The rise of regulatory RNA
K.V. Morris1,2 and J.S. Mattick1,3,4
1School of Biotechnology and Biomedical Sciences, University of New South Wales, Sydney,
NSW 2052, Australia
2Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
3Garvan Institute of Medical Research, 384 Victoria St. Darlinghurst NSW 2010, Australia
4St Vincent’s Clinical School, University of New South Wales, Sydney, NSW 2052, Australia
Abstract
Discoveries over the last decade portend a paradigm shift in molecular biology. Evidence suggests
that RNA is not only functional as a messenger between DNA and protein but also in the
regulation of genome organization and gene expression, which is increasingly elaborated in
complex organisms. Regulatory RNAs appear to operate at many levels, but in particular to play
an important role in the epigenetic processes that control differentiation and development. These
discoveries suggest a central role for RNA in human evolution and ontogeny. Here we survey the
emergence of the previously unsuspected world of regulatory RNAs from an historical
perspective.
Introduction
RNA has long been at the centre of molecular biology and was likely the primordial
molecule of life, encompassing both informational and catalytic functions. It is thought that
its informational functions were subsequently devolved to the more stable and easily
replicable DNA, and its catalytic functions to the more chemically versatile polypeptides1.
The idea that the contemporary role of RNA is to function as the intermediary between the
two had its roots in the early 1940s with the entry of chemists into biology, notably Beadle
and Tatum2, whose work underpinned the “one gene-one enzyme” hypothesis, which later
morphed into the more familiar phrase “one gene-one protein”, and gained currency despite
the prescient misgivings of experienced geneticists, notably McClintock3. The concept that
genes encoded (solely) the functional components of cells (the ‘enzymes’) itself had deeper
roots in the mechanical zeitgeist of the era, being decades before the widespread
understanding of the use of digital information for systems control.
Although the one gene-one protein hypothesis has long been abandoned, due to the
discovery of alternative splicing in the 1970s, the protein-centric view of molecular biology
has persisted, aided by phenotypic and ascertainment bias towards protein-coding mutations
in genetic studies and by the assumption that regulatory mutations affected cis-acting
The authors declare no conflict of interest.
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regulatory protein binding sites4. However, this view is challenged by the discovery of
nuclear introns and the phenomenon of RNA interference (RNAi), as well as by the advent
of high throughput sequencing, which led to the identification of large numbers and different
types of large and small RNAs, whose functions are still under exploration.
Here we examine the history and chart the shift in thinking that is still underway about the
role of RNA in cell and developmental biology, especially in animals. The emerging
evidence suggests that there may be more genes encoding regulatory RNAs than encoding
proteins in the human genome, and that the amount and type of gene regulation in complex
organisms has been substantially misunderstood for most of the past 50 years.
Early ideas for the role of RNA
RNA, the central dogma and gene regulation
After the elucidation of the double-helical structure of DNA in 19535, the following years
were preoccupied with deciphering the ‘genetic code’ and establishing the mechanistic
pathway between gene and protein: the identification of a transitory template (mRNA), an
adaptor (tRNA) and the ‘ribosome’ factory comprised of ribosomal proteins and RNA
(rRNA) for the translation of the code into a polypeptide. In 1958, Crick published the
celebrated ‘central dogma’ to describe the flow of genetic information (DNA → RNA →
protein), which has proved remarkably accurate and durable, including the prediction of
reverse transcription6. Nonetheless, in conceptual terms, RNA was tacitly consigned to be
the template (and infrastructural platform – ribosomal and transfer RNAs) for protein
synthesis or, at least, has been interpreted in this way by most people since that time.
The link between rRNA (which is highly expressed in virtually all cells) and the structures
termed ribosomes as the platform for protein synthesis was established in the mid 1950s7.
The roles of tRNA and mRNA were experimentally confirmed in 19588 and 19619,
respectively, the latter the same year that Jacob and Monod published their classic paper on
the lac operon of Escherichia coli10, the first locus to be characterized at the molecular
genetic level. These studies confirmed that (at least some, but presumed most) genes
encoded proteins, and supported the emerging idea that gene expression was controlled by
regulating the transcription of the gene, as indicated by the locus encoding the lac repressor
– the repressor-operator model. At the time Jacob and Monod did not know the chemical
identity of the repressor, speculating en passant that it “may be a polyribonucleotide” (i.e.,
RNA)10. However, Gilbert later showed that the repressor was a polypeptide that
allosterically bound the substrate lactose, and the brief idea faded11.
These studies reinforced and extended the conception that proteins are not only ‘enzymes’
but also the primary analogue components and control factors that comprise the cellular
machinery. This in turn has led to the prevailing ‘transcription factor’ paradigm of gene
regulation, including the derived assumption that combinatoric interactions would provide
factorially ‘explosive’ regulatory combinations12, more than enough to supervise human
ontogeny. However, this assumption has not been substantiated theoretically or
mechanistically, and the observed scaling of regulatory genes and the extent of the
regulatory challenge in programming human developmental architecture appears to be quite
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different from these expectations13. In this context it is noteworthy that the genome-wide
association studies have shown that most haplotype blocks influencing complex diseases fall
outside the known boundaries of protein-coding genes14.
Small nuclear / spliceosomal RNAs and small nucleolar RNAs
Following the discovery and functional description of tRNAs and rRNAs, other new classes
of common small nuclear RNAs were identified by biochemical fractionation15. Many of the
small RNAs were found to be part of ribonucleoprotein complexes (RNPs) (reviewed in 16).
One class, the small nuclear RNAs (snRNAs, Figure 1), were later found to be central cofactors
in RNA splicing (see below)17, hence their newer designation as ‘spliceosomal’
RNAs. The snRNAs U1, U2, U4, U5 and U6 participate in a number of RNA-RNA and
RNA-protein interactions in the assembly and function of canonical spliceosomes,
recognizing the 5′-end splice site (U1) and the branch point (U2), followed by the
recruitment of U4, U5 and U6, which displace U1 and interact with U2 (via U6) as well as
the 5′ and 3′ splice sites (via U5)18. A set of less abundant snRNAs (U11, U12, u4atac and
U6atac) along with U5 are found in a variant ‘minor’ spliceosome, termed U12-type19.
Other small RNAs were found to be localized to nucleoli and to guide the methylation (the
box CD subclass) and pseudouridylation (the box H/ACA subclass) of ribosomal RNAs
(rRNAs), transfer RNAs (tRNAs), and snRNAs20–22 (Figure 1). The chemical modifications
of rRNAs, tRNAs and snRNAs proved to be essential in ribosomal and cellular function, in
particular tRNA and mRNA maturation and pre-mRNA splicing (U2). Notably, the
disruption of snoRNAs was found to lead to a loss of processing of the 18S, 5.8S, and
25S/28S rRNAs20. Although some snoRNAs are subject to parental imprinting and/or
differentially expressed, for example in the brain23,24, and appear to target a wider range of
RNAs including mRNAs25, suggesting a regulatory role, and there are related small RNAs
(scaRNAs) in other subnuclear structures called Cajal bodies (which also process telomerase
RNA)26, none of these early studies suggested anything other than that the role of RNAs
was limited to protein synthesis.
The emergence of heterogeneous nuclear RNAs
The first hint that RNA may have additional roles in complex organisms was the discovery
of ‘heterogenous’ nuclear RNA (hnRNA)27 and the observation that the complexity of this
population, as determined by denaturation-renaturation hybridization kinetics, was much
greater in the nucleus than the cytoplasm. The existence of hnRNA and the concomitant
discovery of the large amount of ‘repetitive’ sequences (different classes of retrotransposed
sequences with similar composition that occupy large fractions of plant and animal
genomes) led Britten & Davidson to speculate in 1969 that animal cells might contain
extensive RNA-based regulatory networks28–30. While this hypothesis attracted a great deal
of interest at the time, it also quickly lapsed, with the proponents not re-visiting it even after
the subsequent discovery of introns (see below), instead focussing on regulatory networks
controlled by transcription factors31,32 or the significance of transposons in protein
evolution33.
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The discovery of introns
The discovery of introns in 197734,35 was perhaps the biggest surprise in the history of
molecular biology36, as no one expected that the genes of higher organisms would be
mosaics of coding and noncoding sequences, all of which are transcribed. However, the
prevailing conception of the flow of genetic information was not overly disturbed as the
removal of the intervening sequences (‘introns’) and the reconstruction of a mature mRNA
by RNA splicing preserved the conceptual status quo. That is, genes still made proteins. In
parallel, it was assumed that the excised intronic RNAs were simply degraded, although the
technology of the time was too primitive to confirm this. In any case, introns were
immediately and universally dismissed as genomic debris, and their presence rationalized as
evolutionary remnants involved in the prebiotic modular assembly of protein-coding RNAs
that have lingered (and been expanded by transposition) in complex organisms37. This was
consistent, superficially at least, with the implications of the C-value enigma that eukaryotes
contained varying amounts of DNA baggage, and the accompanying conclusion that
retrotransposon sequences, often pejoratively referred to ‘repeats’, which occupy much of
the genomic real estate in plants and animals, are largely selfish DNA that are parasitic co-
travellers38,39.
RNA as a catalyst
A few years later, Cech, Altman and colleagues showed that RNA itself was capable of
enzymatic catalysis (‘ribozymes’)40,41, which provided evidence in support of the ‘RNA
early’ hypothesis, and showed that RNA catalysis exists and has persisted in particular
contexts, notably at the core of RNA splicing42 and mRNA translation43. This reinforced the
mechanical conception of molecular biology, and the role of RNA as the platform for
protein synthesis, but did not give any hint of RNA as a widespread regulatory factor,
although that possibility is perfectly feasible. Indeed there is increasing evidence that
catalytic RNA exists in animal and plant cells, in introns, UTRs and elsewhere, and may
play a variety of roles including, for example, in regulating post-transcriptional cleavage
reactions44,45.
The small RNA revolution
The discovery of microRNAs
In 1993 Ambros and colleagues showed the first evidence for small (~22 nt) regulatory
RNAs, by the discovery of the genetic loci lin-4 and let-7, which regulate the timing of
Caenorhabditis elegans development46,47. Although let-7 is highly conserved from
nematodes to humans48, very few miRNAs have been discovered genetically49,50, and these
RNAs remained interesting idiosyncrasies until the discovery of RNAi (see below), which
led to the targeted cloning after size selection of many more51–53 and the demonstration that
these ‘microRNAs’ (miRNAs) act, at least in part, by imperfect base pairing with (usually)
the 3′UTRs of target mRNAs to inhibit their translation and accelerate their degradation54.
Currently, there are large numbers of evolutionarily widespread miRNAs in the databases55,
almost all of which had evaded prior detection by genetic screens (but many subsequently
validated by reverse genetics). While many miRNAs can be identified by conservation, it is
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also evident that many are tissue- and lineage-specific56,57, and that there may be many
more to be discovered.
It has also become evident that many if not most protein-coding transcripts are targets for
miRNA regulation58,59, that miRNAs can, in some cases, regulate large numbers of target
mRNAs60, and reciprocally that many mRNAs contain target sites for many miRNAs61,
although the implied regulatory logic of this complex multiplex arrangement has not been
explained. The targets of miRNAs are usually thought to be mRNAs, but may also include
other RNAs62. Biologically, miRNAs have been shown to regulate many physiological,
developmental and disease processes, including, for example, pluripotency63, epithelialmesenchymal
transition and metastasis64, testis differentiation65, diabetes66, and neural
plasticity and memory67, among others68.
The RNA interference pathway
MicroRNAs are just one facet of the phenomenon of ‘RNA interference’ (RNAi), which
causes silencing of gene expression after the introduction of sense-antisense RNA pairs,
discovered in 1998 in plants69 and C. elegans70. These discoveries were presaged by the
curious phenomenon of transgene silencing, mainly in plants71,72, linked to antisense RNA
and small RNA-directed DNA methylation of transgenes, indicating transcriptional as well
as post-transcriptional silencing73,74. Mechanistic analysis of these silencing mechanisms
showed that exogenous double-stranded RNA was processed into short fragments (short
interfering RNAs or siRNAs) with a similar size to miRNAs, suggesting that miRNAs may
represent a similar endogenous system.
This was confirmed and led to the elucidation of natural double-stranded precursors in stemloop
structures75 and the identification of key genes and enzymes involved in their
biogenesis and function, notably Drosha76, Dicer77 and multiple Argonaute (Ago)
proteins78, the latter of which were already known to play central roles in differentiation and
development,79 but are now known to also be involved in defence against RNA viruses in
many organisms80. Drosha and Exportin 5 are involved in the cleavage and export of
double-stranded RNA (dsRNA) precursors from the nucleus to the cytoplasm76, where they
are further processed by Dicer to small (21–24 nt) dsRNA moieties, one strand of which is
loaded into Ago component of the RNA-induced silencing complex (RISC), which also
contains other proteins77. The RISC is guided by the small RNA strand to complementary
RNA targets, which are subsequently silenced by translational repression and/or RNA
destabilization81,82 (Figure 2).
While still under discussion, the current view is that siRNAs (and ‘short-hairpin RNAs’,
shRNAs), which seem to naturally occur more commonly in plants, act primarily by perfect
base-pairing and by Ago-mediated cleavage of complementary target RNAs (and hence are
used widely as experimental tools and potential therapeutic agents83), whereas miRNAs
have incomplete homology with their targets and act primarily at the translational level81,82
(Figure 2).
MiRNAs and siRNAs are thought to act post-transcriptionally and cytoplasmically, but the
existence of Ago in the nucleus84–87 and the role of the RNAi pathway in epigenetic
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modulation88 suggests that the system is more complex and multifaceted than expected, with
(for example) demonstrations that miRNA isoforms are developmentally regulated89 that the
target ‘seed’ sequence is only one factor in target recognition90,91, and that miRNAs can
also act to impose transcriptional silencing92 (Figure 2). There is also increasing evidence of
intersecting pathways, such as RNA editing and modification, in these networks93–96.
Piwi-associated small RNAs
While most Ago proteins are expressed ubiquitously and associate with miRNAs and
siRNAs, there is a subclade of Argonaute proteins, termed Piwi, that is required for germ
cell development97–100. Piwi and Piwi-like proteins associate with a distinctive class of
small RNAs (26–30 nt; piRNAs), which act to epigenetically and post-transcriptionally
silence transposons in germ cells101–110. Piwi is found predominantly in the nucleus111, colocalizes
in an RNA-dependent manner with Polycomb group proteins112, and appears to be
expressed in other tissues, including the brain113, suggesting a role beyond genome
protection in epigenetic processes114,115.
Other classes of small RNAs in eukaryotes
The molecular genetics, biochemistry and structural biology of the RNAi system are still
being unravelled, but indicate an ancient, widespread and multilaterally adapted system that
controls many cellular processes, whose dimensions are still being explored. These include
potentially lineage-specific variations, such as the ‘21U’ RNAs in C. elegans116.
Surprisingly, it appears that all snoRNAs from fission yeast to human produce at least 3
different subclasses of small RNAs117, one of which has the same size and functions as a
miRNA118, and another that is similar in size to piRNA117. There are also intriguing and
recurring reports of fragments of tRNAs produced in tissue-specific patterns119 and
associated with Ago proteins120.
More recently, deep sequencing of small RNA populations has revealed the existence of
another class of small RNAs in animals but not plants, which are 17–18 nt in length and
associated with transcription initiation (‘tiRNAs’)121 and splice sites (‘spliRNAs’)122
(Figure 2). The origin and function of these RNAs is uncertain, but preliminary evidence
suggests that they may play a role in nucleosome positioning123 and/or be involved in other
levels of chromatin organization124. There are also other reports of less distinct classes of
promoter-associated RNAs called PASRs125, TSSa-RNAs126 and PROMPTS127, some of
which may play a role in RNA-directed transcriptional gene silencing128.
Regulatory RNAs in prokaryotes
Many small regulatory RNAs (sRNAs) have been identified in bacteria, which regulate a
wide variety of adaptive responses. Bacterial sRNAs generally function by simple antisense
mechanisms to regulate translation or stability of target mRNAs by altering their secondary
structure to expose or sequester cis-acting sites129,130. Studies in bacteria have also
identified cis-acting regulatory RNA sequences (‘riboswitches’), which act allosterically by
binding metabolites to regulate gene expression131,132, and which almost certainly exist as
part of the RNA regulatory landscape in all kingdoms of life.
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Very recently, the prokaryotic kingdom has once again surprised us with the sophistication
of its molecular machinery. Many bacterial and most archaeal genomes possess loci
comprised of regularly spaced repeats interspersed by other DNA sequences derived from
viruses133–136. These loci, now termed CRISPRs (Clustered Regularly Interspaced Short
Palindromic Repeats) act as an innate immune system by incorporating fragments of viral
DNA between the repeats, which are then transcribed and processed to produce small guide
RNAs (linked to their effector complexes via the repeat sequence) that target and destroy
viral DNA137–140 or RNA141). This system has recently been adapted for RNA
programmable sequence-specific genome manipulation in eukaryotes, including
mammals142–145, with extraordinary versatility including targeted gene excision and fusion,
and modified CRISPRs capable of recruiting silencing and activating proteins to target
loci146–150. Moreover, the biological arms race continues, with bacteriophages encoding
their own CRISPR system to evade host innate immunity151.
Long noncoding RNAs
The eukaryotic transcriptome and long non-coding RNAs
Noting that the density and size of introns (and, as it turned out later, intergenic sequences)
expanded with developmental complexity, Mattick posited in 1994 that introns had evolved
to express an expanding repertoire of trans-acting regulatory RNAs, that some genes
subsequently evolved to express only (intronic or exonic) regulatory RNAs, and that this
RNA-based regulatory system was the essential prerequisite for the emergence of
developmentally complex organisms152. Subsequently, the application of genome tiling
array technology and deep sequencing to the characterization of the transcriptome showed
that there are tens of thousands of loci in mammals that express large transcripts that do not
encode proteins, located intergenic, intronic and antisense to protein-coding genes. The
initial findings153–155 were confirmed in 2005156–159 and extended by the ENCODE
project160–162, all of which showed that the vast majority (at least 80%) of the human and
mouse genomes are differentially transcribed in one context or another, with other studies
reporting similar findings in all organisms examined. Indeed, it seems most intergenic (and
by definition intronic) sequences are differentially transcribed, and therefore that the extent
of the transcriptome expands with developmental complexity163.
Using more focussed deep sequencing methodologies, it has become evident that the full
repertoire of the protein-coding and non-protein-coding transcriptome is still vastly undersampled164.
In addition, many transcripts are not polyadenylated, and represent a largely
different sequence class156,165, some of which appear relevant to development (e.g.,
OCT4166,167). Moreover, 95% of human transcription initiation sites are not associated with
mRNAs, but rather mainly with non-polyadenylated noncoding transcription168. These nonpolyadenylated
transcripts are as yet largely uncharacterized because of the historical use of
polyA tails to remove the overwhelming rRNA contamination in RNA preparations, which
is being alleviated by more sophisticated approaches, including cap trapping169,
oligonucleotide subtraction170 and array capture164,171.
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Defining long noncoding RNAs
Long noncoding RNAs (lncRNAs) are operationally defined as any non-protein-coding
RNA >200 nt in length, which corresponds to a convenient cut-off in biochemical
fractionation and excludes all known classes of small RNAs172. Transcripts are judged to be
‘noncoding’ if they lack a long open reading frame (traditionally >100 codons) and/or do not
show codon conservation, although with initially limited genomic and transcriptomic data
for comparison, there was considerable uncertainty. However, recent analyses provide
strong evidence that most annotated lncRNAs do not encode proteins, although some specify
small proteins that had previously fallen under the bioinformatic radar173–175.
These noncoding transcripts can be parsed into intronic, antisense or intergenic (‘lincRNA’)
subsets in experimental studies and databases159,176,177, partly because of mechanistic
expectations178 coupled with a desire to reduce ambiguity and overlap with protein-coding
loci in functional analyses179–181. However, there is no evidence of any intrinsic difference
between RNAs that are intronic, intergenic, antisense or overlap with protein-coding genes,
for example in their interaction with chromatin-activating or -repressive complexes (Figure
1 and below), although subclasses will inevitably exist and be defined, some of which may
have be biased in relation to genomic origin.
Exploration of lncRNA functions
The unexpected discovery of large numbers of noncoding transcripts in eukaryotes, some of
which span tens or hundreds of kilobases182, has led to debates about their functionality (see
e.g. 183,184), particularly since many have relatively low evolutionary conservation and low
levels of expression, leading some to posit that they represent ‘transcriptional noise’ and/or
redundant transcripts with no biological significance. This is a possibility, at least in part.
However, it is clear that lncRNAs actually show a wide range of evolutionary conservation,
from those that are ultraconserved185 to those that are primate-specific186–188, which can be
explained as the result of different structure-function constraints and lineage-specific
adaptive radiation189. Indeed there is now considerable evidence that lack of primary
sequence conservation in lncRNAs does not indicate lack of function190,191, and that many
lncRNAs show evidence of structural conservation192,193.
Moreover, those loci expressing lncRNAs show all of the hallmarks of bona fide genes4,
including conservation of promoters169, indicative chromatin structure194 and regulation by
conventional morphogens and transcription factors195. LncRNAs have been found to have a
similar range of cellular half-lives as mRNAs196 and to be differentially expressed in a
tissue-specific manner158,197, with higher resolution in the brain198. The latter showed that
while the level of expression of many lncRNAs superficially appears to be lower than
mRNAs in whole tissues, lncRNAs are highly expressed and easily detectable in particular
cells198, and it appears that they have, on average, higher cell-specificity than
proteins165,199, consistent with their proposed role in architectural (as opposed to ‘celltype’)
regulation, where each cell has a unique positional identity in precisely sculpted
organs, bones and muscles200.
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Many lncRNAs are alternatively spliced201, further evidence of the precision of their
expression and hard to reconcile with the suggestion that they are simply transcriptional
noise. It should also be noted that some functionally validated lncRNAs can have isoforms
that encode proteins202 and reciprocally that some (perhaps many) mRNAs may also have
intrinsic functions as trans-acting regulatory RNAs203–205, that in some contexts 3′UTRs
can be separately expressed and convey genetic functions in trans204, and that both may be
further processed to produce subsidiary species206.
LncRNAs have been shown to be dynamically expressed in a range of differentiating
systems, including embryonal stem cell207, muscle208 T-cell209, breast210,211, erythroid211
and neuronal differentiation212–214, as well as in cancer and other diseases (see
e.g.210,215–222), at least partly controlled by conventional transcription factors195,213.
The validation of lncRNA function has to date mainly relied on knockdown of candidate
lncRNAs. It has proven surprisingly easy to knockdown lncRNA expression by si/shRNAmediated
approaches, and thereby to detect phenotypic changes in cultured cells, where
most analyses have been carried out. By 2009, ~50 lncRNAs had been shown to be
functional4 and hundreds more are now published or en route to publication, a large enough
sample to draw the conclusion that these transcripts are generally functional.
Roles in development and differentiation
Some lncRNAs play a role in general or differentiation-specific cell biological processes.
These include: Template RNAs that guide chromosomal rearrangements in ciliates223; Terra
RNAs, involved in telomere biology224; 7S RNA, an essential component of the signal
recognition particle involved in protein export225; 7SK, a highly expressed structured RNA
that is the scaffold to assemble a multimeric protein complex containing SR splicing
proteins and P-TEFb, a cyclin-dependent kinase required for transcriptional elongation by
RNA polymerase II and other factors 226; Neat1, an essential component of paraspeckles,
enigmatic subnuclear organelles that appear in differentiated but not stem cells in
mammals227,228; the nuclear-localized MALAT-1, which regulates alternative splicing229
and cell cycle progression230; Gomafu, which is expressed in an unknown subnuclear
structure, possibly a specialized spliceosome, in a subset of neurons231, and has recently
been implicated in schizophrenia232; and others of unknown function associated with bipolar
structures in the nuclei of Purkinje cells198.
Not surprisingly, given their expression patterns, most functionally analyzed lncRNAs
appear to play roles in the regulation of differentiation and development233. These include,
based on studies in cell culture, the regulation of apoptosis and metastatic
processes211,218,220,221,234, retinal and erythroid development211,235, breast
development210,236, and epidermal differentiation237, among many others.
Antisense knockdown of lncRNAs in zebrafish and deletion of sequences specifying
lncRNAs in mouse have shown that some confer visible developmental
defects181,191,238,239, although others do not, including knockouts of the widely expressed
Neat1 required for paraspeckle function240 and some of the most highly conserved
sequences in the mammalian genome241. This suggests that more sophisticated phenotypic
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screens may be required, especially in relation to cognitive function, since most mammalian
lncRNAs are expressed in the brain198 and many are mammal- or primate-specific242,243. A
good example is the retrotransposon-derived lncRNA BC1, which is widely expressed in the
brain but whose knockout causes no visible anatomical abnormality, but leads to behavioural
changes that would be lethal in the wild244.
Epigenetic roles of noncoding RNAs
Consistent with their roles in differentiation and development, a range of genetic and
biochemical evidence suggests that a major function of lncRNAs and many small RNAs is
the regulation of epigenetic processes245,246, likely by guiding chromatin-modifying
enzymes to their sites of action and/or acting as scaffolding for chromosomal
organization179,246–249 (Figure 3).
RNAs were first shown to induce transcriptional gene silencing in plants74,250, fungi251 and
human cells88, with an intimate involvement of small RNAs and the RNA interference
pathway in the epigenetic processes involved251–253, consistent with the observation that
small RNAs interact with Polycomb254 and that Ago proteins occur in the nucleus86,87
(Figure 3).
In parallel, dating back to 1990, antisense RNAs have also been shown to affect gene
expression, again initially in plants73 and later in animals159,166,255–257. Some lncRNAs,
similar to small ncRNAs258, have been shown to control alternative splicing259,260. Other
naturally occurring lncRNAs had been shown to control epigenetic processes in vivo,
notably in X chromosome dosage compensation261–265 and parental imprinting in
mammals266–268 and vernalization in plants269, with subsequent studies showing that
intergenic and antisense RNAs bind to polycomb chromatin repressive
complexes194,270–272, to trithorax chromatin activating complexes and activated forms of
histones207, and to DNA methyltransferases201,273,274. These observations were writ large in
2009 when it was shown that approximately 20% of ~3,300 lncRNAs examined were bound
by PRC2, with others bound by other chromatin-modifying complexes, that siRNAmediated
knockdown of lncRNAs associated with PRC2 led to changes in gene expression,
with the up-regulated genes being enriched for those normally silenced by PRC2179, and that
Polycomb binds RNA with high affinity but low specificity275, consistent with the idea that
many RNAs are Polycomb interactors.
One of the notable lncRNAs to emerge from the studies of Rinn, Chang and colleagues,
HOTAIR, is derived from the HOXC locus and regulates HOXD in trans194, is involved in
cancer metastasis220 and, when inactivated, results in homeotic transformation in vivo276,
Chang and colleagues also showed that lncRNAs could act as scaffolds for the assembly of
histone modification complexes277, with the widespread alternative splicing of these RNAs
suggesting that the cargo and/or target specificity can be varied in a context-dependent and
differentiation-specific manner.
LncRNAs may also be involved in orchestrating the highly dynamic spatial structure of
chromatin during differentiation and development164,278, which would explain their often
highly cell-specific expression patterns200. Developmental enhancers, as well as polycombMorris
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and trithorax-response elements, are transcribed in the cells in which they are
active203,279–284, and are likely not only scaffolds for the recruitment of epigenetic
regulators285 but also the physical mediators of the complex genetic phenomena of
transvection and transinduction245. Moreover, many lncRNAs display the properties of
enhancers180. These RNAs may well guide the physical looping that occurs between
enhancers, target promoters and exons, with precise positioning of nucleosomes286–290, to
control transcription and alternative splicing248,291,292, again modulated by alternative
splicing. Indeed the emerging picture is of a chromatin and transcriptional landscape that is
exquisitely and precisely controlled in 4 dimensions by a suite of regulatory RNAs that
assemble relatively generic (albeit often cell- or differentiation state-specific) enzyme
complexes and isoforms to their sites of action in a context-dependent manner249.
A substantial proportion of lncRNAs reside within, or are dynamically shuttled, to the
cytoplasm indicating roles in other cellular processes, including the regulation of protein
localization293, mRNA translation294 and mRNA stability295.
RNA editing, modification, retrotransposition and inheritance
Regulatory RNAs may also be influenced by environmental signals and be transmitted
between cells and generations, which has important implications for understanding geneenvironment
interactions and evolution. There is evidence that plasticity has been
superimposed on RNA-directed epigenetic networks by the expansion of RNA editing,
especially during cognitive evolution296,297, and by retrotransposon utilization and
mobility114,298–301, which harks back to the insights of McClintock and Britten & Davidson.
The raw material for evolution is gene duplication and transposition, the latter having the
advantage of being able to mobilize functional cassettes in regulatory networks302, which
appears to be the main driver of adaptive radiation245,303. Indeed many lncRNAs may have
originated from retrotransposons and the evolution of mRNAs and lncRNAs may have been
accelerated by retrotransposition of functional modules304–308.
Moreover, apart from snoRNA-directed modifications, there are well over 100 other
documented modifications of RNA309,310, including cytosine and adenosine methylation
which have known physiological and cognitive effects311–314, indicating a new additional
layer of RNA informational code and epitranscriptomics, an exciting field that is just
beginning to emerge315,316.
There is evidence for systemic transmission of RNA317,318 and RNA-mediated epigenetic
inheritance in plants and animals319–323. There is also the intriguing possibility of RNAdirected
DNA recoding, which may place RNA at the centre not only of gene regulation in
the developmental ontogeny of higher organisms, but also of both hard- and soft-wired
somatic and germline evolution324–326.
Conclusions and outlook
The past two decades have seen an explosion in our understanding of the previously hidden
and unanticipated world of RNA regulation. Indeed, in retrospect, it appears that we may
have fundamentally misunderstood the nature of the genetic programming in complex
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organisms because of the assumption that most genetic information is transacted by proteins.
This maybe largely true in simpler organisms, but is turning out not to be the case in more
complex organisms, whose genomes appear to be progressively dominated by regulatory
RNAs that orchestrate the epigenetic trajectories of differentiation and development.
The picture that emerges is of an extraordinarily complex transcriptional landscape in
mammals and other multicellular organisms, comprised of overlapping, intergenic and
intronic sense and antisense small and large RNAs with interlaced exons327,328, whose
promoters, splicing patterns, polyadenylation sites and regional repertoire varies in different
cells and developmental contexts (see below). Since there appear to be few distinct
boundaries to genes in humans, it seems better to change the focus of analysis to the
transcript, with genetic loci redefined as fuzzy transcription clusters165,328,329 albeit
semantically anchored or related to an enclosed or nearby protein-coding locus. However,
this can only be stretched so far, and non-protein-coding loci raise problems for existing
schema of human genome nomenclature.
Indeed even the notion of a (simple) protein-coding sequence needs to be reassessed. It is
becoming evident not only that mRNAs can have multiple functions205, but also that
protein-coding sequences themselves can have other embedded functions, as suggested by
constraints on synonymous codon usage330,331, including regulatory functions as epigenetic
modulators203, tissue-specific enhancers331,332 and transcription factor binding sites333. The
possibility, if not likelihood, is that there is a very complex functional and evolutionary
interplay between the protein-coding and regulatory functions of RNAs200, and that some
lncRNAs may have evolved, at least in part, from protein-coding genes, as appears to have
occurred with Xist, by duplication or pseudogenization followed by the emergence of
paralogous regulatory and/or coding functions201,334. Conversely, it appears that new
protein-coding capacity may also appear in lncRNAs174.
The sheer number and diversity of RNAs juxtaposed with their extraordinarily complex
molecular functions (Figure 3) in regulating epigenetic processes, subcellular organelles,
protein-coding and non-coding gene transcription, translation, RNA turnover, chromosomal
organization and integrity, and genome defence, among others, suggests that we have a long
way to go to understand the structure and functions of what is surely a highly interconnected
system. There are literally tens of thousands, if not more, of individual noncoding RNAs
whose roles in cell and developmental biology, as well as brain function, remain to be
determined. Moreover, many if not most regulatory RNAs, especially in complex organisms,
remain to be identified, including new classes such as the circular RNAs and others that may
function as miRNA ‘sponges’62,335–340, which will require targeted deep sequencing of
small and large RNAs that are derived from different genomic locations in different cells,
using targeted techniques such as RNA CaptureSeq164,171.
RNA is not a linear molecule, but can rather fold into complex and allosterically responsive
3-dimensional structures that can both recruit generic effector proteins and guide the
resulting complexes sequence-specifically to other RNAs and DNA, via duplex or triplex
formation. There are many important questions. These include the identification of
functional domains in RNA and their interacting partners, so that we can predict and parse
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RNA functional interactions in the same way that is already done by recognition of wellcharacterized
motifs and domains in proteins. One way to do this, already underway in many
laboratories, is to combine immunoprecipitation of different types of RNA binding proteins
(chromatin-modifying proteins, transcription factors, and RNA transport proteins, among
others) with deep sequencing of the associated RNAs (RIP-Seq) followed by analysis of
primary and predicted secondary structures, and ultimately by biochemical validation and
characterization.
Determination of the structure of RNAs, RNA-RNA, RNA-DNA and ribonucleoprotein
complexes will be a rapidly growing field, requiring the development of new technologies,
such as RNA footprinting with high-throughput sequencing341, as well in vivo studies using
RNA-based genetic techniques like CRISPR-mediated mutation143. Other objectives include
determination of whether small RNA pathways are used in viral defence in humans80, the
functions of ti/spliRNAs and snoRNA-derived small RNAs, the roles of piRNAs in
retrotransposon dynamics and the remodelling of the genome by retrotransposons in the
brain114, the mechanisms and extent of RNA-mediated trans-generational epigenetic
inheritance342, the locations of RNA binding sites (RNA-DNA duplexes and RNADNA:DNA
triplexes) in DNA, the cross-talk between different types of regulatory RNAs,
the logic and hierarchy of RNA- and protein-mediated regulation of gene expression, and
the extent, mechanisms and information content of RNA-mediated communication between
cells, both within318 and between organisms (‘social RNA’)343. Indeed it appears that RNA
is the computational engine of cell biology, developmental biology, brain function and
perhaps even evolution itself325. The complexity and interconnectedness of these systems
should not be cause for concern but rather the motivation for exploring the vast unknown
universe of RNA regulation, without which we will not understand biology.
Acknowledgments
This work was supported by NIH PO1 AI099783-01 and ARC Future Fellowship FT130100572 (KVM) and by
NHMRC Australia Fellowship 631688 (JSM).
Glossary
Antisense RNA A single stranded RNA that is complimentary to a messenger RNA or
a gene
ENCODE Encyclopaedia of DNA elements is a consortium of international
collaborators involved in building a comprehensive list of functional
elements in the human genome160,161
hnRNA Heterogenous nuclear RNA. Similar to messenger RNA, or premRNAs,
but retained predominantly in the nucleus
Intron A term first coined by Walter Gilbert to describe those nucleotide
regions in RNA that are removed, by being spliced out, to produce
messenger RNAs34,35
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lncRNA Long non-coding RNAs, first used to differentiate between smaller
forms of non-coding RNA, e.g. greater than 200bp in size358
piRNA Piwi associated RNAs. Small RNAs associated with the Piwi protein
complex and emanating from transposable like elements100
Pseudogene Relics of genes that have lost their protein coding potential but remain
transcribed and integrated within the genome359
PTGS Post-transcriptional gene silencing. Silencing a gene at the messenger
RNA or translational level, after transcription has occurred210
RNA-directed
DNA
methylation
An epigenetic process whereby processed double stranded small RNAs
(21–24bp) guide the methylation of homologous DNA loci52
siRNA Small interfering RNAs, double stranded RNAs that can be used to
suppress homology containing transcripts in a transcriptional and posttranscriptional
manner352
Transposons Mobile genetic elements262, with evolutionary links to retroviruses
tiRNAs Transcription initiation RNAs are small RNAs associated with
promoters with peak density ~10–30 nucleotides downstream of
transcriptional start sites121. Similar RNAs are derived from splice
sites (spliRNAs)122
TGS Transcriptional gene silencing. The regulation of a gene at the
transcriptional level
UTRs Untranslated regions, referring to either side, 5′ (leader sequence) or 3′
(trailer sequence) of a coding sequence on a strand of messenger RNA
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Figure 1. Complex expression of the genome and examples of non-coding RNA expression
Graphical representation of the mammalian transcriptional landscape with genes expressing
rRNAs, tRNAs, snRNAs, snoRNAs, various protein coding and non-coding transcripts
(mRNAs and lncRNAs), as well as small regulatory RNAs including miRNAs, piRNAs,
tiRNAs and spliRNAs, snoRNA-derived small RNAs, and tRNA-derived small RNAs.
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Figure 2. Functional pathways of small regulatory RNAs
(A) miRNA precursors are expressed as stem-loop structures75, which (B) interact with
Drosha76 and DGR8, where they are processed then exported from the nucleus by Exportin
5. (C) These transcripts are further processed by Dicer to small (21–23 nt) dsRNAs, one
strand of which is loaded into AGO component of the RNA-induced silencing complex
(RISC). Exogenously introduced siRNAs can also be processed by RISC. Either the
endogenous miRNA or exogenously added siRNAs can then to target (D) the repression of
translation and/or (E) cleavage of homology containing transcripts81,82. Some small RNAs
are functional in the nucleus. (F) Exogenously introduced small antisense RNAs (asRNAs)
can target epigenetic silencing of targeted loci88,344,345, a pathway that miRNAs may also
utilize in the nucleus92. (G) tiRNAs and spliRNAs121,122 are also expressed through an
unknown pathway that may involve RNAPII backtracking and TFIIS cleavage123, with the
tiRNAs shown to modulate CTCF chromatin localization and to be associated with
nucleosome position124.
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Figure 3. Various roles for lncRNAs in cellular regulation
(A) Long non-coding RNAs are expressed from many loci in the genome, sense and
antisense, intronic, overlapping and intergenic with respect to nearby protein-coding loci,
and function both in cis and trans. (B–E) Some lncRNAs interact with proteins to control
the access of chromatin to cellular components and/or guide epigenetic regulatory
complexes to target loci resulting in both (B) transcriptional suppression201 and (C)
activation or suppression (bimodal control)194. Proteins involved in chromatin modification
such as DNMT3a, EZH2 and PRC2 complexes have been associated with epigenetic
targeted lncRNA regulation194,201,277. (D) Some lncRNAs function to tether distal enhancer
elements with their promoters346,347. (E) LncRNAs can also function by binding proteins to
sequester them away from their sites of action (decoy lncRNAs)274 while other lncRNAs
can interact with each other and/or function to sequester small regulatory RNAs such as
miRNAs and therefore RISC targeting complexes away from protein-coding
mRNAs201,339,340. (G) LncRNAs can also act as translational inhibitors by binding and
sequestering mRNAs away from the translational machinery348 while other lncRNAs (H)
appear to regulate splicing232.
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Table 1
Timeline Table:
1941 One gene-one enzyme2
1953 Double helical structure of DNA described349
1958 Central dogma proposed by Francis Crick6
1961 mRNA confirmed as intermediate between protein and DNA9
1961 Jacob and Monod speculate that the lac repressor is an RNA10
1966 Discovery of heterogenous nuclear RNA27
1969 Model proposed for RNA acting in intermediate fashion in gene regulation28
1972 hnRNAs, chromosomal RNAs shown to be functional without making protein350
1977 Intron ncRNA elements defined34,35
1982–3 Self Splicing catalytic RNAs40,41
1989 Transgene silencing observed in plants71,72
1990 Transgene silencing linked to antisense RNA73
1990 H19 ncRNA discovered351
1992 Xist ncRNA discovered261, 262
1993 Lin-4 miRNA discovered46
1994 Regulatory RNAs proposed to be central to animal evolution and development152
1994 RNA directed DNA methylation observed in plants74
1998 RNA interference described in plants69 and animals352
1999 Tsix, antisense transcript to Xist described264
1999 Small RNA required for PTGS in plants353
2000 Let-7 miRNA discovered47
2001 Dicer described involved in RNAi77
2001 RNAi (PTGS) found functional in human cells354
2001 Regulatory RNA networks proposed to control epigenetic processes245,355
2002 First reports of large numbers of noncoding RNAs in animals153–155
2002 AIR antisense RNA involved in imprinting267
2003 Drosha described in miRNA processing76
2004 Small RNA shown to epigenetically control transcription (TGS) in human cells88
2004 Argonuate 2 directs catalysis in RNAi in mammals356
2005 piRNAs described100
2005 Confirmation of large numbers of long noncoding RNAs in mammals156,158,159
2005 ~70% of sense transcripts have antisense counterparts, some show function159
2005 Discovery of the CRISPR system of bacterial RNA-based defence134–136
2006 Antisense RNA TGS shown to require DNMT3a, EZH2, HDAC-1344
2006 Argonautes 1 and 2 found involved in RNA-directed TGS in human cells86,87
2006 ncRNAs involved in trithorax regulation285.
2007 HOTAIR shown to play a role in development and associate with polycomb194
2008 Long antisense RNAs found to epigenetically regulate sense counterparts256,257
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2008 LncRNAs shown to interact with trithorax and activated chromatin207
2008 Hundreds of lncRNAs shown to have specific expression in brain198
2009 tiRNAs reported at transcription start sites in mammals121
2009 PRC2 found to interact with a large number of lncRNAs179
2009 Long antisense RNA shown to direct vernalization in plants269
2010 Pseudogene lncRNAs found to regulate protein-coding genes166,340
2012 ENCODE reports ~80% of the genome is transcribing ncRNAs162
2013 Enhancer RNAs shown in oestrogen-dependent transcriptional activation357
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