-
neously generate the 39 end of the mature MALAT1 tran-
script and the 59 end of mascRNA. Additional enzymes
involved in tRNA biogenesis, including RNase Z and the
CCA-adding enzyme, then further process the small RNA
prior to its export to the cytoplasm. While MALAT1 is
very stable, mascRNA is fairly rapidly degraded—so not
only do the two ncRNAs localize to separate subcellular
locations, they also have vastly different half-lives and
presumably distinct functions. MEN b, a >20-kb ncRNA
that is retained in the nucleus in paraspeckles, is pro-
cessed at its 39 end by a similar mechanism (Sunwoo et al.
2009). Based on genome-wide studies (Kapranov et al.
2007a; Fejes-Toth et al. 2009), it is likely that many
genetic loci generate multiple ncRNA transcripts, which
localize to different subcellular compartments and may
not fit into already known and characterized classes of
The RNAi machinery has well-characterized roles in
the generation of miRNAs and siRNAs that regulate gene
expression post-transcriptionally (for review,see Lee et al.
2006; Jaskiewicz and Filipowicz 2008). However, a recent
report implicates these enzymes in the processing of long
transcripts to small RNAs that likely do not function
as miRNAs (Ganesan and Rao 2008). In mice, a 2.4-kb
unspliced, polyadenylated nuclear-retained ncRNA known
as mrhl is processed by Drosha to yield an 80-nt small
RNA (Ganesan and Rao 2008). Interestingly, the 80-nt
transcript is not further processed by Dicer in vivo,
probably because it is retained in the nucleus in associ-
ation with chromatin.
Last, there is emerging evidence that Xist and Tsix, two
long ncRNAs that regulate mammalian X-chromosome
inactivation, may also be processed to yield small RNAs
(Ogawa et al. 2008). Developmentally regulated small
RNAs between 25 and 42 nt in length map to the Xist and
Tsix loci. Because Xist and Tsix form a dsRNA duplex in
vivo and expression of the small RNAs is diminished
to generate small RNAs (Fejes-Toth et al. 2009). A cap structure (denoted by a red star) is then added to the 59 ends of many of these
small RNAs. In addition, capped small RNAs, known as PASRs, map near the transcription start site (TSS) of many genes. (B) The
nascent MALAT1 transcript is processed to yield two ncRNAs that localize to different subcellular compartments. Cleavage by RNase
P simultaneously generates the 39 end of the mature MALAT1 transcript and the 59 end of mascRNA (Wilusz et al. 2008).
Long ncRNAs are processed to yield small RNAs. (A) Many long processed transcripts can be post-transcriptionally cleaved
Wilusz et al.
1498GENES & DEVELOPMENT
when Dicer is deleted, it was suggested that processing of
dsRNA generates these small RNAs (Ogawa et al. 2008).
However, Dicer is not currently known to cleave RNAs
to small transcripts in this size range (25–42 nt), suggest-
ing that Dicer may play an indirect role in the biogenesis
of these small RNAs and that Xist and Tsix are processed
via other mechanisms. In addition, two recent reports
suggest that X inactivation does not depend on Dicer
(Nesterova et al. 2008; Kanellopoulou et al. 2009). Al-
though miRNAs and piRNAs have received the most
attention of late, it is becoming increasingly clear that
long RNA transcripts are processed to yield many other
classes of small RNAs with likely very different and
Long ncRNAs affect the processing of other RNAs
Long ncRNAs can be processed to yield small RNAs, but
they can also affect how other transcripts are processed;
for example, by modulating their ability to be cut into
small RNAs or changing their pre-mRNA splicing pat-
terns. The 800-nt spliced and polyadenylated C. elegans
rncs-1 (RNA noncoding, starvation up-regulated) ncRNA
inhibits the production of small RNAs from other tran-
scripts in trans (Hellwig and Bass 2008). Despite contain-
ing an almost perfectly double-stranded helix of ;300
base pairs, this transcript is not a Dicer substrate because
branched structures flanking the central double-stranded
helix inhibit its processing. Instead, rncs-1 functions in
trans to inhibit Dicer activity. Upon overexpressing or
deleting rncs-1 in vivo, the expression levels of certain
siRNAs were found to decrease or increase, respectively,
with a corresponding change in the mRNA levels of their
gene targets (Hellwig and Bass 2008). Therefore, it has
been proposed that rncs-1 binds to Dicer or accessory
dsRNA-binding proteins to compete with other dsRNAs
involved in gene silencing.
Certain long ncRNAs are likely able to base-pair with
small RNAs to modulate their activities. For example, by
interacting with miRNAs, long noncoding transcripts
could competitively inhibit the ability of miRNAs to
interact with their mRNA targets, analogous to how
artificial miRNA sponges function (Ebert et al. 2007).
This target mimicry mechanism is used by the long
ncRNA IPS1 (INDUCED BY PHOSPHATE STARVA-
TION 1) in Arabidopsis thaliana (Franco-Zorrilla et al.
2007). The ;550-nt IPS1 ncRNA is poorly evolutionarily
conserved except for a short 23-nt motif that is highly
complementary to miR-399, a miRNA that is induced
in response to phosphate starvation, although the base-
pairing is interrupted by a mismatched loop at the ex-
pected miRNA cleavage site. This interruption in base-
pairing causes the IPS1 ncRNA to be noncleavable and
instead allows IPS1 to sequester miR-399, resulting in
increased expression of miR-399 target genes (Franco-
Zorrilla et al. 2007).
Several recent reports indicate that RNA transcripts
derived from pseudogenes can surprisingly cause mRNAs
from the functional protein-coding copy of the gene to be
processed to small RNAs (Tam et al. 2008;Watanabe etal.
2008). This is because long antisense transcripts produced
from pseudogenes are able to hybridize to their corre-
sponding spliced mRNAs, resulting in the formation of
dsRNAs that are cleaved by Dicer to endogenous siRNAs
(endo-siRNAs). The coding mRNA is thus consumed
to generate endo-siRNAs that may direct RISC (RNA-
induced silencing complex) to cleave additional copies of
the mRNA transcript,resulting infurther down-regulation
of the protein-coding gene (Tam et al. 2008; Watanabe
et al. 2008). Thus, these results show that pseudogenes
are not simply nonfunctional elements that eventually
will be lost, but instead are key regulators of gene ex-
pression when transcribed as long ncRNAs.
Like pseudogenes, some natural antisense transcripts
(NATs) are able to hybridize to overlapping genes and
generate endo-siRNAs (Czech et al. 2008; Ghildiyal et al.
2008; Okamura et al. 2008; Watanabe et al. 2008). In
addition, there are numerous examples of NATs modu-
lating the alternative splicing patterns of their overlap-
ping genes (Krystal et al. 1990; Munroe and Lazar 1991;
Yan et al. 2005), such as at the Zeb2/Sip1 gene locus.
Zeb2/Sip1 is a transcriptional repressor of E-cadherin
whose expression is tightly regulated during epithelial–
mesenchymal transition (EMT) (Beltran et al. 2008).
Translation of the Zeb2/Sip1 protein requires an internal
ribosome entry site (IRES); however, in epithelial cells,
the region containing the IRES is spliced out of the
mature mRNA. Upon EMT, a NAT is produced that is
complementary to the 59 splice site of this intron, thus
blocking the spliceosome from removing the IRES from
the mature mRNA and enabling expression of the Zeb2/
Sip1 protein (Beltran et al. 2008).
Long ncRNAs serve as structural RNAs
The mammalian cell nucleus is not only compartmen-
talized such that it is separate from the cytoplasm, but
also such that it contains many membraneless compart-
ments that serve specialized functions (for review, see
Spector 2001, 2006). How many of these subcellular bodies
form and are maintained is unclear, but recent work
suggests that long ncRNAs may, in some cases, serve as
of RNA-binding proteins, including paraspeckle protein
component 1 (PSPC1, also known as PSP1a), NONO (also
known as p54/nrb), and the 68-kDa subunit of cleavage
factor Im, as well as a nuclear-retained mRNA (CTN-
RNA), localize to paraspeckles (Fox et al. 2002, 2005;
Dettwiler et al. 2004; Prasanth et al. 2005). Although the
exact function of paraspeckles is unclear, they have been
suggested to function as storage sites for nuclear-retained
RNAs (Prasanth et al. 2005). Interestingly, RNase A treat-
ment disrupts the structural integrity of paraspeckles
(Fox et al. 2005; Prasanth et al. 2005), suggesting that
RNA(s) may be a critical component of these nuclear
structures. Three recent reports, including one from our
group, have identified the MEN e/b long ncRNAs as these
critical RNA components of paraspeckles (Clemson et al.
2009; Sasaki et al. 2009; Sunwoo et al. 2009). Transcribed
from the same RNA polymerase II promoter, MEN e (also
Long noncoding RNAs
GENES & DEVELOPMENT1499
known as NEAT1 [Hutchinson et al. 2007]) and MEN b
differ in the location of their 39 ends, but both are retained
in the nucleus in paraspeckles (Sasaki et al. 2009; Sunwoo
et al. 2009). Unlike depleting CTN-RNA expression
(Prasanth et al. 2005), depletion of MEN e/b results in
the disruption of paraspeckles, arguing that these long
ncRNAs are required for paraspeckle establishment and
maintenance (Fig. 3; Clemson et al. 2009; Sasaki et al.
2009; Sunwoo et al. 2009).
RNA has also been found to serve a structural role in
the organization and maintenance of the cellular cyto-
skeleton as well as the mitotic spindle. In Xenopus
oocytes, the proper organization of the cytokeratin cyto-
skeleton is dependent on two RNAs, the Xlsirts ncRNA
and the VegT mRNA, which are integrated within the
cytoskeleton (Kloc et al. 2005, 2007). Depletion of either
transcript using antisense oligonucleotides disrupts the
cytokeratin network, but not the actin cytoskeleton.
Interestingly, although VegT is a protein-coding mRNA,
blocking its translation had no effect on the cytokeratin
network (Heasman et al. 2001; Kloc et al. 2005), arguing
that the RNA itself is functioning to maintain the
cytoskeleton. Likewise, a large number of RNAs, partic-
ularly ribosomal RNAs as well as a number of uncharac-
terized transcripts, have been found to associate with the
mitotic spindle (Blower et al. 2005). RNase A treatment
disrupts spindle assembly and causes the spindle to
collapse, although treatment with translation inhibitors
have no effect, arguing that these RNAs play a trans-
lation-independent role in spindle assembly in M phase
(Blower et al. 2005).
Considering the great variety of mRNA localization
patterns that are observed during early Drosophila em-
bryogenesis (Lecuyer et al. 2007), it is tempting to specu-
late that many more RNAs (especially ncRNAs) may have
structural and organization roles in the cell. For example,
Xist (Zhang et al. 2007) and Kcnq1ot1 (Mohammad et al.
2008) both cause silenced chromatin to localize to the
perinucleolar region during S phase of the cell cycle.
Not so fast—short ORFs can be translated
Many transcripts are classified as noncoding on the basis
of not having ORFs longer than 50–100 amino acids.
However, the Drosophila tarsal-less (tal) gene provides
a telling example of the importance of validating these
sorts of bioinformatic predictions. The tal gene expresses
a 1.5-kb transcript that contains only ORFs of <50 amino
acids and was, therefore, originally classified as a long
ncRNA. However, several 33-nt ORFs within the tal gene
are actually translated into 11-amino-acid-long peptides
that control key tissue morphogenesis and pattern for-
mation events during Drosophila development (Galindo
et al. 2007; Kondo et al. 2007; Pueyo and Couso 2008).
Due to practical and statistical reasons, ORFs as short as
these in tal are generally systematically eliminated from
gene annotations, but clearly need to be considered when
addressing the function of an unannotated transcript.
Additionally, some ncRNA genes, such as SRA (Steroid
receptor RNA activator), appear to yield multiple RNA
isoforms, some of which can be translated (for review, see
Leygue 2007), thus allowing a gene to have functions
carried out by both RNA and protein.
It is quickly becoming clear that long ncRNAs can have
numerous molecular functions, including modulating
transcriptional patterns, regulating protein activities,
serving structural or organizational roles, altering RNA
processing events, and serving as precursors to small
RNAs (Fig. 1). Although only a very small portion of
known long ncRNAs has been thoroughly characterized
to date, future work will likely identify many more
transcripts that fit into these and other functional para-
digms. In addition, future work will further address the
question of whether the act of ncRNA transcription itself
is enough to have functional consequences, or if many of
the resulting ncRNAs actually have functions in cis that
cannot be recapitulated by ectopic expression in trans.
A major current challenge is to understand how the
molecular functions of these long ncRNAs affect the
organism. For example, long ncRNAs have been impli-
cated in numerous developmental events (for review, see
Amaral and Mattick 2008), such as the formation of
photoreceptors in the developing retina (Young et al.
2005) and the regulation of cell survival and cell cycle
progression during mammary gland development (Ginger
et al. 2006). The generation of knockout animal models
will likely reveal many insights and definitively show
that many long ncRNAs are not transcriptional ‘‘noise,’’
but are instead required for normal development.
Numerous long ncRNAs are misregulated in various
diseases, especially cancer (for review, see Costa 2005;
Prasanth and Spector 2007), and some have been found to
integrity of paraspeckles. The MEN e/b ncRNAs localize to
paraspeckles in HeLa cells as shown by the colocalization of an
RNA FISH probe with PSPC1, a known protein component of
paraspeckles, fused to EYFP. Upon treating the cells with an
antisense oligonucleotide (ASO) complementary to MEN e/b to
deplete expression of the ncRNAs, paraspeckles are disrupted. In
contrast, a control ASO has no effect on paraspeckle integrity.
Bar, 10 mm.
MEN e/b ncRNAs are essential for the structural
Wilusz et al.
1500GENES & DEVELOPMENT
be very sensitive and specific markers of tumors, such as
DD3 (also known as PCA3) in prostate tumors (de Kok
et al. 2002). However, the mechanisms by which these
transcripts may affect tumor initiation and/or progres-
sion are currently unknown. Long ncRNAs thus remain
a relatively unexplored area in disease research, which
may allow us to identify new therapeutic targets. Recent
work on Alzheimer’s disease has identified a ncRNA
antisense to the b-secretase (BACE1) gene, which gener-
ates amyloid b (Ab), that may aid in driving the disease
(Faghihi et al. 2008). The ;2-kb ncRNA is induced in
response to numerous cell stressors, including serum
starvation and Ab peptides and, unfortunately, increases
the stability of the BACE1 mRNA, thus leading to even
more Ab peptides and the deleterious feed-forward cycle
of disease progression. However, treatment with siRNAs
against the ncRNA reduces the levels of Ab peptides,
suggesting that this noncoding transcript may serve as an
attractive drug target candidate for Alzheimer’s disease.
Although it has been classically assumed that most
genetic information is expressed as and transacted by
proteins, it is now clear that transcription is pervasive
throughout the eukaryotic genome, yielding many func-
tional ncRNAs with key regulatory roles. Many surprises
have surfaced over the past few years, and it is certain
that future research will provide many more unexpected
insights into the functions of ncRNAs.
We thank members of the Spector laboratory for many helpful
discussions and comments. Research in the Spector laboratory
is supported by NIH/GM42694, 5P01CA013106-38, and NIH/
EY18244. J.E.W. is supported by a Beckman Graduate Student-
ship at the Watson School of Biological Sciences.
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