Journal of Pathology

J Pathol 2010; 220: 126139

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
of Queensland, Brisbane QLD
4072, Australia.
E-mail: j.mattick@imb.uq.edu.au
No conflicts of interest were
declared.
Received: 14 September 2009
Revised: 25 September 2009
Accepted: 26 September 2009
For 50 years the term gene has been synonymous with regions of the genome encoding
mRNAs that are translated into protein. However, recent genome-wide studies have shown
that the human genome is pervasively transcribed and produces many thousands of
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
RNAs fulfil critical roles as transcriptional and post-transcriptional regulators and as guides
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
Wiley & Sons, Ltd.
Keywords: non-coding RNAs; small RNAs; microRNAs; RNA interference; genome-wide
association study

Introduction to all complex genetic phenomena in the eukary-

Over the past decade there has been an explosion
of large-scale genome sequencing, which has led to
both great insights and unexpected conundrums. Con-
trary to the original expectation that more complex
organisms would have a greater number of genes,
it is now clear that humans and mice have approxi-
mately the same number of protein-coding genes as
the microscopic roundworm Caenorhabditis elegans,
most of which are orthologous, and that all multicel-
lular organisms sequenced to date have fewer protein-
coding genes than some simple unicellar eukaryotes
[1]. An explanation for this apparent paradox comes
from two unexpected findings: (a) that biological com-
plexity generally correlates with the proportion of the
genome that is non-protein-coding [1]; and (b) that,
while only 2% of the mammalian genome encodes
mRNAs, the vast majority is transcribed, largely as
long and short non-protein-coding RNAs (ncRNAs)
[210]. These findings have directly challenged the
traditional view of RNA as simply an intermedi-
ary between DNA and protein, and imply that the
vast majority of the genome long regarded as
junk - encodes functional RNA species that orches-
trate the development of complex organisms [11,12].
Indeed it appears that RNA signalling is central
otes, including transcriptional and post-transcriptional
gene silencing [1322], hybrid dysgenesis [15,23], X-
chromosome dosage compensation and allelic exclu-
sion [24,25], germ cell reprogramming [26] and para-
mutation [27,28] all of which involve epigenetic
processes (see below).
The expanding RNA world
Small regulatory RNAs
A fundamental and general role for regulatory RNA
in eukaryotic biology was dramatically demonstrated
in the late 1990s by the finding that double-stranded
RNA introduced into C. elegans is cleaved by the
bidentate ribonuclease Dicer into 21 nucleotide
(nt) small RNAs that induce widespread and her-
itable gene silencing [2931]. Although this phe-
nomenon, termed RNA interference (RNAi), was orig-
inally thought to be restricted to exogenous dsRNAs,
it soon became clear that plants and animals pro-
duce a dazzling array of endogenous small interfering
RNAs (siRNAs), microRNAs (miRNAs) and PIWI-
interacting RNAs (piRNAs) [1417,3237]. Recent
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-
cent to transcription start sites [38,39], including
promoter-associated small RNAs (PASRs) [9,40] and
transcription initiation RNAs (tiRNAs) [41], species
that are derived from centromeres and telomeres
[42,43], and tiny species processed from other short
RNAs [44] (Figure 1, Table 1). Indeed, over the last
decade we have witnessed a near-exponential growth
of manuscripts devoted to regulatory RNAs (Figure 2).
Of the classes identified to date, miRNAs, siR-
NAs and piRNAs, which guide effector Argonaute
proteins to genomic loci or target RNAs in a sequence-
specific manner, have been most thoroughly inves-
tigated. In humans there are at least 700 miRNAs,
hundreds of siRNAs and millions of unique piRNA
sequences [1517,45], suggesting that small RNAs
are a substantial portion of the RNA output of cells
and that they comprise a diverse, widespread and
basal regulatory system. Indeed, several recent studies
have shown that miRNAs and piRNAs are detectable
in the most primitive multicellular organisms [46]
and that once acquired, they are seldom, if ever,
lost [4749]. Although exogenous siRNAs were dis-
covered a decade ago, endogenous siRNAs (endo-
siRNAs) have only recently been identified in fruit-
flies and mammals [5058], where their biogene-
sis is dependent on Dicer processing of duplexes
formed by overlapping transcripts or long perfect
hairpin structures. Work from other animals, includ-
ing nematode and fruitfly, yeast and plants, indi-
cates that that these endo-siRNAs are involved in
anti-viral defence, transposon silencing, chromatin
remodelling and post-transcriptional gene regulation
through Argonaute-mediated cleavage of target tran-
scripts (reviewed in [16,17]). The longest small RNA
class, piRNAs, which are 2530 nt in length, are
also largely derived from, and involved in, trans-
poson defence (reviewed in [15,19]) but are largely
restricted to the germline, where active transposons
could severely disrupt embryogenesis. Intriguingly, in
a departure from the Dicer biogenesis pathway that
defines siRNAs and miRNAs, piRNAs are produced
by successive waves of Argonaute-cleavage of long
non-coding transcripts (reviewed in [15,59]), which
may suggest that other Dicer-independent small RNA
species are still to be discovered.
A detailed examination of miRNA biogenesis and
function is beyond the scope of this work and has
recently been covered in detail in several excellent
reviews [16,17,60]. However, miRNA mechanisms of
action, and the autoregulatory feedback loops that
increasingly characterize small RNA biogenesis, are
well illustrated by one of the first miRNAs discov-
ered, let-7 (Figure 3). The let-7 family of miRNAs
is highly conserved throughout the Metazoa and func-
tions as a master temporal regulator of development
and differentiation, both in early embryos and com-
plex adult tissues such as brain, in nematode, fruitfly,
zebrafish and mouse [6167]. Indeed, let-7 targets
well-established cell-cycle regulators, including Cdk6
and Ras [68,69]. Like the majority of miRNAs, the let-
7 precursor hairpin, or pre-miRNA, is processed from
a long RNA polymerase II transcript by the nuclear
RNase Drosha, which is then exported to the cytosol
and processed to a 22 nt mature miRNA by Dicer
[70].
Each of these steps of let-7 biogenesis is tightly
regulated (Figure 3a). For example, while differentia-
tion factors such as Notch [71] induce transcription,
pluripotency factors (i.e. those that support an undif-
ferentiated cellular state), such as c-Myc, repress tran-
scriptional activation [7274]. Likewise, the pluripo-
tency factor LIN28 can bind to the conserved loop
of the primary let-7 transcript (pri-let-7) to directly
inhibit the Drosha cleavage steps [7577] and can
inhibit Dicer cleavage directly or by facilitating pre-
miRNA degradation [78,79]. Completing the feed-
back loop, let-7 targets LIN28 [75,80,81], c-Myc
[82,83] and the c-Myc-activating gene IMP-1 [80].
let-7 also forms a separate overlapping loop with the
TRIMNHL family of proteins that negatively reg-
ulate c-Myc and enhance let-7 activity [66,8486].
These let-7 targets are canonical, i.e. the miRNA
seed sequence (nucleotides 28) binds to a target
mRNA 3 UTR and (generally) represses translation
(Figure 3b).

J Pathol 2010; 220: 126139 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
RNAs (lncRNAs)
Small interfering RNAs (siRNAs)
microRNAs (miRNAs)
PIWI-interacting RNAs (piRNAs)
Promoter-associated RNAs
(PARs)
Small nucleolar RNAs
(snoRNAs)
Other recently described classes
X-inactivation RNAs (xiRNAs)
Sno-derived RNAs (sdRNAs)
microRNA-offset RNAs
(moRNAs)
tRNA-derived RNAs
MSY2-associated RNAs
(MSY-RNAs)
Telomere small RNAs
(tel-sRNAs)
Centrosome-associated RNAs
(crasiRNAs)
The broadest class, lncRNAs, encompass all non-protein-coding RNA Reviewed in [97,98]
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 RNAs 2122 nt long, produced by Dicer cleavage of Reviewed in [1517,227]
complementary dsRNA duplexes. siRNAs form complexes with
Argonaute proteins and are involved in gene regulation, transposon
control and viral defence
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
Dicer-independent small RNAs 2630 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
A general term encompassing a suite of long and short RNAs, including Reviewed in [38,39]
promoter-associated RNAs (PASRs) and transcription initiation RNAs
(tiRNAs) that overlap promoters and TSSs. These transcripts may regulate
gene expression
Traditionally viewed as guides of rRNA methylation and Reviewed in [228]
pseudouridylation. However, there is emerging evidence that they also
have gene-regulatory roles
Dicer-dependent small RNAs processed from duplexes of two lncRNAs, 131
Xist and Tsix, which are responsible for X-chromosome inactivation in
placental mammals
Small RNAs, some of which are Dicer-dependent, which are processed 229231
from small nucleolar RNAs (snoRNAs). Some sdRNAs have been shown
to function as miRNA-like regulators of translation
Small RNAs 20 nt long, derived from the regions adjacent to 232,233
pre-miRNAs. Their function is unknown
tRNAs can be processed into small RNA species by a conserved RNase Reviewed in [44]
(angiogenin). They are able to induce translational repression
MSY-RNAs are associated with the germ cell-specific DNA/RNA binding 234
protein MSY2. Like piRNAs, they are largely restricted to the germline and
are 2630 nt long. Their function is unknown
Dicer-independent 24 nt RNAs principally derived from the G-rich 43
strand of telomeric repeats. May have a role in telomere maintenance
A class of 3442 nt small RNAs, derived from centrosomes that show 42
evidence of guiding local chromatin modifications

However, let-7 also targets the Dicer coding
sequence (CDS) [87] (Figure 3b), consistent with
what appears to be an emerging theme of non-
canonical miRNA targets in developmentally regulated
genes [8892]. Additionally, let-7 was also recently
shown to regulate HMGA2, an oncofetal gene and
pluripotency factor, in a cell cycle-dependent manner.
HMGA2 translation is up-regulated upon cell cycle
arrest but inhibited in proliferating cells [93]. Taken
as an example, let-7 provides a compelling illustration
of the complexity of small RNA biogenesis and func-
tion, and points more generally towards small RNAs
having a wide range of regulatory functions facilitated
by sequence-specific interactions, any of which may
malfunction to cause disease.
Long non-coding RNAs
Genome-wide transcriptomic studies have now shown
that the mammalian genome is abundantly transcribed
[210] and that at least 80% of this transcription is
exclusively associated with long non-coding RNAs
(lncRNAs) [9]. Although lncRNAs have frequently
been disregarded as artifacts of chromatin remod-
elling or transcriptional noise [94,95], there is sub-
stantial evidence to suggest that they mirror protein-
coding genes. Indeed, they are frequently long (gen-
erally >2 and some >100 kb) [96], spliced and con-
tain canonical polyadenylation signals [97,98]. Addi-
tionally, lncRNA promoters are bound and regulated
by transcriptional factors, including Oct3/4, Nanog,
CREB, Sp1, c-myc, Sox2, NF-B and p53 [99102]
and epigenetically marked with specific histone mod-
ifications [102,103]. Overall, there are at least tens of
thousands of lncRNAs that show signatures of selec-
tion many of which, like small RNAs, are tissue
and developmental stage-specific [97,104108].
Long ncRNAs have a variety of functions, but one
of their primary roles appears to be as epigenetic

J Pathol 2010; 220: 126139 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
Figure 2. The rise in the number of non-coding RNA publi-
cations per year. The number of PubMed indexed publications
with title, abstract or keywords matching each class of ncRNA
is plotted. Records were retrieved using an in-house Perl script
and the PubMed eFetch utility. The search terms used were:
piRNA, PIWI RNA, PIWI-associated RNA, PIWI-interacting
RNA, siRNA, small interfering RNA, endo-siRNA, endogenous
siRNA, miRNA, microRNA, non-coding RNA, non-coding RNA,
non-protein-coding RNA, ncRNA and lincRNA. Note that 2009
data were only gathered through August 2009
regulators of protein-coding gene expression [109].
For example, Hox genes are associated with hundreds
of lncRNAs that define domains of differential his-
tone methylation and RNA polymerase accessibility
along the spatial and temporal axes of human devel-
opment [110]. A lncRNA transcribed from the HOXC
locus, termed HOTAIR, regulates the chromatin methy-
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-
modifying complexes [111], with evidence that other
lncRNAs operate to activate gene expression through
Trithorax-group complexes [108]. Long ncRNAs are
also frequently associated with the phenomenon of
genomic imprinting, which ensures that one of the two
parental alleles of certain autosomal genes are epige-
netically silenced [112116].
Long ncRNAs can also directly modulate gene
transcription and protein degradation. For example,
the lncRNA Evf2 activates the homeobox transcrip-
tion factor Dlx2 and recruits it to an ultraconserved
genomic element, which induces transcription of Dlx5
[117]. Mutant mice lacking Evf2 show reduced num-
bers of GABAergic interneurons early in development
and reduced synaptic inhibition in adulthood [118],
suggesting that lncRNA-dependent processes are fun-
damental to the central nervous system. Indeed, a
large fraction of lncRNAs is expressed in very precise
patterns in the brain [106]. Similarly, the archetypal
Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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
/ 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 [124127]. 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-
ically studied and classified separately, it is impor-
tant to note that they frequently overlap both phys-
ically and functionally. Indeed, the dynamic inter-
play between lncRNAs and small RNAs is evi-
dent during the process of X-chromosome inac-
tivation (XCI), which occurs in female mammals
to ensure dosage compensation for X-linked genes
between the sexes. The antisense lncRNAs Xist and
Tsix are not only responsible for the chromatin
modifications that maintain XCI [24,129,130], but
also form dsRNA duplexes in vivo that are pro-
cessed by Dicer into 2542 nt X-inactivation RNAs
(xiRNAs) [131]. Given the abundance of mam-
malian antisense transcripts with the potential to
form dsRNA structures [132,133], such relationships
between lncRNAs and small RNAs may be common-
place.
Non-coding RNAs in disease
Small regulatory RNAs
Small RNAs have roles in virtually all develop-
mental processes, including stem cell and germline
maintenance, development and differentiation, tran-
scriptional and post-transcriptional gene silencing and
subcellular localization (see above, and reviewed in
[1417,20,21]). Not surprisingly, therefore, their dis-
ruption has been linked to human disease. For exam-
ple, miRNAs are aberrantly expressed in: liver, pan-
creatic, oesophageal, stomach, colon, haematopoietic,
ovarian, breast, pituitary, prostate, thyroid, testicular
and brain cancers [134138]; central nervous system
disorders (e.g. schizophrenia and Alzheimers disease)
[139]; and cardiovascular disease [140,141]. MiRNAs
are also enriched at fragile sites in the human genome
and are associated with oncogenic viral integration
sites [142,143].
J Pathol 2010; 220: 126139 DOI: 10.1002/path
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 TRIMNHL 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 28, 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-
ciated with PraderWilli syndrome (PWS), a disor-
der caused by the loss of imprinting on chromo-
some 15q11-q13 and characterized by hyperphagia,
hypogonadism and cognitive impairment. A recent
study has shown that a single microdeletion involv-
ing several small nucleolar RNA clusters (HBII-85
and HBII-52) results in PWS, suggesting that loss
of small RNAs is a causal determinant of the dis-
ease [144]. Consistent with this hypothesis, knockout

J Pathol 2010; 220: 126139 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
mice lacking the relevant snoRNAs largely recapitu-
late the PWS phenotype [145]. Interestingly, HBII-52
forms an antisense duplex with the serotonin recep-
tor 2C (5HT2C ) mRNA and negatively regulates its
post-transcriptional editing [146,147], strongly impli-
cating it in PWS-associated and autistic neurological
defects [148]. Taken together, these studies suggest
that the loss of small RNA loci plays an important
role in human illness.
Like protein-coding genes, small RNAs can func-
tion either as activators or inhibitors of disease. Con-
sistent with its role as a differentiation factor, let-7
is a well-established tumour suppressor [61,149151]
whose reduced expression is associated with poor sur-
vival in human lung cancers [152]. Likewise, mir-29b
expression is associated with disease-free survival in
patients with ovarian serous carcinoma [153], poten-
tially due to regulation of the de novo methyl trans-
ferases Dnmt3a and Dnmt3b [154]. Indeed, altered
expression of a broad suite of miRNAs that, dependent
on their targets can either act as tumour suppressors
or oncogenes (so-called oncomiRs), has been detected
in virtually all cancer types examined (for reviews
and tables of cancer-associated miRNAs and their
targets, see [134,151,155,156]). Similar relationships
are apparent in cardiovascular illnesses [140,141].
For example, miR-92a controls functional recovery
of ischaemic tissues in mice [157], and miR-145 and
miR-143, which have recently been implicated in dif-
ferentiation of progenitors into cardiac myocytes, are
down-regulated in injured and atherosclerotic vessels
[158]. MicroRNAs may even play a direct role in viral
defence. A study of human T lymphocytes has shown
that miR-29a targets the HIV-1 3 UTR and directs it to
P bodies, where it is suppressed by the RNA-induced
silencing complex (RISC) [159].
Small RNA dysregulation occurs for multiple rea-
sons and reflects the processes involved in their
biogenesis, regulation and targeting. MicroRNA loci
and individual components of the miRNA biogen-
esis pathway are frequently lost or amplified in a
wide range of cancers [160,161], and there is now
widespread evidence that miRNAs that act as differ-
entiation factors (e.g. let-7, above) are globally down-
regulated in cancers [134,150,151]. Indeed, ovarian
cancer patients who show decreased expression of
Dicer and Drosha, the RNases involved in miRNA
production (see above), are associated with poor prog-
noses, suboptimal surgical cytoreduction and advanced
tumour stages [162]. Likewise, a mutation resulting in
premature termination of DICER1 results in pleuropul-
monary blastoma, a rare paediatric lung tumour [163].
Consistent with these findings, studies in mice have
shown that mammalian systems are highly sensitive
to Dicer activity. Complete loss of Dicer results in the
disruption of the developmental programme and early
embryonic lethality [164].
Elements associated with the regulation of miRNA
processing can also be associated with various patholo-
gies. A recent study has shown that over-expression of
Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
131
LIN28 and LIN28B, negative regulators of let-7 bio-
genesis, correlates with repression of let-7, occurs in
at least 15% of human malignancies and is associ-
ated with more advanced disease states [165]. Simi-
larly, and consistent with let-7 s role in developmental
regulation, genetic variants of the LIN28 locus have
recently been associated with altered timing of human
pubertal growth and development [166].
Single nucleotide polymorphisms (SNPs) in mature
and precursor miRNAs have been robustly associ-
ated with schizophrenia and autism [167,168], and
a pathogenic SNP in the seed sequence of miR-
96 is responsible for progressive hearing loss [169]
(Figure 3b). A SNP in the 3 UTR of K-Ras, a
well-characterized GTPase-regulated oncogene and
target of let-7, inhibits let-7 translational suppres-
sion and results in reduced survival in oral cancers
[170]. Indeed, SNPs in the 3 UTRs of mRNAs
that abolish or create target sites may be common
in miRNA-associated diseases (reviewed in [171]).
As examples, SNP-induced illegitimate miRNA bind-
ing sites are associated with muscular hypertrophy in
sheep, Tourettes syndrome and cardiovascular disease
[171173]. More generally, allele-specific polymor-
phisms in miRNA target sites have been shown to play
a role in the tissue-specific miRNA regulation of hun-
dreds of genes, suggesting that such genetic subtleties
may be a widespread underlying cause of individual
phenotypic variability [174].
Long non-coding RNAs
The data gathered to date strongly implicate lncRNAs
in the basal regulation of protein-coding genes, includ-
ing those central to normal development and oncoge-
nesis, at both the transcriptional (e.g. epigenetic) and
post-transcriptional (e.g. subcellular dynamics) levels,
and an increasing number have been functionally val-
idated to affect different cellular and developmental
pathways (see [107]). It is not surprising, then, that
the dysregulation of lncRNAs appears to be a primary
feature of many complex human diseases, including
leukaemia [175], colon cancer [176], prostate cancer
[177], breast cancer [178], hepatocellular carcinoma
[175,179], psoriasis [180], ischaemic heart disease
[181,182], Alzheimers disease [183] and spinocere-
bellar ataxia type 8 [184].
In some cases, the mechanisms by which lncRNAs
contribute to disease have been carefully dissected. For
example, the dsDNA-binding protein PSF constitu-
tively silences the proto-oncogene GAGE6. However,
at least five lncRNAs can bind to PSF, which results
in deactivation of PSF-induced silencing, expression
of GAGE6 and enhanced tumorigenicity [185]. Long
ncRNAs overlapping or antisense to protein-coding
gene promoters may also contribute to oncogenesis.
A transcript antisense to the p15 tumour suppressor
gene, first identified in a human leukaemia, regu-
lates the chromatin and DNA methylation status of
the p15 locus [186]. A lncRNA antisense to p21 was
J Pathol 2010; 220: 126139 DOI: 10.1002/path
132
also recently shown to behave similarly [187]. These
results, combined with the observation that antisense
transcripts are present at thousands of protein-coding
genes, have led to speculation that antisense lncR-
NAs generally control the expression of their cog-
nate protein-coding genes through epigenetic modifi-
cations [188,189]. This model has profound ramifi-
cations for our understanding of disease, particularly
cancer dysregulation of a lncRNA regulating the
expression of a tumour suppressor or oncogene, and
not the protein-coding sequence itself, may be one of
the hits that leads to oncogenesis.
The hidden layer of non-coding variation
These examples likely represent the tip of a very big
iceberg. The same technologies that have revealed a
breadth of ncRNA expression are also driving a revo-
lution in genome sequencing that will ultimately iden-
tify variations in the human genome that underpin dis-
ease susceptibility and aetiology. However, given the
focus on mutations in protein-coding exons that cause
most of the high-penetrance simple genetic disorders,
the variation that occurs in non-protein-coding regions
of the genome has, to date, largely been ignored or at
least not been considered [107,190]. This is changing:
the emergence of genome-wide association studies to
identify variant loci affecting complex diseases and
traits and an increased awareness of ncRNA biology
have prompted a reconsideration of the underlying
protein-centric assumptions and provided a number
of novel insights into disease-causing mechanisms.
For example, many pathological mechanisms are now
known to involve aberrant regulation (and in many
cases ncRNAs) rather than alterations to the protein-
coding sequences themselves. This is perhaps not sur-
prising, given that the primary engine of phenotypic
radiation and higher complexity has been the expan-
sion and divergence of the regulatory architecture that
controls the deployment of protein components during
differentiation and development [191], much of which
may be embedded within ncRNAs [12,105]. Indeed,
the same forces that drive evolutionary innovations
can result in deleterious variations.
Genome-wide association studies are beginning to
identify novel ncRNAs as candidate disease-associated
genes. For example, the lncRNA MIAT is associated
with myocardial infarction [181], and a novel lncRNA
induced by a chromosomal deletion that truncates the
polyadenylation site of the LUC7L gene [192] results
in aberrant methylation and silencing of the neighbour-
ing HBA2 gene, leading to the onset of -thalassemia.
Indeed, many disease variants map far from protein-
coding genes and, given the level of genome-wide
transcription, are therefore likely to interrupt lncRNAs.
For example, a disease-causing 7.4 kb deletion asso-
ciated with blepharophimosis syndrome occurs over
250 kb upstream from the nearest gene, FOXL2 [193],
and this mutation interrupts a lncRNA of unknown
J Pathol 2010; 220: 126139 DOI: 10.1002/path
Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
RJ Taft et al
function, PISRT1, which has also been identified as a
candidate gene in a goat model of this disease [193].
The potential role of lncRNAs in long-range
enhancer function, and therefore dysfunction, is illus-
trated by the Evf2 lncRNA (see above), which
may contribute to split-hand/split-foot malformation
1 (SHFM1). Although the region associated with
this developmental disorder encompasses three genes,
DLX5, DLX6 and DSS1, none are directly mutated in
patients [194]. Instead, exhibition of the limb pheno-
type requires the expression of both the Dlx5 and Dlx6
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-
sion of both these genes, it warrants investigation as a
candidate SHFM1 disease locus.
Similarly, two lncRNAs, SOX2OT and SOX2DOT,
exhibit enriched expression in the lens of eye and over-
lap a known myopia susceptibility locus (Figure 4)
[196,197]. These transcripts, one of which is tran-
scribed from a distal ultraconserved enhancer, also
overlaps the SOX2 gene, itself an important regula-
tor of ocular development. Given that developmental
genes are significantly enriched for a proximal asso-
ciation with lncRNAs, it may well be that the under-
standing of developmental disorders is likely to benefit
from an appreciation of lncRNA biology. The future
convergence of lncRNA identification by deep RNA
sequencing with the increased resolution of disease
variants afforded by genomic sequencing will, we sug-
gest, prove a potent combination in elucidating the
functional contribution of ncRNAs to disease.
Non-coding RNAs as diagnostics
and therapeutics
The growing body of research showing that ncRNAs
may be primary genetic regulators in complex animals
has led to the corresponding realization that this may
make them ideal diagnostic markers. For example, in
some cases the expression profiles of miRNAs, in con-
trast to those of protein-coding mRNAs, are able to
accurately identify the origin of poorly differentiated
tumours and carcinomas [198,199]. Indeed, a signa-
ture of as few as 200 miRNAs may be sufficient for
cancer classification [198], and it appears that some of
the difficulties of early detection associated with colon
and other occult cancers may be overcome by pro-
filing miRNAs obtained from patient serum, plasma,
saliva and tissues [200,201]. Likewise colon, lung and
breast cancer prognosis is strongly associated with a
small suite of miRNAs (reviewed in [202]), suggesting
that assays designed to query ncRNAs may eventually
become core components of the pathologists toolkit.
This will undoubtedly be facilitated by recent advance-
ments in massively parallel sequencing technologies
[203,204], which allow rapid and sensitive profiling of
both long and short ncRNAs, and will almost certainly
make personal genomics a reality in the next 5 years
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, 415182, 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-
tion with other datasets (e.g. protein interaction and
genome-wide association studies) will pose a consid-
erable, but tractable, challenge well into the future.
The link between endogenous ncRNAs and dis-
ease, and the perfection of RNAi-based techniques to
silence genes in simple animals, has led to specula-
tion that RNA molecules can be employed as ther-
apeutic agents. Indeed, it may be both easier and
more productive to adjust the regulatory software
(i.e. ncRNAs) than to try and correct the hardware
(i.e. protein-coding genes). Hopes for RNA-based and
RNA-targeted therapies were bolstered by early suc-
cesses using siRNAs in human in vitro culture systems
[206] and in targeting HIV-1 and human BCL2 with
siRNA-like molecules [207209]. Like gene therapy,
however, RNA therapeutics face considerable hur-
dles, including development of reliable delivery sys-
tems, dosage regimes and techniques to ameliorate off-
target effects [210,211]. Nonetheless, multiple modes
of administration have been developed, including viral,
liposome and nanoparticle delivery systems, and there
are currently multiple ongoing clinical trials targeting
age-related macular degeneration, respiratory syncytial
virus, acute renal failure, hepatocellular carcinoma and
congenital pachyonychia, among others (reviewed in
[212]).
There is also an increasing interest in RNA ther-
apeutics that mimic or regulate miRNA activity in
human cancers (reviewed in [213]). This could be
facilitated by exogenous expression of a repressed
miRNA (using the same delivery systems as siRNA
therapeutics), by the introduction of antagomirs [214]
that are complementary and bind to miRNAs, or
the use of sponges that contain multiple artificial
miRNA-binding sites [215]. Artificial expression of
specific miRNAs in vivo may be a powerful therapeu-
tic mechanism, particularly given recent reports that
over-expression of a single miRNA, miR-302, is capa-
ble of inducing stemness [216,217].
A series of recent studies has suggested that an
equally fruitful target may be gene promoters. Indeed,

J Pathol 2010; 220: 126139 DOI: 10.1002/path

Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
134
there is a growing body of work showing that exoge-
nous small RNAs can activate or suppress transcription
by interfering with epigenetic marks and chromatin
formation, and thereby disrupting transcription initi-
ation [40,187,188,218225]. Moreover, siRNAs have
been shown to effectively modulate alternative splic-
ing [226], suggesting that, once viable, RNA therapeu-
tics may have a wide diversity of possible uses.
Conclusions
The absolute number of protein-coding genes encoded
by a genome is essentially static across all animals
from simple nematodes to humans [1], indicating
that additional genetic elements must be involved in
the development of the increasingly complex cellular,
physiological and neurological systems. Non-coding
RNAs are likely candidates, as they are adaptively
plastic, capable of regulating processes both broadly
and sequence-specifically, and are now known to be
components of nearly all cellular and developmen-
tal systems. It is becoming clear that a comprehen-
sive understanding of human biology must include
both small and large non-coding RNAs, and that it
is perhaps only through inclusion of these elements in
the biomedical research agenda, including studies to
determine the mechanistic basis of the causative vari-
ations identified by genome-wide association studies,
that complex human diseases will be completely deci-
phered.
Acknowledgements
This study was supported by the Australian National Health
and Medical Research Council, the Menzies Foundation, the
AustralianAmerican Fulbright Commission and the Royal
Australasian College of Physicians (KCP), the Australian
Research Council, the University of Queensland and the
Queensland State Government (JSM).
Teaching materials
PowerPoint slides of the figures from this review
are supplied as supporting information in the online
version of this article.
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