Molecular Mechanisms of Long Noncoding RNAs
Kevin C. Wang1and Howard Y. Chang1,*
1Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological
functions. Here we discuss the emerging archetypes of molecular functions that lncRNAs execute—as
signals, decoys, guides, and scaffolds. For each archetype, examples from several disparate biological
contexts illustrate the commonality of the molecular mechanisms, and these mechanistic views provide
useful explanations and predictions of biological outcomes. These archetypes of lncRNA function may be
a useful framework to consider how lncRNAs acquire properties as biological signal transducers and hint
at their possible origins in evolution. As new lncRNAs are being discovered at a rapid pace, the molecular
mechanisms of lncRNAs are likely to be enriched and diversified.
The conventional view of gene regulation in biology has centered
around protein-coding genes via the central dogma of DNA /
mRNA / protein. However, over the past decade, evidence
from numerous high-throughput genomic platforms suggests
that the evolution of developmental processes regulating the
complexity of the organism is mainly due to the expansion of
regulatory potential of the noncoding portions of the genome
(Mattick, 2004). In fact, the portion of the genome responsible
for protein coding constitutes approximately 1.5%, while many
noncoding regulatory elements are transcribed into noncoding
RNA (ncRNA), the implication being that ncRNAs could play
significant regulatory roles in complex organisms. Indeed, the
recent explosion in knowledge demonstrating the importance
of ncRNAs in the regulation of multiple major biological pro-
cesses impacting development, differentiation, and metabolism
have brought these heretofore neglected molecular players to
the forefront (Mercer et al., 2009; Ponting et al., 2009; Wilusz
et al., 2009).
The complexity of mammalian transcriptome has been high-
lighted by recent high-throughput studies, which have revealed
that tens of thousands of sites are transcribed to produce
transcripts with little protein-coding potential—this was most
recently demonstrated throughabinitioreconstruction (Guttman
et al., 2010). In contrast to the small ncRNAs such as siRNAs,
miRNAs, and piRNAs, which are highly conserved and in-
volved in transcriptional and posttranscriptional gene silencing
through specific base pairing with their targets, long ncRNAs
(lncRNAs)—defined as transcribed RNA molecules greater
than 200 nt in length—are poorly conserved and regulate gene
expression by diverse mechanisms that are not yet fully under-
stood (Bernstein and Allis, 2005; Bracken and Helin, 2009;
Faghihi and Wahlestedt, 2009; Mercer et al., 2009; Whitehead
et al., 2009; Wilusz et al., 2009).
well characterized to date, they have been shown to control
every level of the gene expression program (Wapinski and
Chang, 2011). For example, they have long been implicated in
posttranscriptional gene regulation through controlling pro-
cesses like protein synthesis, RNA maturation, and transport
lating the chromatin structure (Bernstein and Allis, 2005; White-
head et al., 2009). Structurally different RNAs engage diverse
mechanisms that lead to different regulatory outcomes.
Although there is no conservation at the primary sequence level
between these RNAs, there are several similarities in their mode
of action. Some of the lncRNAs converge on chromatin structure
to silence multiple genes located on the overlapping and
nonoverlapping sides. They interact with DNA and/or chro-
matin-modifyingproteins andrecruitthemtotheir target regions.
The exact physical association between these lncRNAs and
chromatin modifiers and/or gene promoter chromatin remains
to be elucidated. Given the large number of lncRNAs whose
functions are still to be uncovered, there is clear potential for
widespread regulation of chromatin modifications and gene
In this review, we distill the myriad functions of lncRNAs into
four archetypes of molecular mechanisms. Each archetype is
illustrated with examples drawn from diverse systems and
organisms, and we explore similarities and differences between
the archetypes to demonstrate the changes in functional com-
plexity (Figure 1). As it will soon become clear, an individual
lncRNA may fulfill several archetypes; thus, these archetypes
are not meant to be mutually exclusive. Rather, we aim to illus-
trate how apparently complex functions can be constructed
from combinatorial usage of archetypal molecular mechanisms.
An understanding of the possible commonalities of the under-
lying mechanisms may facilitate instructive and predictive
models of lncRNA function.
Archetype I: Signals
The majority of lncRNAs are transcribed by RNA polymerase II,
asevidenced byPolIIoccupancy, 50caps, histone modifications
associated with Pol II transcriptional elongation, and polyadeny-
lation (Guttman et al., 2009). LncRNAs show cell type-specific
expression and respond to diverse stimuli, suggesting that
their expression is under considerable transcriptional control.
As such, lncRNAs can serve as molecular signals, because tran-
scription of individual lncRNAs occurs at a very specific time and
place to integrate developmental cues, interpret cellular context,
Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.
or respond to diverse stimuli. Some lncRNAs in this archetype
possess regulatory functions, while others are merely by-prod-
ucts of transcription—it is the act of initiation, elongation, or
termination that is regulatory. In either case, one can conve-
niently infer the chromatin state of regulatory elements merely
by the expression of their associated lncRNAs. Furthermore,
theadvantage of usingRNAasa medium suggeststhat potential
regulatory functions can be performed quickly without protein
translation. Several examples below illustrate lncRNA as signals
marking space, time, developmental stage, and expression for
gene regulation. Specifically, the lncRNAs in this archetype can
act as markers of functionally significant biological events.
Allele Specificity: KCNQ1ot1, Air, and Xist
Imprinting is an epigenetic regulatory mechanism that best
illustrates the concept of allele specificity. Mammals are diploid
organisms carrying two alleles of each autosomal gene, one in-
herited from the mother and one from the father. Whereas in
most cases both parental alleles are expressed equally, a subset
of genes show imprinting in which expression is restricted by
an epigenetic mechanism to either the maternal or the paternal
Recent emerging evidence indicates that lncRNAs such as
Kcnq1ot1 and Air, which map to the Kcnq1 and Igf2r imprinted
gene clusters, respectively, mediate the transcriptional silencing
of multiple genes by interacting with chromatin and recruiting
the chromatin-modifying machinery. For example, in mouse
placenta, lncRNAs such as Air and Kcnq1ot1 accumulate at
promoter chromatin of silenced alleles and mediate repressive
histone modifications in an allele-specific manner (Mohammad
et al., 2009). Kcnq1ot1 is a 90 kb lncRNA expressed from the
paternal allele that directs silencing of a cluster of genes in the
imprinted Kcnq1 domain (Pandey et al., 2008). Kcnq1ot1 inter-
acts with the histone methyltransferases G9a and PRC2, effec-
tively forming a repression domain in cis to its transcription site
through recruitment of polycomb complexes—the RNA itself
seems to play a critical role in the bidirectional silencing of genes
in the Kcnq1 domain, thus resembling the mechanisms of action
of Xist RNA.
Similarly, the ncRNA Air is imprinted and expressed only from
the paternal allele, and its transcription is required for repression
of several imprinted genes on the paternal chromosome in
a tissue- and allele-specific manner. In the placenta, similar to
the recruitment of chromatin-modifying activities of Kcnq1ot1,
the Air transcription unit initiates in the second intron of the
mouse Igf2r gene and recruits G9a to its target promoter to bring
about gene silencing (Nagano et al., 2008). However, in embry-
onic tissues, Air exerts its effects via a different mechanism,
where its own transcription plays a critical role in the silencing
of the overlapping gene (Sto ¨ger et al., 1993).
X inactivation (XCI) is a closely related process that equalizes
gene expression between mammalian males and females by
inactivating one X in female cells. Xist is a well-known lncRNA
that plays an essential role in XCI (reviewed by Pontier and Grib-
nau, 2011). During female development, Xist RNA is expressed
from the inactive X and ‘‘coats’’ the X chromosome from which
it is transcribed, leading to chromosome-wide repression of
gene expression. An overlapping antisense lncRNA called Tsix
represses Xist expression in cis, while the lncRNA Jpx, whose
expression accumulates during XCI, activates Xist on the inac-
tive X (Tian et al., 2010).
cates active silencing at their respective genomic locations.
Anatomic Specific Expression: HOTAIR and HOTTIP
Another example of the tight relationship between time and
space is illustrated by two ncRNAs from the mammalian Hox
loci. In mammals, the homeobox transcription factors (HOX)
are organized into four chromosomal clusters and expressed
in a segmental fashion that is collinear between gene position
Figure 1. Schematic Diagram of the Four Archetypes of LncRNA
Archetype I: as signals, lncRNA expression can faithfully reflect the combi-
natorial actions of transcription factors (colored ovals) or signaling pathways
to indicate gene regulation in space and time. Archetype II: as decoys,
lncRNAs can titrate transcription factors and other proteins away from chro-
matin or titrate the protein factors into nuclear subdomains. A further example
of decoys is lncRNA decoy for miRNA target sites (not shown on schematic).
Archetype III: as guides, lncRNAs can recruit chromatin-modifying enzymes to
target genes, either in cis (near the site of lncRNA production) or in trans to
distant target genes. Archetype IV: as scaffolds, lncRNAs can bring together
multiple proteins to form ribonucleoprotein complexes. The lncRNA-RNP
may act on chromatin as illustrated to affect histone modifications. In other
instances, the lncRNA scaffold is structural and stabilizesnuclear structures or
Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.
within the cluster and spatial position along the anterior-poste-
rior anatomic axis of the body (reviewed by Wang et al., 2009).
Numerous lncRNAs were found to be transcribed from within
the human HOX clusters (Rinn et al., 2007) that were expressed
in a temporal and site-specific fashion. The lncRNAs were found
to be also collinear with the overall anatomic expression pattern
of the HOX loci, implying that they probably used the same
enhancers as the HOX genes: for example, HOTAIR, a lncRNA
of the HOXC locus that is expressed in cells with distal and
posterior positional identities, and Frigidair, another HOXC
lncRNA, which has an anterior pattern of expression. In contrast,
HOTTIP, another lncRNA found at the distal end of the human
HOXA cluster, is also expressed in distal cells. In addition to
serving as signals of anatomic position, both lncRNAs have
additional biological functions, as detailed below.
Induction by DNA Damage: LincRNA-p21 and PANDA
LncRNAs that act to integrate contextual and environmental
cues can be found not only during development, but also during
times of organismal stress. Huarte et al. (2010) showed that
lncRNAs play a key regulatory role in the p53 transcriptional
response. One of the direct p53 targets in response to DNA
damage, a lncRNA called lincRNA-p21 located upstream of
CDKN1A gene, was found to act as a transcriptional repressor
in the canonical p53 pathway and to play a role in triggering
apoptosis. p53 regulates lincRNA-p21 by directly inducing its
expression, likely through direct binding to the lincRNA-p21
promoter, while reduction of large intergenic ncRNA (lincRNA)-
p21 increases expression of numerous p53-repressed tran-
scripts. Furthermore, lincRNA-p21 repressed p53-regulated
genes through its binding to and modulation of heterogeneous
nuclear ribonucleoprotein K (hnRNP-K) localization (Huarte
et al., 2010).
Another example of modulation of gene activity in response to
external stimuli is found again in the mammalian CDKN1A
promoter, where upon DNA damage several lncRNA are tran-
scribed (Hung et al., 2011). One such lncRNA, named PANDA,
is also induced in a p53-dependent manner. PANDA cannot be
activated by DNA damage in the absence of p53. After DNA
damage, p53 directly binds to the CDKN1A locus and activates
PANDA; PANDA then interacts with the transcription factor
NF-YA to limit expression of proapoptotic genes and enables
cell-cycle arrest, suggesting potentially widespread roles for
lncRNAs in cell-growth control.
These results reveal insights into a prototypical transcriptional
response and raise the possibility that lncRNAs may act as key
regulatory nodes in multiple transcriptional pathways, serving
as both a signal and a convenient means of tracking the tran-
scriptional activity of promoters in response to stimuli.
Pluripotency and Reprogramming: ROR LncRNA
Pluripotency-associated lincRNAs were initially discovered in
mouse embryonic stem cells (ESCs) (Guttman et al., 2009), but
evidence to support their direct functional relevance has been
lacking. Loewer and colleagues show that somatic cell repro-
gramming to induced pluripotent stem cells (iPSCs) is accom-
panied by enriched expression of lincRNAs (Loewer et al.,
2010). One of these reprogramming-induced lincRNAs, named
lincRNA-RoR, was shown to be directly targeted by the key plu-
ripotency factors Oct4, Sox2, and Nanog through colocalization
of the three factors close its promoter region (Loewer et al.,
2010). RoR was downregulated upon Oct4 depletion, as well
as during differentiation of iPSCs, implicating coregulation of
specific lincRNAs by key pluripotency factors.
Induction by Cold: COLDAIR and COOLAIR
The phenomenon of combinatorial transcriptional regulation by
lncRNAs is also found in plants. The transition from vegetative
to reproductive development is a highly regulated process
that, in many plant species, is sensitive to environmental cues
that provide seasonal information to initiate flowering during
optimal times of the year. One environmental cue is the cold of
winter. Vernalization is an environmentally induced epigenetic
switch in which prolonged exposure to winter cold triggers
epigenetic silencing of floral repressors and provides com-
petence to flower in spring (Kim et al., 2009). In the plant
Arabidopsis thaliana, winter cold triggers enrichment
trimethylated histone H3K27 at the chromatin of the floral
repressor, FLOWERING LOCUS C (FLC), and results in epige-
netically stable repression of FLC. This epigenetic change is
mediated also through the PRC2 complex. One of the earliest
events in this silencing is a large increase in abundance of the
antisense transcripts, aptly named COOLAIR, that silences
sense FLC transcription and promotes polycomb occupancy
(Swiezewski et al., 2009). The COOLAIR promoter is cold
inducible and sufficient to induce cold-dependent silencing of
a heterologous reporter construct (Swiezewski et al., 2009).
transcript that triggers FLC silencing (Liu et al., 2010).
ncRNA termed COLD-ASSISTED INTRONIC NONCODING RNA
(COLDAIR) is required for the vernalization-mediated epigenetic
repression of FLC, via the establishment of stable repressive
chromatins at FLC through its physical association with and
recruitment ofPRC2to thelocus.Incontrast toCOOLAIR, where
directly recruits the silencing factors. COLDAIR is transcribed in
the sense direction from an intron of its target gene, FLC. FLC
harbors a cryptic promoter for COLDAIR ncRNA within its first
intron, and this promoter becomes active when FLC is being
repressed (Heo and Sung, 2011). The demonstration of a similar
interaction of a lncRNA with PRC2 in plants suggests that
the ncRNA-PRC2 relationship appears to be an evolutionarily
conserved mechanism of gene repression. In addition, both
COOLAIR and COLDAIR appear to serve as signals of tran-
scriptional activity with spatial and temporal specificity.
Coordinated Activity as Exemplified by eRNAs
It is well known that regulatory proteins exert their functions by
binding stretches of noncoding DNA, either close to a gene’s
mRNA transcription start site at a promoter or further away on
the genome at an enhancer. Enhancers, in turn, act by helping
to recruit the RNA polymerase to the promoter. Recently,
a new class of ncRNAs—enhancer or eRNAs—have been
described that are produced by activity-dependent RNA Pol II
binding of specific enhancers (Kim et al., 2010). The level of
eRNA expression at these enhancers positively correlates with
the level of messenger RNA synthesis at nearby genes, suggest-
ing that eRNA synthesis occurs specifically at enhancers that
are actively engaged in promoting mRNA synthesis (Kim et al.,
Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.
2010; Wang et al., 2011a). These findings suggest that
enhancers have a more active ‘‘promoter-like’’ role in regulating
Approaching the same question from a different angle,
another group identified a new class of lncRNAs with an
enhancer-like function in various human cell lines (Ørom et al.,
2010). Depletion of these lncRNAs led to decreased expression
of their neighboring protein-coding genes, including several
master regulators of cellular differentiation. Like classical
enhancers, lncRNAs are orientation independent and require
a minimal promoter in their target genes to enhance their
transcription. Although the precise molecular mechanism is yet
to be defined, this group of lncRNAs illustrates that eukaryotic
transcription is very tightly regulated by overlapping mecha-
nisms. Both examples above provide evidence that RNAs can
serve as markers of active regulatory pathways.
Repeat Detector: RNA Decay and Staufen
Yet another unforeseen function for lncRNAs was uncovered
when a group identified several lncRNAs that are important
in downregulating subsets of Staufen 1 (STAU1)-mediated
messenger RNA decay (SMD) (Gong and Maquat, 2011). SMD
regulates diverse classes of mRNAs in mammalian cells that
have STAU1 binding sites in their 30untranslated region (30
UTR). It was thought that the STAU1 binding site is a cis element
with specific secondary structures within the 30UTR of SMD
targets. Interestingly, a group of cytoplasmic lncRNAs, named
the half-STAU1-binding site RNAs (1/2-sbsRNAs), was found to
facilitate the formation of STAU1 binding sites. This was accom-
plished through imperfect base pairing, based on Alu repeats,
between the lncRNA and the 30UTR of certain mRNAs, resulting
in the degradation of the mRNAs via SMD (Gong and Maquat,
2011). Individual members of this functional class of lncRNAs
can downregulate the same SMD target. These findings reveal a
novel role of lncRNAs in mRNA metabolism, specifically as a
mechanism of temporal- and spatial-specific recruitment of pro-
teins to mediate mRNA decay. The idea in which partially com-
plementary lncRNA/mRNA duplexes can form Staufen binding
sites is likely to also apply to the regulation by other double-
stranded RNA-binding protein (RBP)-dependent pathways.
Thus, the signal archetype of lncRNAs includes not only
markers of downstream transcriptional elements but also detec-
tors of transcript abundance/repetition. Taken together, the first
archetype of lncRNAs all function as indicators of transcriptional
activity, independent of additional functional roles, in a surpris-
ingly straightforward one-to-one relationship.
Archetype II: Decoys
The pervasive transcription of enhancers and promoters
(Guenther et al., 2007) hints at a central role for lncRNAs in regu-
which such ncRNAs regulate transcription are expanding to
encompass a diversity of mechanisms, a major one of which is
to act as molecular decoys. This archetype of lncRNAs is tran-
scribed and then binds and titrates away a protein target, but
does not exert any additional functions. The RNAs act as
a ‘‘molecular sink’’ for RBPs, which are themselves transcription
factors, chromatin modifiers, or other regulatory factors.
LncRNAs that fit into this functional archetype would presum-
ably act by negatively regulating an effector. Thus, the logical
operation is RNA inhibits effector X from executing effector func-
of the protein partner, and loss of function of both the lncRNA
and the effector would be result in a rescue phenotype.
Alternative promoters within the same gene are a general
phenomenon in gene expression (Ayoubi and Van De Ven,
1996). Mechanisms of their selective regulation vary from one
gene to another and are just beginning to be uncovered. The
human dihydrofolate reductase (DHFR) gene is one such locus,
and it has been shown to possess an RNA-dependent mecha-
nism of transcriptional repression (Martianov et al., 2007). The
lncRNA initiated from the upstream minor promoter of the
DHFR gene inhibits assembly of the preinitiation complex at the
major promoter by forming a stable ncRNA-DNA complex with
promoter sequences, as well as through direct interactions with
the general transcription factor IIB (TFIIB). When the lncRNA
was specifically degraded through siRNA knockdown, the
occupancy of TFIIB on the major promoter remained high
(Martianov et al., 2007). This is a highly dynamic process that
presents a specific mechanism that may contribute to promoter
targeting and repression and highlights the importance of inter-
genic ncRNAs in regulation of neighboring gene expression, by
acting as a decoy.
Telomeres, the DNA-protein complexes located at the physical
ends of eukaryotic chromosomes that are essential for chromo-
some stability, have been found to be transcribed into telomeric
repeat-containing RNA (TERRA), a lncRNA, which forms an
integral part of telomeric heterochromatin (Azzalin et al., 2007). It
had been hypothesized that the existence of TERRA RNA
hints at a new level of regulation and protection of chromosome
ends—this is indeed the case, as TERRA has now been demon-
strated to physically interact with the telomerase through a
repeat sequence complementary to the template sequence of
telomerase RNA (Redon et al., 2010). In addition, TERRA also
contacts the telomerase reverse transcriptase (TERT) protein
subunit independently of the telomerase template RNA moiety.
Of note, telomeric heterochromatin-bound TERRA is thought to
telomerase near the telomeric 30end while inhibiting its action
(Redon et al., 2010). Additionally, TERRA levels change in a cell-
cycle-dependent manner—accumulating in early G1, continu-
ously decreasing in S phase, and reaching its lowest expression
levels at the transition between late S and G2 (Porro et al., 2010;
Flynn et al., 2011). Downregulation of TERRA in S phase might
unleash telomerase and allow extension of the telomeric strand
in a cell-cycle-dependent manner. Thus, telomerase regulation
by the telomere substrate may be mediated via its transcription;
this is also an example of a natural RNA ligand acting as a direct
regulator of enzymatic activity, without being a substrate.
The lncRNA PANDA, discussed above as a p53-dependent
transcript, also appears to possess decoy function. DNA
damage can result in apoptosis or cell-cycle arrest. PANDA is
very sensitive to DNA damage, and its expression is induced
Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.
temporally ahead of thatof CDKN1A. PANDA inhibits expression
of apoptotic genes to favor cell-cycle arrest through direct
binding to and sequestration of NF-YA, a nuclear transcription
factor that activates the apoptotic program upon DNA damage
(Hung et al., 2011), resulting in promotion of cell survival (in the
context of low-level DNA damage) through repression of the
apoptotic gene expression program. Depletion of PANDA
substantially increased NF-YA occupancy at target genes, while
concomitant knockdown of NF-YA and PANDA substantially
attenuated induction ofapoptotic genesandapoptosis.Interest-
ingly, a subset of human breast cancers overexpress PANDA,
and PANDA depletion can sensitize cells to chemotherapeutic
agent, hinting at a possible clinical application.
Gas5: Glucocorticoid Receptor
Recently, the lncRNA Gas5 (growth arrest-specific 5) has been
of relative glucocorticoid resistance (Kino et al., 2010). Gas5
represses the glucocorticoid receptor through formation of an
RNA motif from one of its stem-loop structures, mimicking the
DNA motif equivalent to that of hormone response elements
found in the promoter regions of glucocorticoid-responsive
genes. Gas5 then competes for binding to the DNA binding
domain of the glucocorticoid receptor, acting as a molecular
decoy, and effectively precludes its interaction with the chromo-
some (Kino et al., 2010). This may turn out to be an integral
component of the regulatory machinery for modulating steroid
hormone activity in target tissue.
LncRNA Decoys for miRNAs and Splicing Factors:
Pseudogenes, miRNA Target Mimics, and MALAT1
miRNAs, a large class of small ncRNAs, have emerged as a crit-
ical element in gene regulation by interacting with incompletely
complementary sequences in a target messenger RNA (Baek
et al., 2008; Bartel, 2009). miRNAs function by annealing to
complementary sites on the coding sequences or 30UTRs of
target gene transcripts, where they promote recruitment of
protein complexes that impair translation and/or decrease the
stability of mRNA, leading to a decrease in target protein abun-
dance (Baek et al., 2008; Bartel, 2009). There is now evidence
that the converse mechanism may also be in play—i.e., mRNA
expression can affect the distribution of miRNAs. Recent work
on the tumor suppressor pseudogene PTENP1, previously
considered biologically inconsequential, has brought forth the
idea that it may have biological function as ‘‘decoys’’ through
sequestration of miRNAs to affect their regulation of expressed
genes (Poliseno et al., 2010). Specifically, the 30UTR of PTENP1
RNA was found to bind the same set of regulatory miRNA
sequences that normally target the tumor-suppressor gene
its translation into the tumor-suppressor protein PTEN. In addi-
tion to the decoy idea for ncRNAs, this finding nicely illustrates
another possible regulatory role for mRNAs in addition to their
between other cancer-related genes and their pseudogenes
(Song et al., 2011). Similarly, in the plant A. thaliana, the ncRNA
IPS1 binds to and sequesters the phosphate starvation-induc-
ible miR-399 through near perfect sequence complementarity
and, as a result, blunts miR-399 action and alters shoot phos-
phate content (Franco-Zorrilla et al., 2007).
One of the most abundant nuclear lncRNAs in mammalian
cells is MALAT1 (metastasis-associated lung adenocarcinoma
transcript 1), which is localized in nuclear speckles. MALAT1
binds to and sequesters several serine/arginine (SR) splicing
factors to nuclear speckles. Depletion of MALAT1 alters splicing
factor localization and activity, leading to altered pattern of alter-
native splicing for a set of pre-mRNAs (Tripathi et al., 2010). In
hippocampal neurons, MALAT1 regulation of SR splicing factors
is important for synapse formation (Bernard et al., 2010). Thus,
lncRNA decoys can function in nuclear subdomains as well as
illustrate that lncRNA decoys can titrate away proteins and small
regulatory RNAs and likely function in multiple kingdoms of life.
Archetype III: Guides
The third archetype of lncRNA is the guide—RNA binds
protein(s), then directs the localization of ribonucleoprotein
complex to specific targets. As is evident from the discussions
so far, lncRNAs can guide changes in gene expression either in
cis (on neighboring genes) or in trans (distantly located genes)
in a manner that is not easily predicted based on lncRNA
sequence. The myriad distinctive and likely widespread roles
of these RNAs in transcriptional regulation dictates the necessity
that certain local changes in chromatin structure can have not
only local consequences, but structural repercussions at a
distance. Indeed, lncRNAs such as Air and eRNAs appear to
exert their effects in cis by spreading from focal sequence
elements of transcriptional control such as promoters or en-
hancers. In contrast, for lncRNAs such as HOTAIR and
lincRNA-p21, long-range actions of gene regulation require
additional ability of components of the interacting partners to
can guide chromatin change in cis in a cotranscriptional manner
(tethered by RNA polymerase) or as a complementary target
for small regulatory RNAs; guidance in trans can occur by
lncRNA binding to target DNA as a RNA:DNA heteroduplex, as
RNA:DNA:DNA triplex, or RNA recognition of complex surface
of specific chromatin features (reviewed by Hung and Chang,
2010; Bonasio et al., 2010).
The gene regulatory components brought on by the lncRNAs
include both repressive (e.g., polycomb) and activating (MLL)
complexes, as well as transcription factors (TFIIB). However,
no matter the distance or mechanism (either cis or trans), the
principle remains the same: to convey regulatory information
across an intervening stretch of DNA to control target gene
expression, bringing about changes in the epigenome.
There isfurthercomplexity built into the archetype in that there
are several possible functional classes of effector molecules:
activating complexes such as the trithorax group proteins
(TxG), repressive complexes such as the polycomb group
proteins (PcG), as well as the usual collection of transcription
factors. Additionally, the effectors can be localized both in cis
and in trans. Taking the idea a step further, some lncRNAs may
be ‘‘tethers’’ that recruit several chromatin modifications to their
sites of synthesis (Lee, 2009), while other lncRNAs may act on
distantly located genes as ‘‘guides’’ to affect desired chromatin
states. One can then speculate with regards to additional levels
of regulation, such as binding of lncRNA by small molecules
Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.
(e.g., ions, enzyme cofactors, and nucleotide analogs), similar to
classic riboswitch interactions (Wachter, 2010), that results in
modulation of the RNA chromatin/RNA chromatin-remodeling
Key predictions for this archetype of lncRNAs are as follows:
knockdown of the lncRNA would change/interfere with the
proper localization of the effector molecule or may phenocopy
loss of function of the effector itself; a double knockdown of
both the lncRNA and the effector will probably result in exacer-
bation of the phenotype instead of a rescue, as would be ex-
pected from the decoy archetype.
Guides In cis: Xist, Air, COLDAIR, rDNA Transcript,
CCND1, and HOTTIP
Work over the past several years has shed light on the advan-
tages that RNA offers in delivering allelic, cis-limited, and
locus-specific control. Perhaps the most intensely studied and
best understood cis mechanism of regulation by lncRNAs is
the mammalian X inactivation center (Xic), a genetic locus that
specifies a number of ncRNAs, including Xist (Plath et al.,
2002; Lee, 2010). Xic controls the silencing of one of the two X
chromosomes in female mammals, to achieve dosage compen-
sation between the sexes. One of the first changes to occur
during the chromosome-wide silencing step of the extra X chro-
mosome is the recruitment of polycomb repressive complex 2
(PRC2); PRC2 is brought in cis by RepA RNA, a 1.6 kb ncRNA
originating fromthe 50end of Xist(Wutz etal., 2002). RepA-medi-
ated PRC2 recruitment and H3K27 trimethylation of the Xist
promoter result in the creation of a ‘‘heterochromatic state’’
(Sun et al., 2006) that is required for transcriptional induction of
Xist. Spreading of Xist is accompanied by the recruitment of
polycomb and their associated chromatin modifications to the
inactive X chromosome (Xi). More recently, a matrix protein,
hnRNP U, was shown to be required for the accumulation of
Xist RNA on the Xi (Hasegawa et al., 2010). Xist RNA and hnRNP
U interact, and depletion of hnRNP U causes Xist to detach from
the Xi and to localize diffusely in the nucleoplasm. Thus, XCI
represents a prominent example of the recruitment of chro-
matin-modifying activities by lncRNAs and provides a prototype
model for a cis localization mechanism.
Similar mechanisms of action appear to be in play for other
lncRNAs with transcriptional repression activities. The lncRNA
Air silences transcription of its target gene on the paternal
chromosome via a specific interaction between the ncRNA and
chromatin at its promoter (Nagano et al., 2008); accumulated
Air at the promoter recruits G9a and leads to targeted H3K9
methylation and allelic silencing. The cold-induced plant lncRNA
COLDAIR, described above, is required to establish and main-
tain stable repressive chromatin. COLDAIR plays a critical role
in guiding PRC2 complexes to the chromatin of FLC, a strong
floral repressor, during vernalization, affecting gene repression
through trimethylation of H3K27 (Heo and Sung, 2011). Similarly
in yeast, antisense lncRNAs at numerous gene loci act to silence
sense transcription by affecting histone acetylation and methyl-
ation states (Camblong et al., 2007; van Dijk et al., 2011). Taken
together, these findings suggest a mechanism by which the
lncRNAs can function through specific interaction with chro-
matin to mediate targeted recruitment of repressive histone-
modifying activities in cis to epigenetically silence transcription.
The potential of RNA to bind to complementary DNA
sequences has led many to hypothesize that RNAs may play
important guiding roles in the establishment and transmission
of chromatin states. Grummt and colleagues have now shown
that this could indeed be a major mechanism by showing that
pRNA (promoter-associated RNA), an ncRNA that is comple-
mentary to the ribosomal DNA promoter, can form an RNA-
DNA triplex structure at the site of binding of TTF-1, the major
transcription factor for ribosomal RNA (rRNA) transcription by
polymerase I (Schmitz et al., 2010). The triplex was shown to
prevent TTF-1 binding while at the same time recruiting
DNMT3b, a DNA methylase that facilitates promoter methylation
and leads to transcriptional silencing of the rRNA gene. This may
represent a more general means of epigenetic regulation, by
promoter-specific targeting of chromatin-modifying enzymes
through triplex-forming ncRNAs, with the ncRNAs serving as
LncRNAs are also involved in the regulation of gene expres-
sion programs by transcriptional coactivator and corepressor
complexes such as CREB-binding protein (CBP) and p300
histone acetyltransferase (Wang et al., 2008). The RBP TLS
(translocated in liposarcoma), known to be involved in chromo-
somal translocations in sarcomas and leukemias, is recruited
to chromatin via a tethered lncRNA produced at the cyclin D1
promoter inresponseto ionizingradiation. BindingofthelncRNA
to TLS,in turn,inducesaconformation change inTLSthatallows
its amino terminus to inhibit the histone acetyl transferase
activity of p300 and CBP and thereby inhibit gene expression
(Wang et al., 2008). Although the extent to which this particular
mechanism is used remains to be established, the identification
of highly conserved lncRNAs and the presence of RNA binding
domains in a large number of transcriptional coregulators raise
the possibility that promoter-specific lncRNA/coregulator inter-
actions play broad roles in the regulation of gene expression in
cis. The recent identification of HOTTIP lncRNA from the human
HoxA cluster (described above; Wang et al., 2011b) adds an
additional dimension to cis regulation by lncRNAs by defining
a central role for chromosome looping in delivering a lncRNA
to its site of action. By serving as key intermediates that transmit
information from higher-order chromosomal looping into chro-
matin modifications, lincRNAs may organize chromatin domains
to coordinate long-range gene activation. It is interesting to note
that application of genome-wide chromosome conformation
capture (3C) technology has recently revealed a vast network
of physical interactions between PcG protein target sites (Ban-
tignies et al., 2011). In theory, the chromosome configuration
scriptional activity of multiple contiguous loci—a phenomenon
termed locus control—such as in the HOX clusters.
Guides In trans: HOTAIR, LincRNA-p21, Jpx, and Other
In contrast to the group of cis-regulatory lncRNAs, there are
a couple of examples of lncRNAs that exert their transcriptional
effects across chromosomes in trans. Expression of the Hox
lncRNA HOTAIR has recently been associated with cancer
metastasis (Gupta et al., 2010). Elevated expression of HOTAIR
is observed in primary and metastatic breast cancer. Further-
more, depletion of HOTAIR from cancer cells leads to a reduced
Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.
invasiveness of cells that express a high level of polycomb
proteins (PRC2) (Gupta et al., 2010). These findings suggest
that ncRNA-mediated targeting of polycomb complexes is
a crucial event in breast tumorigenesis. Specifically, the implica-
tion is that lncRNAs such as HOTAIR are able to alter and
regulate epigenetic states in cells through their targeting of chro-
activity in trans. In support of this idea, multiple lncRNAs ex-
pressed in various cell types bind PRC2, and siRNA-mediated
depletion of a number of these lncRNAs led to enrichment for
genes normally repressed by PRC2, akin to a partial PRC2
knockdown phenotype (Khalil et al., 2009; Zhao et al., 2010).
Jpx, the lncRNA important for activation of Xist RNA on the
inactive X, is developmentally regulated and accumulates during
XCI (Tian et al., 2010). Deleting Jpx blocks XCI, and posttran-
scriptional knockdown of Jpx recapitulates the knockout pheno-
type. Moreover, supplying Jpx in trans rescues the Jpx knockout
(Tian et al., 2010). The mechanism by which Jpx transactivates
Xist RNA is not yet understood, but perhaps involves loading
of the polycomb complex onto the Xist promoter to create
a permissive state for Xist transactivation.
LincRNA-p21 is able to exert its effects on chromatin structure
and gene expression across multiple sites in the genome (Huarte
et al., 2010). Ectopic expression of lincRNA-p21 induces gene
expression changes and apoptosis, bypassing the upstream
regulator p53. It remains to be determined how the repressive
complex associated with lincRNA-p21 recognizes targeted
gene loci and how this complex silences transcription.
Archetype IV: Scaffolds
LncRNAs can serve as central platforms upon which relevant
molecular components are assembled; in many diverse biolog-
ical signaling processes, this characteristic of precise control is
vital to the precise control of the specificity and dynamics of
intermolecular interactions and signaling events (Spitale et al.,
2011). Traditionally, proteins were thought to be the major
players in various scaffolding complexes (Good et al., 2011).
Recent evidence, however, raises the possibility that lncRNAs
may also play a similar role.
The fourth archetypal class of lncRNAs is the scaffolds. This is
perhaps the most functionally intricate and complex class where
the lncRNA possesses different domains that bind distinct
effector molecules. The lncRNA would bind its multiple effector
partners at the same time and by doing so brings the effectors,
which may have transcriptional activating or repressive activi-
ties, together in both time and space. Once a greater under-
standing of how these scaffolding complexes are assembled
and regulated is achieved, it would then be possible to design
strategies to selectively utilize specific signaling components
be analogous to assembly of an integrated circuit board for
molecular scaffolds that functions to dictate flow of information.
Key predictions for this archetype of lncRNAs would include
the following: knockdown of the lncRNA would change/interfere
with the proper localization of the effector molecule or may
phenocopy loss of function of the component effector itself,
through dismantling of the lncRNA-effector scaffold such that
the components no longer assemble together. A double knock-
down of both the lncRNA and the effector(s) will be expected to
result in exacerbation of the phenotype instead of a rescue, as
would be expected from the decoy archetype. Finally, disruption
of distinct regions of the lncRNA should affect different effector
partners and function.
TERC: Assembles Telomerase
The telomerase is a specialized reverse transcriptase conserved
in almost all eukaryotes and plays a fundamental role in mainte-
lost from chromosome ends. The protein and RNA subunits of
telomerase fold and function in a codependent manner to
establish a high fidelity of telomeric repeat synthesis (Lustig,
2004). Telomerase catalytic activity requires the association of
two universal telomerase subunits: an integral RNA subunit,
synthesis, and a catalytic protein subunit, the TERT, as well as
ular also possesses structures that contribute to TERT binding
and catalytic activity, in addition to those that play major roles
in stability of the complex (Collins, 2008).
In particular, RNA domains have been identified that affect
template usage (Chen and Greider, 2003; Lai et al., 2003) as
well as TERT association (Ly et al., 2003). Mutations that alter
the equilibrium between different conformational states of
TERCs result in disease states such as dyskeratosis congenita
(Chen and Greider, 2004), presumably through disruptions of
the RNA scaffold structure into which are plugged modular
binding sites for telomeric regulatory proteins. Thus, the primary
functional role for TERC appears to be that of a scaffold. In an
elegant demonstration of this concept, Zappulla and Cech
engineered functionally active minitelomerase RNAs that reas-
sembled the telomerase complex in budding yeast by stitching
together minimal RNA motifs (Zappulla and Cech, 2004). This
evidence suggests that the telomerase RNA serves as a loosely
ordered flexible scaffold for its protein subunits.
HOTAIR: PRC2 and LSD1, Resolution of Bivalent
On the basis of their dynamic patterns of expression (Guttman
et al., 2009), specific lncRNAs can potentially integrate and
direct complex patterns of chromatin states at specific target
loci in spatially and temporally specific manner during both
organismal development and disease.
As mentioned above,the lncRNA HOTAIR binds the polycomb
complex PRC2, which methylates histone H3 on K27 to promote
gene repression (Rinn et al., 2007); the fragment responsible for
PRC2 binding was recently identified to be the first 300 nt in the
HOTAIR was found to also interact with a second complex
containing LSD1, CoREST, and REST that demethylates histone
H3 on K4 to antagonize gene activation (Tsai et al., 2010). This
finding shows that multiple chromatin-modifying complexes
are targeted by HOTAIR, suggesting that HOTAIR acts as a scaf-
fold and bridges between PRC2 and the LSD1/CoREST/REST
complex—in one package, the HOTAIR/PRC2/LSD1 complex
can suppress gene expression via multiple mechanisms at the
same time. Indeed HOTAIR expression can induce the interac-
tion of PRC2 and LSD1 complexes, while depletion of HOTAIR
led to loss of occupancy of both complexes from target genes.
Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.
Notably, many additional lincRNAs can interact with both PRC2
andLSD1 complexes in several cell types (Khalilet al.,2009). Itis
quite possible that other lincRNAs may also contain multiple
binding sites where distinct protein complexes can assemble
to more specifically bring forth specific combinations of histone
modifications on target gene chromatin (Guttman et al., 2011).
KCNQ1ot1 may perform an analogous function for PRC2 and
G9a, mediating H3K27me3 and H3K9me3 (Pandey et al., 2008).
ANRIL: PRC2 and PRC1
The molecular interplay between lncRNAs and chromatin-modi-
fying complexes can also be found in the transcriptional repres-
sion of the well-studied INK4a locus. Expression of the INK4b/
ARF/INK4a tumor suppressor locus in normal and cancerous
bythe PcG proteins (Gil and Peters, 2006). The antisense ncRNA
ANRIL, which emanates from the same INK4b/ARF/INK4a locus,
is also important for expression of the protein-coding genes in
cis. Work over the past few years has demonstrated a direct
interaction between ANRIL and components from both PRC1
and PRC2 complexes (Kotake et al., 2011; Yap et al., 2010).
Binding to ANRIL contributes to the functions of both PRC1
and PRC2 proteins, and disruption of either interaction impacts
to HOTAIR, ANRIL represents a prototype of an lncRNA that is
always present at the locus and recruits multiple sets of chro-
matin-modifying complexes to the target gene for silencing,
serving as a molecular scaffold to dynamically modulate tran-
scriptional activity. In addition, ANRIL provides a powerful
system with which to further investigate the biological signifi-
cance of RNA-mediated targeting of polycomb. XCI may also
involve RNA scaffolding of both PRC1 and PRC2 (Bernstein
et al., 2006; Zhao et al., 2008).
Pericentric Heterochromatin: Alpha Satellite Repeat
In recent years, a role for ncRNA in heterochromatin establish-
ment has emerged. Heterochromatin at pericentric satellites,
characterized by a specific chromatin signature and chromo-
center organization, is of paramount importance for genome
function (Probst and Almouzni, 2011). Maison and colleagues
provide evidence for the presence of long nuclear noncoding
transcripts corresponding to major satellite repeats at the
periphery of pericentric heterochromatin and that major tran-
scripts of these lncRNAs in the forward orientation specifically
associate with small ubiquitin-like modifier (SUMO)-modified
heterochromatin protein 1 (HP1) proteins (Maison et al., 2011).
The forward RNA associates with and provides specificity to
the initial targeting of the SUMO-HP1 complex at pericentric
heterochromatin to seed further HP1 localization. Thus, the
lncRNA acts as a molecular scaffold for the targeting and local
accumulation of HP1. It would be interesting to investigate if
similar principles are exploited in biological situations where
major nuclear reorganization occurs.
Not surprisingly, several lncRNAs possess characteristics
from multiple archetypes that, in combination, are critical to
their eventual biological function. For example, COLDAIR and
COOLAIR are transcribed in response to the environmental
cue of cold temperature—their transcription serves as a signal
of a significant biological event, in this case the preparations
for competence to flower after a prolonged winter. Epigenetic
repression of floral repressors is then achieved through binding
of PRC2 by COLDAIR, with the lncRNA serving as a guide to
affect silencing at the FLC locus to bring about the biological
effect of vernalization. The lncRNA HOTTIP is another example
ofa ‘‘signalplusguide’’combination archetype—it istranscribed
in a temporal and spatial manner along with the rest of the distal
HOXA genes to convey positional identity and functions by
binding to and targeting the trithorax protein complex mixed
lineage in leukemia-1 (MLL-1) to the 50HOXA locus to drive
histone methylation and gene transcription.
Another combinatorial archetype is exemplified by HOTAIR.
Like HOTTIP, HOTAIR is transcribed in posterior and distal cells,
acting as a signal for anatomic specificity. By binding to both the
PRC2 and LSD1 complexes, HOTAIR serves as a modular scaf-
fold, and by targeting PRC2 to its proper genomic locations, it
acts as a guide. Thus, the desired biological outcome—posi-
tional identity and appropriate chromatin modifications leading
to proper gene expression—is ultimately achieved through a
functional multifunctional lncRNA.
One emerging theme from the analysis of the four lncRNA
archetypes is that of stepwise complexity. When one considers
each of the archetype classes from an evolutionary perspective,
it is a strikingly simple process of incremental modifications that
confer alterations in molecular utility. The simple signal arche-
type lncRNA, such as eRNA, merely requires the transcription
of a regulatory DNA element. If the lncRNA that is produced
also binds a protein due to the formation of an RNA motif
mimicking its DNA counterpart, as is the case for Gas5, then
the lncRNA develops into a molecular decoy. If the lncRNA
then gains the ability to target the bound effectors to a specific
DNA sequence either in cis or trans, it transitions to become
a guide. With nucleic acid duplication, fusion, and recombination
events, it is not farfetched to imagine that lncRNAs may subse-
quently acquire multiple effector binding sites to turn into a
scaffold. This stepwise scenario is potentially quite likely
because the regulatory DNA being transcribed, such as en-
hancers, by definition possess high-affinity transcription factor
binding sequences, often in tandem and combinatorial arrange-
ments. Thus, the primordial lncRNA ‘‘signals’’ may often contain
functional seeds to become decoys, guides, and scaffolds.
In fact, experimental evolution hints at the feasibility of
evolving many new lncRNA regulators of gene expression.
Work by Kehayova and Liu (2007) highlights the value of RNA
evolution and argues that the polyanionic characteristic, in
combination with the great structural and functional diversity of
RNA, makes it especially well suited to mediate processes that
involve proteins with cationic patches (Kehayova and Liu,
easy to evolve, on the order of 104, in contrast to in vitro selec-
tions for RNA aptamers for a specific ligand or protein, in which
the rate of active RNAs among random library members is
approximately 1 in 1010?1014(Kehayova and Liu, 2007).
Polymorphisms and mutations in regulatory regions are
increasingly shown to be associated with human disease.
However, currently, we are only observing the tip of the iceberg.
Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.
It is becoming clear that many common disease-association
studies are identifying noncoding region variants as the under-
lying cause of these later-onset disorders. It will be exciting
and potentially useful for disease management and treatment
to see what aspects of fine tuning are altered in different anom-
alies. Areas for future exploration will include the mechanisms
through which physiological and environmental changes are
translated into altered gene function through lncRNAs and their
regulatory networks. We hope that we have provided logic and
experimental evidence to support the archetypal classifications
of lncRNAs as a useful framework. As more examples of regula-
tion by lncRNA are uncovered, one might predict that the large
transcripts will eventually rival small RNAs and proteins in their
versatility as regulators of genetic information.
We apologize to colleagues whose work was not discussed due to space
constraints. We thank R. Flynn and O. Wapinski for critical reading of the
manuscript and assistance with the figure. This work is supported by NIH
and California Institute for Regenerative Medicine (H.Y.C.). H.Y.C. is an Early
Career Scientist of the Howard Hughes Medical Institute.
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