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Non-coding RNAs: Regulators of disease

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Journal of Pathology
J Pathol 2010; 220: 126–139
Published online 30 October 2009 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/path.2638

Invited Review

Non-coding RNAs: regulators of disease

Ryan J Taft,1 Ken C Pang,2 Timothy R Mercer,1 Marcel Dinger1 and John S Mattick1 *
1 Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia
2 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA

*Correspondence to: Abstract

John S Mattick, Institute for
Molecular Bioscience, University For 50 years the term ‘gene’ has been synonymous with regions of the genome encoding
of Queensland, Brisbane QLD mRNAs that are translated into protein. However, recent genome-wide studies have shown
4072, Australia. that the human genome is pervasively transcribed and produces many thousands of
E-mail: [email protected] regulatory non-protein-coding RNAs (ncRNAs), including microRNAs, small interfering
RNAs, PIWI-interacting RNAs and various classes of long ncRNAs. It is now clear that these
No conflicts of interest were
RNAs fulfil critical roles as transcriptional and post-transcriptional regulators and as guides
declared.
of chromatin-modifying complexes. Here we review the biology of ncRNAs, focusing on the
fundamental mechanisms by which ncRNAs facilitate normal development and physiology
and, when dysfunctional, underpin disease. We also discuss evidence that intergenic regions
associated with complex diseases express ncRNAs, as well as the potential use of ncRNAs as
diagnostic markers and therapeutic targets. Taken together, these observations emphasize
the need to move beyond the confines of protein-coding genes and highlight the fact
that continued investigation of ncRNA biogenesis and function will be necessary for a
comprehensive understanding of human disease.
Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John
Received: 14 September 2009 Wiley & Sons, Ltd.
Revised: 25 September 2009
Keywords: non-coding RNAs; small RNAs; microRNAs; RNA interference; genome-wide
Accepted: 26 September 2009
association study

Introduction to all complex genetic phenomena in the eukary-

otes, including transcriptional and post-transcriptional
Over the past decade there has been an explosion gene silencing [13–22], hybrid dysgenesis [15,23], X-
of large-scale genome sequencing, which has led to chromosome dosage compensation and allelic exclu-
both great insights and unexpected conundrums. Con- sion [24,25], germ cell reprogramming [26] and para-
trary to the original expectation that more complex mutation [27,28] — all of which involve epigenetic
organisms would have a greater number of genes, processes (see below).
it is now clear that humans and mice have approxi-
mately the same number of protein-coding genes as
the microscopic roundworm Caenorhabditis elegans, The expanding RNA world
most of which are orthologous, and that all multicel-
lular organisms sequenced to date have fewer protein- Small regulatory RNAs
coding genes than some simple unicellar eukaryotes
[1]. An explanation for this apparent paradox comes A fundamental and general role for regulatory RNA
from two unexpected findings: (a) that biological com- in eukaryotic biology was dramatically demonstrated
plexity generally correlates with the proportion of the in the late 1990s by the finding that double-stranded
genome that is non-protein-coding [1]; and (b) that, RNA introduced into C. elegans is cleaved by the
while only 2% of the mammalian genome encodes bidentate ribonuclease Dicer into ∼21 nucleotide
mRNAs, the vast majority is transcribed, largely as (nt) small RNAs that induce widespread and her-
long and short non-protein-coding RNAs (ncRNAs) itable gene silencing [29–31]. Although this phe-
[2–10]. These findings have directly challenged the nomenon, termed RNA interference (RNAi), was orig-
traditional view of RNA as simply an intermedi- inally thought to be restricted to exogenous dsRNAs,
ary between DNA and protein, and imply that the it soon became clear that plants and animals pro-
vast majority of the genome — long regarded as duce a dazzling array of endogenous small interfering
‘junk’ - encodes functional RNA species that orches- RNAs (siRNAs), microRNAs (miRNAs) and PIWI-
trate the development of complex organisms [11,12]. interacting RNAs (piRNAs) [14–17,32–37]. Recent
Indeed it appears that RNA signalling is central work has seen this repertoire increase even further

Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
www.pathsoc.org.uk
Non-coding RNAs: regulators of disease 127

Epigenetic Genome integrity / Transcriptional Post-transcriptional Viral defence

modifications transposon defence regulation regulation

endo-siRNAs

piRNAs miRNAs
PARs
(e.g. tiRNAs) antisense lncRNA snoRNAs
sdRNA

[repeat element] [overlapping 3′ UTRs]

Figure 1. Simplified representation of regulatory ncRNAs and their functions. Generalized gene models are presented in dark
grey and light orange and overlap the double-strand DNA structure (light grey), representative of the genome. Each class of
regulatory RNA is defined by a colour. Functions are ascribed to each class of ncRNA by colour below the text at the top of the
figure. Bars and arrows indicate the direction of transcription. For more detail please see the text and references therein. PARs,
promoter-associated RNAs; lncRNAs, long non-coding RNAs; miRNAs, microRNAs; snoRNAs, small nucleolar RNAs; sdRNAs,
sno-derived RNAs; endo-siRNAs, endogenous siRNAs; piRNAs, PIWI-interacting RNAs; tiRNAs, transcription initiation RNAs

to include a host of short transcripts that sit adja- non-coding transcripts (reviewed in [15,59]), which
cent to transcription start sites [38,39], including may suggest that other Dicer-independent small RNA
promoter-associated small RNAs (PASRs) [9,40] and species are still to be discovered.
transcription initiation RNAs (tiRNAs) [41], species A detailed examination of miRNA biogenesis and
that are derived from centromeres and telomeres function is beyond the scope of this work and has
[42,43], and tiny species processed from other short recently been covered in detail in several excellent
RNAs [44] (Figure 1, Table 1). Indeed, over the last reviews [16,17,60]. However, miRNA mechanisms of
decade we have witnessed a near-exponential growth action, and the autoregulatory feedback loops that
of manuscripts devoted to regulatory RNAs (Figure 2). increasingly characterize small RNA biogenesis, are
Of the classes identified to date, miRNAs, siR- well illustrated by one of the first miRNAs discov-
NAs and piRNAs, which guide effector Argonaute ered, let-7 (Figure 3). The let-7 family of miRNAs
proteins to genomic loci or target RNAs in a sequence- is highly conserved throughout the Metazoa and func-
specific manner, have been most thoroughly inves- tions as a master temporal regulator of development
tigated. In humans there are at least 700 miRNAs, and differentiation, both in early embryos and com-
hundreds of siRNAs and millions of unique piRNA plex adult tissues such as brain, in nematode, fruitfly,
sequences [15–17,45], suggesting that small RNAs zebrafish and mouse [61–67]. Indeed, let-7 targets
are a substantial portion of the RNA output of cells well-established cell-cycle regulators, including Cdk6
and that they comprise a diverse, widespread and and Ras [68,69]. Like the majority of miRNAs, the let-
basal regulatory system. Indeed, several recent studies 7 precursor hairpin, or pre-miRNA, is processed from
have shown that miRNAs and piRNAs are detectable a long RNA polymerase II transcript by the nuclear
in the most primitive multicellular organisms [46] RNase Drosha, which is then exported to the cytosol
and that once acquired, they are seldom, if ever, and processed to a ∼22 nt mature miRNA by Dicer
lost [47–49]. Although exogenous siRNAs were dis- [70].
covered a decade ago, endogenous siRNAs (endo- Each of these steps of let-7 biogenesis is tightly
siRNAs) have only recently been identified in fruit- regulated (Figure 3a). For example, while differentia-
flies and mammals [50–58], where their biogene- tion factors such as Notch [71] induce transcription,
sis is dependent on Dicer processing of duplexes pluripotency factors (i.e. those that support an undif-
formed by overlapping transcripts or long perfect ferentiated cellular state), such as c-Myc, repress tran-
hairpin structures. Work from other animals, includ- scriptional activation [72–74]. Likewise, the pluripo-
ing nematode and fruitfly, yeast and plants, indi- tency factor LIN28 can bind to the conserved loop
cates that that these endo-siRNAs are involved in of the primary let-7 transcript (pri-let-7) to directly
anti-viral defence, transposon silencing, chromatin inhibit the Drosha cleavage steps [75–77] and can
remodelling and post-transcriptional gene regulation inhibit Dicer cleavage directly or by facilitating pre-
through Argonaute-mediated cleavage of target tran- miRNA degradation [78,79]. Completing the feed-
scripts (reviewed in [16,17]). The longest small RNA back loop, let-7 targets LIN28 [75,80,81], c-Myc
class, piRNAs, which are ∼25–30 nt in length, are [82,83] and the c-Myc-activating gene IMP-1 [80].
also largely derived from, and involved in, trans- let-7 also forms a separate overlapping loop with the
poson defence (reviewed in [15,19]) but are largely TRIM–NHL family of proteins that negatively reg-
restricted to the germline, where active transposons ulate c-Myc and enhance let-7 activity [66,84–86].
could severely disrupt embryogenesis. Intriguingly, in These let-7 targets are ‘canonical’, i.e. the miRNA
a departure from the Dicer biogenesis pathway that ‘seed’ sequence (nucleotides 2–8) binds to a target
defines siRNAs and miRNAs, piRNAs are produced mRNA 3 UTR and (generally) represses translation
by successive waves of Argonaute-cleavage of long (Figure 3b).

J Pathol 2010; 220: 126–139 DOI: 10.1002/path

Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
128 RJ Taft et al

Table 1. Classes of non-coding RNA in mammals

NcRNA class Characteristics References

Established ncRNA classes

Long (regulatory) non-coding The broadest class, lncRNAs, encompass all non-protein-coding RNA Reviewed in [97,98]
RNAs (lncRNAs) species >∼200 nt, including mRNA-like ncRNAs. Their functions include
epigenetic regulation, acting as sequence-specific tethers for protein
complexes and specifying subcellular compartments or localization
Small interfering RNAs (siRNAs) Small RNAs ∼21–22 nt long, produced by Dicer cleavage of Reviewed in [15–17,227]
complementary dsRNA duplexes. siRNAs form complexes with
Argonaute proteins and are involved in gene regulation, transposon
control and viral defence
microRNAs (miRNAs) Small RNAs ∼22 nt long, produced by Dicer cleavage of imperfect RNA Reviewed in [16,17,70]
hairpins encoded in long primary transcripts or short introns. They
associate with Argonaute proteins and are primarily involved in
post-transcriptional gene regulation
PIWI-interacting RNAs (piRNAs) Dicer-independent small RNAs ∼26–30 nt long, principally restricted to Reviewed in [15,17]
the germline and somatic cells bordering the germline. They associate with
PIWI-clade Argonaute proteins and regulate transposon activity and
chromatin state
Promoter-associated RNAs A general term encompassing a suite of long and short RNAs, including Reviewed in [38,39]
(PARs) promoter-associated RNAs (PASRs) and transcription initiation RNAs
(tiRNAs) that overlap promoters and TSSs. These transcripts may regulate
gene expression
Small nucleolar RNAs Traditionally viewed as guides of rRNA methylation and Reviewed in [228]
(snoRNAs) pseudouridylation. However, there is emerging evidence that they also
have gene-regulatory roles
Other recently described classes
X-inactivation RNAs (xiRNAs) Dicer-dependent small RNAs processed from duplexes of two lncRNAs, 131
Xist and Tsix, which are responsible for X-chromosome inactivation in
placental mammals
Sno-derived RNAs (sdRNAs) Small RNAs, some of which are Dicer-dependent, which are processed 229–231
from small nucleolar RNAs (snoRNAs). Some sdRNAs have been shown
to function as miRNA-like regulators of translation
microRNA-offset RNAs Small RNAs ∼20 nt long, derived from the regions adjacent to 232,233
(moRNAs) pre-miRNAs. Their function is unknown
tRNA-derived RNAs tRNAs can be processed into small RNA species by a conserved RNase Reviewed in [44]
(angiogenin). They are able to induce translational repression
MSY2-associated RNAs MSY-RNAs are associated with the germ cell-specific DNA/RNA binding 234
(MSY-RNAs) protein MSY2. Like piRNAs, they are largely restricted to the germline and
are ∼26–30 nt long. Their function is unknown
Telomere small RNAs Dicer-independent ∼24 nt RNAs principally derived from the G-rich 43
(tel-sRNAs) strand of telomeric repeats. May have a role in telomere maintenance
Centrosome-associated RNAs A class of ∼34–42 nt small RNAs, derived from centrosomes that show 42
(crasiRNAs) evidence of guiding local chromatin modifications

However, let-7 also targets the Dicer coding [2–10] and that at least 80% of this transcription is
sequence (CDS) [87] (Figure 3b), consistent with exclusively associated with long non-coding RNAs
what appears to be an emerging theme of non- (lncRNAs) [9]. Although lncRNAs have frequently
canonical miRNA targets in developmentally regulated been disregarded as artifacts of chromatin remod-
genes [88–92]. Additionally, let-7 was also recently elling or transcriptional ‘noise’ [94,95], there is sub-
shown to regulate HMGA2, an oncofetal gene and stantial evidence to suggest that they mirror protein-
pluripotency factor, in a cell cycle-dependent manner. coding genes. Indeed, they are frequently long (gen-
HMGA2 translation is up-regulated upon cell cycle erally >2 and some >100 kb) [96], spliced and con-
arrest but inhibited in proliferating cells [93]. Taken tain canonical polyadenylation signals [97,98]. Addi-
as an example, let-7 provides a compelling illustration tionally, lncRNA promoters are bound and regulated
of the complexity of small RNA biogenesis and func- by transcriptional factors, including Oct3/4, Nanog,
tion, and points more generally towards small RNAs CREB, Sp1, c-myc, Sox2, NF-κB and p53 [99–102]
having a wide range of regulatory functions facilitated and epigenetically marked with specific histone mod-
by sequence-specific interactions, any of which may ifications [102,103]. Overall, there are at least tens of
malfunction to cause disease.
thousands of lncRNAs that show signatures of selec-
tion — many of which, like small RNAs, are tissue
Long non-coding RNAs and developmental stage-specific [97,104–108].
Genome-wide transcriptomic studies have now shown Long ncRNAs have a variety of functions, but one
that the mammalian genome is abundantly transcribed of their primary roles appears to be as epigenetic

J Pathol 2010; 220: 126–139 DOI: 10.1002/path

Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Non-coding RNAs: regulators of disease 129

tumour suppressor p53 mRNA, in addition to being

translated, also functions as an RNA to inhibit the
ubiquitin ligase activity of Mdm2 [119]. Indeed, there
is increasing evidence to suggest that mRNAs contain
extensive RNA structural features [120,121], raising
the possibility that, in addition to their protein-coding
functions, mRNAs may intrinsically act as regulatory
RNAs [122].
Long ncRNAs have also been implicated in
organelle biogenesis and subcellular trafficking. For
instance, the NRON lncRNA is involved in the
cytoplasmic-to-nuclear shuttling of the NFAT tran-
scription factor [123], and the lncRNA NEAT1 /MEN
ε/β is required for the formation of the nuclear sub-
compartment paraspeckles in differentiated cells and
regulates the movement of primate-specific mRNAs
containing inverted Alu repeats [124–127]. Another
lncRNA, Gomafu, is specifically localized in a novel
nuclear domain in a subset of neurons [128].
Although long and short regulatory RNAs are typ-
Figure 2. The rise in the number of non-coding RNA publi- ically studied and classified separately, it is impor-
cations per year. The number of PubMed indexed publications tant to note that they frequently overlap both phys-
with title, abstract or keywords matching each class of ncRNA
is plotted. Records were retrieved using an in-house Perl script ically and functionally. Indeed, the dynamic inter-
and the PubMed eFetch utility. The search terms used were: play between lncRNAs and small RNAs is evi-
piRNA, PIWI RNA, PIWI-associated RNA, PIWI-interacting dent during the process of X-chromosome inac-
RNA, siRNA, small interfering RNA, endo-siRNA, endogenous tivation (XCI), which occurs in female mammals
siRNA, miRNA, microRNA, non-coding RNA, non-coding RNA,
non-protein-coding RNA, ncRNA and lincRNA. Note that 2009
to ensure dosage compensation for X-linked genes
data were only gathered through August 2009 between the sexes. The antisense lncRNAs Xist and
Tsix are not only responsible for the chromatin
modifications that maintain XCI [24,129,130], but
regulators of protein-coding gene expression [109]. also form dsRNA duplexes in vivo that are pro-
For example, Hox genes are associated with hundreds cessed by Dicer into ∼25–42 nt X-inactivation RNAs
of lncRNAs that define domains of differential his- (xiRNAs) [131]. Given the abundance of mam-
tone methylation and RNA polymerase accessibility malian antisense transcripts with the potential to
along the spatial and temporal axes of human devel- form dsRNA structures [132,133], such relationships
opment [110]. A lncRNA transcribed from the HOXC between lncRNAs and small RNAs may be common-
locus, termed HOTAIR, regulates the chromatin methy- place.
lation state of the HOXD locus in trans through the
polycomb repressive complex PRC2 [110]. Addition-
ally, a recent study has shown that more than 20%
of long intergenic ncRNAs associate with chromatin- Non-coding RNAs in disease
modifying complexes [111], with evidence that other
lncRNAs operate to activate gene expression through Small regulatory RNAs
Trithorax-group complexes [108]. Long ncRNAs are
also frequently associated with the phenomenon of Small RNAs have roles in virtually all develop-
genomic imprinting, which ensures that one of the two mental processes, including stem cell and germline
parental alleles of certain autosomal genes are epige- maintenance, development and differentiation, tran-
netically silenced [112–116]. scriptional and post-transcriptional gene silencing and
Long ncRNAs can also directly modulate gene subcellular localization (see above, and reviewed in
transcription and protein degradation. For example, [14–17,20,21]). Not surprisingly, therefore, their dis-
the lncRNA Evf2 activates the homeobox transcrip- ruption has been linked to human disease. For exam-
tion factor Dlx2 and recruits it to an ultraconserved ple, miRNAs are aberrantly expressed in: liver, pan-
genomic element, which induces transcription of Dlx5 creatic, oesophageal, stomach, colon, haematopoietic,
[117]. Mutant mice lacking Evf2 show reduced num- ovarian, breast, pituitary, prostate, thyroid, testicular
bers of GABAergic interneurons early in development and brain cancers [134–138]; central nervous system
and reduced synaptic inhibition in adulthood [118], disorders (e.g. schizophrenia and Alzheimer’s disease)
suggesting that lncRNA-dependent processes are fun- [139]; and cardiovascular disease [140,141]. MiRNAs
damental to the central nervous system. Indeed, a are also enriched at fragile sites in the human genome
large fraction of lncRNAs is expressed in very precise and are associated with oncogenic viral integration
patterns in the brain [106]. Similarly, the archetypal sites [142,143].

J Pathol 2010; 220: 126–139 DOI: 10.1002/path

Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
130 RJ Taft et al

Figure 3. let-7 provides a window into miRNA biogenesis and function. (a) let-7 biogenesis and gene regulation is characterized by
a series of autoregulatory feedback loops. Lines ending in bars indicate inhibitory interactions, while those terminating in arrows
indicate activating interactions. For simplicity, all let-7 family members (of which there are 11 in vertebrates) are considered as a
group. Likewise, mammalian LIN28 homologues (LIN28 and LIN28B) and TRIM–NHL family members (TRIM71 and TRIM32) are
depicted as single elements within the schematic. Mature let-7 mediates its effects through a complex composed of an Argonaute
protein (grey) and GW182 (brown), which is also depicted in simplified form in the lower panel. Consistent with its expression in
late embryogenesis, the principal targets of let-7 are cell cycle regulators, oncofetal genes, pluripotency factors and components
of the miRNA biogenesis pathway. Please see the text for more detail and references for each depicted interaction. (b) A general
schematic of mRNA transcription and miRNA targeting. Canonical miRNA targets (blue) are dependent on base pairing between
nucleotides 2–8, the seed sequence, and the mRNA 3 UTR. Due to the short length of the seed sequence, legitimate interactions
can be abolished and illegitimate targets created by single base changes. Non-canonical targets (orange), e.g. those in coding
sequences (CDSs) or 5 UTRs, are not reliant on the ‘seed’ sequence and generally show more extensive base pairing. Canonical
and non-canonical targets are depicted for let-7a:HMGA2 [235] and let-7b:Dicer [87], respectively

Similarly, loss of specific small RNA loci is asso- study has shown that a single microdeletion involv-
ciated with Prader–Willi syndrome (PWS), a disor- ing several small nucleolar RNA clusters (HBII-85
der caused by the loss of imprinting on chromo- and HBII-52) results in PWS, suggesting that loss
some 15q11-q13 and characterized by hyperphagia, of small RNAs is a causal determinant of the dis-
hypogonadism and cognitive impairment. A recent ease [144]. Consistent with this hypothesis, knockout

J Pathol 2010; 220: 126–139 DOI: 10.1002/path

Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Non-coding RNAs: regulators of disease 131

mice lacking the relevant snoRNAs largely recapitu- LIN28 and LIN28B, negative regulators of let-7 bio-
late the PWS phenotype [145]. Interestingly, HBII-52 genesis, correlates with repression of let-7, occurs in
forms an antisense duplex with the serotonin recep- at least 15% of human malignancies and is associ-
tor 2C (5HT2C ) mRNA and negatively regulates its ated with more advanced disease states [165]. Simi-
post-transcriptional editing [146,147], strongly impli- larly, and consistent with let-7 ’s role in developmental
cating it in PWS-associated and autistic neurological regulation, genetic variants of the LIN28 locus have
defects [148]. Taken together, these studies suggest recently been associated with altered timing of human
that the loss of small RNA loci plays an important pubertal growth and development [166].
role in human illness. Single nucleotide polymorphisms (SNPs) in mature
Like protein-coding genes, small RNAs can func- and precursor miRNAs have been robustly associ-
tion either as activators or inhibitors of disease. Con- ated with schizophrenia and autism [167,168], and
sistent with its role as a differentiation factor, let-7 a pathogenic SNP in the seed sequence of miR-
is a well-established tumour suppressor [61,149–151] 96 is responsible for progressive hearing loss [169]
whose reduced expression is associated with poor sur- (Figure 3b). A SNP in the 3 UTR of K-Ras, a
vival in human lung cancers [152]. Likewise, mir-29b well-characterized GTPase-regulated oncogene and
expression is associated with disease-free survival in target of let-7, inhibits let-7 translational suppres-
patients with ovarian serous carcinoma [153], poten- sion and results in reduced survival in oral cancers
tially due to regulation of the de novo methyl trans- [170]. Indeed, SNPs in the 3 UTRs of mRNAs
ferases Dnmt3a and Dnmt3b [154]. Indeed, altered that abolish or create target sites may be common
expression of a broad suite of miRNAs that, dependent in miRNA-associated diseases (reviewed in [171]).
on their targets can either act as tumour suppressors As examples, SNP-induced illegitimate miRNA bind-
or oncogenes (so-called oncomiRs), has been detected ing sites are associated with muscular hypertrophy in
in virtually all cancer types examined (for reviews sheep, Tourette’s syndrome and cardiovascular disease
and tables of cancer-associated miRNAs and their [171–173]. More generally, allele-specific polymor-
targets, see [134,151,155,156]). Similar relationships phisms in miRNA target sites have been shown to play
are apparent in cardiovascular illnesses [140,141]. a role in the tissue-specific miRNA regulation of hun-
For example, miR-92a controls functional recovery dreds of genes, suggesting that such genetic subtleties
of ischaemic tissues in mice [157], and miR-145 and may be a widespread underlying cause of individual
miR-143, which have recently been implicated in dif- phenotypic variability [174].
ferentiation of progenitors into cardiac myocytes, are
down-regulated in injured and atherosclerotic vessels Long non-coding RNAs
[158]. MicroRNAs may even play a direct role in viral
defence. A study of human T lymphocytes has shown The data gathered to date strongly implicate lncRNAs
that miR-29a targets the HIV-1 3 UTR and directs it to in the basal regulation of protein-coding genes, includ-
P bodies, where it is suppressed by the RNA-induced ing those central to normal development and oncoge-
silencing complex (RISC) [159]. nesis, at both the transcriptional (e.g. epigenetic) and
Small RNA dysregulation occurs for multiple rea- post-transcriptional (e.g. subcellular dynamics) levels,
sons and reflects the processes involved in their and an increasing number have been functionally val-
biogenesis, regulation and targeting. MicroRNA loci idated to affect different cellular and developmental
and individual components of the miRNA biogen- pathways (see [107]). It is not surprising, then, that
esis pathway are frequently lost or amplified in a the dysregulation of lncRNAs appears to be a primary
wide range of cancers [160,161], and there is now feature of many complex human diseases, including
widespread evidence that miRNAs that act as differ- leukaemia [175], colon cancer [176], prostate cancer
entiation factors (e.g. let-7, above) are globally down- [177], breast cancer [178], hepatocellular carcinoma
regulated in cancers [134,150,151]. Indeed, ovarian [175,179], psoriasis [180], ischaemic heart disease
cancer patients who show decreased expression of [181,182], Alzheimer’s disease [183] and spinocere-
Dicer and Drosha, the RNases involved in miRNA bellar ataxia type 8 [184].
production (see above), are associated with poor prog- In some cases, the mechanisms by which lncRNAs
noses, suboptimal surgical cytoreduction and advanced contribute to disease have been carefully dissected. For
tumour stages [162]. Likewise, a mutation resulting in example, the dsDNA-binding protein PSF constitu-
premature termination of DICER1 results in pleuropul- tively silences the proto-oncogene GAGE6. However,
monary blastoma, a rare paediatric lung tumour [163]. at least five lncRNAs can bind to PSF, which results
Consistent with these findings, studies in mice have in deactivation of PSF-induced silencing, expression
shown that mammalian systems are highly sensitive of GAGE6 and enhanced tumorigenicity [185]. Long
to Dicer activity. Complete loss of Dicer results in the ncRNAs overlapping or antisense to protein-coding
disruption of the developmental programme and early gene promoters may also contribute to oncogenesis.
embryonic lethality [164]. A transcript antisense to the p15 tumour suppressor
Elements associated with the regulation of miRNA gene, first identified in a human leukaemia, regu-
processing can also be associated with various patholo- lates the chromatin and DNA methylation status of
gies. A recent study has shown that over-expression of the p15 locus [186]. A lncRNA antisense to p21 was

J Pathol 2010; 220: 126–139 DOI: 10.1002/path

Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
132 RJ Taft et al

also recently shown to behave similarly [187]. These function, PISRT1, which has also been identified as a
results, combined with the observation that antisense candidate gene in a goat model of this disease [193].
transcripts are present at thousands of protein-coding The potential role of lncRNAs in long-range
genes, have led to speculation that antisense lncR- enhancer function, and therefore dysfunction, is illus-
NAs generally control the expression of their cog- trated by the Evf2 lncRNA (see above), which
nate protein-coding genes through epigenetic modifi- may contribute to split-hand/split-foot malformation
cations [188,189]. This model has profound ramifi- 1 (SHFM1). Although the region associated with
cations for our understanding of disease, particularly this developmental disorder encompasses three genes,
cancer — dysregulation of a lncRNA regulating the DLX5, DLX6 and DSS1, none are directly mutated in
expression of a tumour suppressor or oncogene, and patients [194]. Instead, exhibition of the limb pheno-
not the protein-coding sequence itself, may be one of type requires the expression of both the Dlx5 and Dlx6
the ‘hits’ that leads to oncogenesis. genes to be disrupted, suggesting that SHMF1 results
from the ablation of a shared regulatory element [195].
Since it is now known that Evf2 regulates the expres-
The hidden layer of non-coding variation sion of both these genes, it warrants investigation as a
candidate SHFM1 disease locus.
These examples likely represent the tip of a very big Similarly, two lncRNAs, SOX2OT and SOX2DOT,
iceberg. The same technologies that have revealed a exhibit enriched expression in the lens of eye and over-
breadth of ncRNA expression are also driving a revo- lap a known myopia susceptibility locus (Figure 4)
lution in genome sequencing that will ultimately iden- [196,197]. These transcripts, one of which is tran-
tify variations in the human genome that underpin dis- scribed from a distal ultraconserved enhancer, also
ease susceptibility and aetiology. However, given the overlaps the SOX2 gene, itself an important regula-
focus on mutations in protein-coding exons that cause tor of ocular development. Given that developmental
most of the high-penetrance simple genetic disorders, genes are significantly enriched for a proximal asso-
the variation that occurs in non-protein-coding regions ciation with lncRNAs, it may well be that the under-
of the genome has, to date, largely been ignored or at standing of developmental disorders is likely to benefit
least not been considered [107,190]. This is changing: from an appreciation of lncRNA biology. The future
the emergence of genome-wide association studies to convergence of lncRNA identification by deep RNA
identify variant loci affecting complex diseases and sequencing with the increased resolution of disease
traits and an increased awareness of ncRNA biology variants afforded by genomic sequencing will, we sug-
have prompted a reconsideration of the underlying gest, prove a potent combination in elucidating the
protein-centric assumptions and provided a number functional contribution of ncRNAs to disease.
of novel insights into disease-causing mechanisms.
For example, many pathological mechanisms are now
known to involve aberrant regulation (and in many Non-coding RNAs as diagnostics
cases ncRNAs) rather than alterations to the protein- and therapeutics
coding sequences themselves. This is perhaps not sur-
prising, given that the primary engine of phenotypic The growing body of research showing that ncRNAs
radiation and higher complexity has been the expan- may be primary genetic regulators in complex animals
sion and divergence of the regulatory architecture that has led to the corresponding realization that this may
controls the deployment of protein components during make them ideal diagnostic markers. For example, in
differentiation and development [191], much of which some cases the expression profiles of miRNAs, in con-
may be embedded within ncRNAs [12,105]. Indeed, trast to those of protein-coding mRNAs, are able to
the same forces that drive evolutionary innovations accurately identify the origin of poorly differentiated
can result in deleterious variations. tumours and carcinomas [198,199]. Indeed, a signa-
Genome-wide association studies are beginning to ture of as few as 200 miRNAs may be sufficient for
identify novel ncRNAs as candidate disease-associated cancer classification [198], and it appears that some of
genes. For example, the lncRNA MIAT is associated the difficulties of early detection associated with colon
with myocardial infarction [181], and a novel lncRNA and other occult cancers may be overcome by pro-
induced by a chromosomal deletion that truncates the filing miRNAs obtained from patient serum, plasma,
polyadenylation site of the LUC7L gene [192] results saliva and tissues [200,201]. Likewise colon, lung and
in aberrant methylation and silencing of the neighbour- breast cancer prognosis is strongly associated with a
ing HBA2 gene, leading to the onset of α-thalassemia. small suite of miRNAs (reviewed in [202]), suggesting
Indeed, many disease variants map far from protein- that assays designed to query ncRNAs may eventually
coding genes and, given the level of genome-wide become core components of the pathologist’s toolkit.
transcription, are therefore likely to interrupt lncRNAs. This will undoubtedly be facilitated by recent advance-
For example, a disease-causing 7.4 kb deletion asso- ments in massively parallel sequencing technologies
ciated with blepharophimosis syndrome occurs over [203,204], which allow rapid and sensitive profiling of
250 kb upstream from the nearest gene, FOXL2 [193], both long and short ncRNAs, and will almost certainly
and this mutation interrupts a lncRNA of unknown make personal genomics a reality in the next 5 years

J Pathol 2010; 220: 126–139 DOI: 10.1002/path

Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Non-coding RNAs: regulators of disease 133

Figure 4. Long non-coding RNAs, SOX2 distal overlapping transcript (SOX2DOT) and SOX2 overlapping transcript (SOX2OT)
map to the myopia susceptibility locus. A representation of these features, the SOX2 gene, and a highly conserved SOX2DOT
enhancer [236] are shown in the inset schematic representative of a region of the human genome (chr3: 182, 255, 415–182, 945,
055; UCSC Genome Browser hg18). The relative expression of spliced ESTs corresponding to SOX2DOT or SOX2OT is depicted
in the lower half of the figure and shows that these transcripts are highly expressed in the lens of the eye. Adapted from Amaral
et al. [196]

[205]. The analysis and integration of this informa- liposome and nanoparticle delivery systems, and there
tion with other datasets (e.g. protein interaction and are currently multiple ongoing clinical trials targeting
genome-wide association studies) will pose a consid- age-related macular degeneration, respiratory syncytial
erable, but tractable, challenge well into the future. virus, acute renal failure, hepatocellular carcinoma and
The link between endogenous ncRNAs and dis- congenital pachyonychia, among others (reviewed in
ease, and the perfection of RNAi-based techniques to [212]).
silence genes in simple animals, has led to specula- There is also an increasing interest in RNA ther-
tion that RNA molecules can be employed as ther- apeutics that mimic or regulate miRNA activity in
apeutic agents. Indeed, it may be both easier and human cancers (reviewed in [213]). This could be
more productive to adjust the regulatory software facilitated by exogenous expression of a repressed
(i.e. ncRNAs) than to try and correct the hardware miRNA (using the same delivery systems as siRNA
(i.e. protein-coding genes). Hopes for RNA-based and therapeutics), by the introduction of antagomirs [214]
RNA-targeted therapies were bolstered by early suc- that are complementary and bind to miRNAs, or
cesses using siRNAs in human in vitro culture systems the use of ‘sponges’ that contain multiple artificial
[206] and in targeting HIV-1 and human BCL2 with miRNA-binding sites [215]. Artificial expression of
siRNA-like molecules [207–209]. Like gene therapy, specific miRNAs in vivo may be a powerful therapeu-
however, RNA therapeutics face considerable hur- tic mechanism, particularly given recent reports that
dles, including development of reliable delivery sys- over-expression of a single miRNA, miR-302, is capa-
tems, dosage regimes and techniques to ameliorate off- ble of inducing stemness [216,217].
target effects [210,211]. Nonetheless, multiple modes A series of recent studies has suggested that an
of administration have been developed, including viral, equally fruitful target may be gene promoters. Indeed,

J Pathol 2010; 220: 126–139 DOI: 10.1002/path

Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
134 RJ Taft et al

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