1
© The Authors Journal compilation © 2013 Biochemical Society
Essays Biochem. (2013) 54, 1–16: doi: 10.1042/BSE0540001
1
The dark matter rises:
the expanding world of
regulatory RNAs
Michael B. Clark*†1, Anupma Choudhary*, Martin A. Smith*†,
Ryan J. Taft* and John S. Mattick†1
*Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
†Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW 2010, Australia
Abstract
The ability to sequence genomes and characterize their products has begun to
reveal the central role for regulatory RNAs in biology, especially in complex organisms.
It is now evident that the human genome contains not only protein-coding
genes, but also tens of thousands of non–protein coding genes that express small
and long ncRNAs (non-coding RNAs). Rapid progress in characterizing these
ncRNAs has identified a diverse range of subclasses, which vary widely in size,
sequence and mechanism-of-action, but share a common functional theme of
regulating gene expression. ncRNAs play a crucial role in many cellular pathways,
including the differentiation and development of cells and organs and, when misregulated,
in a number of diseases. Increasing evidence suggests that these
RNAs are a major area of evolutionary innovation and play an important role in
determining phenotypic diversity in animals.
1Correspondence may be addressed to either of these authors (email m.clark3@uq.edu.au or j.mattick@garvan.
org.au).
Keywords:
non-coding RNA, regulatory RNA, regulation of gene expression, small RNA.
2 Essays in Biochemistry volume 54 2013
© The Authors Journal compilation © 2013 Biochemical Society
Introduction
Recent advances in molecular biology, led by the large-scale sequencing of genomes and the
characterization of transcriptomes, have revealed that animal genomes are far more complex
and intricate than previously anticipated, containing a great diversity of sequences that can be
effectors of genetic information, i.e. genes. Central to this has been the discovery that many
genes do not encode proteins, but rather produce ncRNAs (non-coding RNAs), sometimes
referred to as genomic ‘dark matter’. Unlike in prokaryotes and most unicellular eukaryotes,
such as yeast, only a small fraction of the mammalian genome encodes protein, yet the vast
majority of the mammalian genome is transcribed, producing tens of thousands of small and
long ncRNAs [1,2].
The properties of RNA molecules, including their ability to form higher-order structures,
to specifically hybridize with other RNAs or DNA, and to assemble RNA–protein complexes,
makes them effective and versatile regulatory molecules that can direct relatively generic effector
proteins to sequence-specific targets [3]. The functional versatility of RNA has led to the
‘RNA world hypothesis’, which postulates that the dual catalytic and informational storage
properties of RNA provided the molecular platform for the evolution of early life [4]. While
proteins and DNA now fulfil most catalytic and information storage roles respectively, RNA
continues to have a variety of functional roles, including as a regulator, in all kingdoms of life
[4]. The emergence of multicellular life, however, has required increasingly complex regulatory
circuits to orchestrate the development and organization of specialized tissues and organs.
Hence, although prokaryotes and unicellular eukaryotes do contain regulatory ncRNAs, the
role of ncRNAs as regulators of gene expression has seemingly expanded in the genomes of
multicellular organisms [5].
The explosion of ncRNA research has revealed that there is an abundance of small and
long ncRNAs involved in regulating almost all steps of gene expression, including, but not limited
to, chromatin modification, transcriptional control, mRNA degradation, translational efficiency
and splicing [6,7]. Hence, ncRNAs function in a wide range of cellular processes, play
crucial roles in development and disease, and may even play a central role in the evolution of
different species and complex organisms [8–10].
The animal genome and the non-coding
universe
For half a century, protein-centric convictions predicated on the central dogma of molecular
biology dominated the discipline. However, the completion of multiple genome sequencing
projects has revealed that protein-coding sequences encompass only a small fraction of animal
genomes and less than 2% of the genome in humans and other mammals [11,12]. Thus either
animal genomes are largely composed of ‘junk’ DNA, or they contain another form of genetic
information that has thus far been overlooked. Several lines of evidence support the latter,
including the positive correlation between the proportion of the genome that is ‘junk’/nonprotein-coding
and developmental complexity [5], the presence of extensive conserved noncoding
sequences outside protein-coding regions [13], and the pervasive differential
transcription of the vast majority of the genome [1,14].
M.B. Clark and others 3
© 2013 Biochemical Society
Characterization of the mammalian transcriptome has revealed that RNA transcripts are
produced from a considerably greater proportion of the genome than the ~40% covered
(including introns and exons) by known genes [1,15,16]. This pervasive transcription of the
genome, defined as occurring when “the majority of (the genome’s) bases are associated with at
least one primary transcript” [17] has been identified by a number of independent techniques,
including genome-wide tiling arrays, large-scale cloning and sequencing of cDNAs, and nextgeneration
RNA sequencing. For example, results from the ENCODE project, which aimed to
identify all functional elements within the human genome, demonstrated that at least 75% of
bases were transcribed [1]. A number of studies have shown that although the vast majority of
the genome is transcribed at some level, most transcription, including novel unannotated transcription,
clusters around known genes [16,18–20]. Such analyses led Kapranov et al. [19] to
propose “a model of genome organization where protein–coding genes are at the center of a
complex network of overlapping sense and antisense (long) RNA transcription, with interleaved
(small) RNAs” (Figure 1).
The net result of pervasive transcription is a complex and interleaved transcriptome producing
approximately 20000 coding genes, along with at least as many, and possibly a much
greater number of, transcripts that do not encode proteins and could function at the RNA level
[2]. These can be separated into two major groups (the small and long ncRNAs) on the basis of
size and mechanism of synthesis.
Small ncRNAs
Small RNAs are generally defined as ncRNAs shorter than 200 nt in length, and are usually
produced by the post–transcriptional processing of longer transcripts by endogenous
Figure 1. Pervasive transcription around a hypothetical protein coding gene
A standard mRNA transcript is shown in blue, other coding or potentially coding transcripts are
shown in green, and non-coding transcripts are in red. Introns are represented by thin lines,
non-coding exons are indicated by medium thickness lines and coding exons are indicated by
the thickest lines. Arrows represent transcription start sites. The absence of an arrow indicates
that the transcripts are generated by processing. An arrow plus a question mark refers to
transcripts where the origin is often unclear, and could involve transcription initiation or
processing. PALR, promoter-associated long RNA [19]; PASR, promoter-associated small
RNA [19]; PROMPT, promoter upstream transcript [51]; snoRNA, small nucleolar RNA; TASR,
termini-associated small RNA [19]; tiRNA, transcription initiation (tiny) RNA [49]; TSS,
transcription start site; uaRNA, 3′-UTR-derived RNAs [118].
4 Essays in Biochemistry volume 54 2013
© The Authors Journal compilation © 2013 Biochemical Society
RNases (i.e. RNA cleavage enzymes). Based primarily on their size, mode of biogenesis and
function, small RNAs can be divided into various subclasses. The three subclasses of small
RNAs that have thus far received the most attention are those that participate in RNAi (RNA
interference) pathways, namely, miRNAs (microRNAs), siRNAs (small interfering RNAs)
and piRNAs (piwi-interacting RNAs), which produce mature RNAs ~20–30 nt in length.
Nucleotide sequence complementarity lies at the heart of the widespread and potent regulatory
control that these small RNAs exert on their targets. In all known RNAi pathways, this
regulatory control is mediated by binding of the small RNA to a complex of proteins, chief
among them being the Argonaute proteins. There are also slightly larger small RNA species
(~100 nt) that have important cellular roles, including snoRNAs (small nucleolar RNAs),
which guide RNA base modification [21] [and can also be processed into other classes of
regulatory small RNA, including miRNAs and sdRNAs (sno-derived RNAs)] [22]; snRNAs
(small nuclear RNAs), which mediate RNA splicing and are important components of the
spliceosome [23]; Y RNAs, which appear to regulate the Ro autoantigen [24]; and vault
RNAs, components of the vault ribonucleoprotein complex [25]. Most small ncRNAs function
as part of RNA–protein complexes to regulate gene expression, with the small RNA
often acting to specify the target for regulation through nucleotide sequence complementarity
(Figures 2A and 2B) [26].
miRNAs are ~22 nt long and bind to short regions of complementary sequence, usually
located in the 3′ UTR (untranslated region), of target mRNAs [27]. miRNAs can bring about
the translational repression or degradation of target transcripts, depending on the extent of
complementarity between them (Figure 2A) [26]. The ability of these small RNAs to modulate
gene expression was first identified 20 years ago with the discovery of lin–4 in the nematode
worm Caenorhabditis elegans [28,29]. Since then, the miRNA field has evolved rapidly and
there are now over 1500 miRNAs annotated in the human genome [30]. Mature miRNAs are
produced as part of a two-step enzymatic process from a longer pri-miRNA (primary miRNA)
that is processed into a pre-miRNA (precursor miRNA) in the nucleus. The pre-miRNA is
then transported to the cytoplasm where it is cleaved to release mature miRNAs [31]. The
mature miRNA is then loaded on to the RISC (RNA-induced silencing complex), which is
composed of the Argonaute2 protein, the miRNA and other auxiliary proteins [26]. The significance
of miRNAs in biological processes is highlighted by the fact that one miRNA can potentially
target, and hence control the expression of, many hundreds of mRNAs [32,33]. miRNAs
can also regulate transcription through mechanisms that are not yet fully understood in mammalian
cells [34]. However, it seems likely that there is more to miRNA function than just the
inhibition of translation. There are increasing reports describing the presence of mature miRNAs
in the nucleus [35,36], suggesting that they may also be directly or indirectly involved in
transcriptional gene silencing.
siRNAs are ~21 nt long and function mainly by degrading transcripts they have perfect
sequence complementarity to. The precursor transcripts of endogenous siRNAs include dsRNAs
(double-stranded RNAs), pseudogenes [37] or transcripts with very long stem-loop structures
[38,39]. Endogenous siRNAs have been proposed to protect eukaryotic cells from dsRNA
viruses and are also important for silencing transposons and other ‘selfish’ genomic elements
[40,41]. Since siRNAs use the same enzymatic machinery as miRNAs to function, synthetic
siRNAs can be introduced into cells to ‘knock-down’ any given gene, a feature that has been
exploited in scientific research and in a number of recently developed therapeutics [42].
M.B. Clark and others 5
© 2013 Biochemical Society
Figure 2. Examples of ncRNA function
(A) Transcriptional and translational regulation by miRNAs (shown in blue), which are expressed
in nearly every tissue and cell type in complex animals, most simple animals, plants and fungi.
Mature miRNAs are loaded into an Argonaute (Ago) protein-containing RISC complex in the
cytoplasm. Argonaute proteins have also been reported to function in the nucleus of various
organisms. (B) piRNA silencing of transposons in germ cells. piRNAs (shown in blue and white)
bind to PIWI proteins (shown in green) in the germ line and cause the degradation of
transposon transcripts. (C) Some potential functions of a lncRNA. Folded lncRNA is shown in
red. Proteins are shown in white or grey. miRNAs are in blue. The series of A on the target
mRNAs represents the polyA tail. DNA is shown as a double helix.
6 Essays in Biochemistry volume 54 2013
© The Authors Journal compilation © 2013 Biochemical Society
piRNAs are a related class of small (28–32 nt) RNAs that are found only in animals and
principally expressed in spermatids [43]. piRNAs are found in clusters in the genome, and
appear to arise from long single-stranded precursor transcripts [44,45]. Unlike miRNAs and
siRNAs, which bind to the Ago subclade of the Argonaute proteins, piRNAs associate with the
Piwi subclass of Argonaute proteins. They have an important role in suppressing the expression
of repetitive elements by guiding DNA methylation, and have been shown to be involved
in gametogenesis (Figure 2B) [46,47], as well as in neuronal plasticity in the sea hare, Aplysia
[48].
Apart from these major categories, other small ncRNAs have been described that originate
from regions adjacent to transcription start-sites, such as tiRNAs (transcription initiation
RNAs). These RNAs are ~17–18 nt long and are abundant at active promoters as well as at loci
with evidence of bidirectional transcription [49], and have been shown to influence the epigenetic
state of the genomic region from which they are derived [50]. RNAs of similar size
[spliRNAs (splice site RNAs)] are also associated with splice sites [36]. Other, but less welldefined,
small RNAs found at or near transcription start-sites that have been reported include
PASRs (promoter-associated small RNAs) [19], PROMPTs (promoter upstream transcripts)
[51], TSSa-RNAs (transcription start-site-associated RNAs) [52] and TASRs (gene terminiassociated
small RNAs) [19].
Consistent with their role in gene regulation, small RNAs are involved in many cellular
processes and their dysfunction is implicated in a number of physiological and developmental
defects [53]. For example, aberrant expression of miRNAs is implicated in a wide variety of
diseases ranging from disorders of the heart to immune diseases and cancer [54]. Additionally,
a snoRNA has been demonstrated to play a central role in Prader–Willi syndrome [55],
whereas another has been associated with autism [56].
lncRNAs (long ncRNAs)
lncRNAs are generally defined as ranging in size from ~200 nt to over 100 kb in length [57,58].
Although the 200 nt cut-off for lncRNAs is quite arbitrary, it has the advantage of excluding
most transcripts accepted to be members of small RNA classes. Unlike small ncRNAs, lncRNAs
cannot be easily divided into different subclasses on the basis of sequence characteristics and
mode-of-action, and this inability to classify lncRNAs into different subtypes has contributed to
their current arbitrary definition. We have previously suggested a more flexible definition that
lncRNAs are “noncoding RNAs that may have a function as either primary or spliced transcripts,
which are independent of processing into known classes of small RNAs, such as miRNAs, piRNAs
and snoRNAs, while also excluding structural RNAs from classical housekeeping families” [59],
such as rRNAs.
The presence of lncRNAs with important functions has been known for some time, with
the characterization of lncRNAs such as Xist (X-inactive specific transcript, which controls the
silencing of one X chromosome in female mammals) in the 1990s [60,61]. However, the first
database of eukaryotic lncRNAs had less than 12 entries by the end of the millennium [62].
The identification of thousands of putative lncRNA transcripts from genome-wide transcriptome
analysis [15,63,64], along with prominent examples of functional lncRNAs [65–67],
demonstrated that lncRNAs such as Xist were not rare genomic oddities, but were instead the
first characterized examples of a large class of novel genes. By the end of 2010 over 100
M.B. Clark and others 7
© 2013 Biochemical Society
lncRNAs had been functionally characterized as part of a surge of research into the lncRNA
world [59]. The subset of lncRNAs transcribed from intergenic regions (i.e. genomic loci some
distance from and not overlapping protein-coding genes) have received the most recent attention
and are known as lincRNAs (long/large intergenic ncRNAs). Along with those from intergenic
loci, lncRNAs are transcribed from many other regions of the genome including
promoters, enhancers, introns, UTRs, as overlapping or non-coding isoforms of coding genes,
antisense to other transcripts and from pseudogenes [15,68–70].
Although often of similar length to mRNAs, there are a number of differences between
mRNAs and lncRNAs beyond the absence of a functional ORF (open reading frame) in the latter.
Analysis of lncRNA expression has shown they have lower expression levels and are more
likely to be expressed in highly tissue- and cell-specific patterns [63,71–73]. Unlike most
mRNAs and many small ncRNAs, lncRNAs are not as highly conserved, although they do
show evidence of conservation in their promoters, primary sequences and splice sites
[15,71,73–75]. Furthermore, many lncRNAs consist of a single exon and those that are spliced
have fewer exons than protein-coding genes [63,72,73]. lncRNAs also commonly contain
transposable elements and other repeats [73]. Sequences from such genetic elements can be
‘domesticated’ during evolution and contribute to lncRNA function by promoting their expression
and providing functional motifs [76].
lncRNAs carry out a diverse range of functions in the cell (Figure 2C). Although few are
reported to function catalytically, many carry out RNA–protein, RNA–DNA and RNA–RNA
interactions. Similar to many small ncRNAs, lncRNAs can regulate gene expression via RNA–
protein (ribonucleoprotein) complexes (Figure 2C) [77]. A common function of lncRNAs
appears to be directing the activity of chromatin-modifying complexes and transcription factors
by specifying their genomic DNA targets and activating or inhibiting their function
[67,78–83]. In these and other contexts, lncRNAs have the ability to act as scaffolds, nucleating
the assembly of larger complexes or cellular structures [84–86]. Other reported lncRNA functions
include acting as miRNA sponges to ‘soak up’ miRNAs, relieving the repression of
mRNAs and so controlling mRNA expression levels and mRNA translation [87,88].
lncRNAs can function both locally and in trans. An example of the former is Airn, which
silences the expression of neighbouring genes to regulate imprinting of the Igf2r (insulin-like
growth factor 2 receptor) locus [57,65]. Trans-acting lncRNAs include HOTAIR, which is
expressed from the HOXC locus and acts to silence gene expression at many genomic locations,
including the HOXD locus, by recruiting repressive chromatin modification complexes [67,89].
Given their range of functions, a number of lncRNAs are also implicated or involved in
disease states, including functioning as oncogenes or tumour suppressors [87,90,91], as well
as being linked to other complex diseases such as myocardial infarction [92] and Alzheimer’s
disease [93].
Regulatory RNAs in prokaryotes
The versatility of RNA as a regulator has also been used by prokaryotes, which contain numerous
ncRNAs. Similar to eukaryotes, prokaryotic ncRNAs are being found to play an increasingly
important role in regulating gene expression [94]. Despite these similarities, few ncRNA
classes are shared between prokaryotes and eukaryotes, with the exception of snoRNAs, which
are present in archaea (although not in bacteria) [95].
8 Essays in Biochemistry volume 54 2013
© The Authors Journal compilation © 2013 Biochemical Society
Many prokaryotic sRNAs (small RNAs; abbreviation only used in prokaryotes) and antisense
RNAs function as ncRNAs. sRNAs are generally defined as transcripts <500 nt and can
be expressed from any region of the genome. Antisense RNAs include both sRNAs and longer
RNAs that are antisense to coding genes, creating some overlap between the sRNA and antisense
RNA classes. ncRNAs are common in prokaryotic genomes, with approximately
170 non-coding sRNAs predicted in the archaea Methanosarcina mazei [96], ~50 in the bacteria
Listeria monocytogenes [97] and 165 in Pseudomonas aeruginosa [98]. In comparison,
Pseudomonas was found to contain 384 antisense RNAs [98], whereas Helicobacter pylori
was reported to have antisense transcription across most of the genome, covering 46% of
ORFs [99].
Prokaryotic regulatory RNAs function by a variety of mechanisms (reviewed in
[94,100]). Cis-antisense ncRNAs commonly act to repress the expression of the sense coding
gene. Repression can occur either at the level of transcription, RNA turnover or by inhibiting
translation, with the extensive nucleotide complementarity between the two important for
many of these mechanisms. Examples include ncRNAs regulating the copy number of mobile
elements such as plasmids and repressing the translation of toxic proteins, such as the SymR
antisense sRNA in Escherichia coli that represses the synthesis of the SymE toxin protein
[101,102].
Trans-encoded sRNAs generally act to repress translation or destabilize target RNA(s),
demonstrating some functional similarity to eukaryotic miRNAs. With more limited complementarity
than cis-antisense RNAs, trans RNAs often require the RNA chaperone protein Hfq
to bind their targets [94]. An example is the association of four sRNAs with Hfq in Vibrio cholerae
to control quorum sensing by destabilizing the mRNA of the quorum-sensing master regulator
[103]. Some sRNAs also bind directly to proteins by mimicking other nucleic acid
sequences. For example, the 6S sRNA mimics the structure of an open promoter to bind RNA
polymerase and regulate transcription [94,104].
Another important class of prokaryotic ncRNAs are CRISPRs (clustered regularly interspersed
short palindromic repeats). First discovered in E. coli [105], CRISPRs are now known
to exist in most bacteria and archaea [106]. CRISPR loci contain short direct repeats interspersed
with spacer regions derived from invading mobile elements. CRISPRs are transcribed
and processed to generate small crRNAs (CRISPR RNAs), which function to protect the cell
from invading bacteriophages and conjugative plasmids [106,107].
The role of ncRNAs in evolutionary
innovation
The sequencing and initial annotation of mammalian genomes provided two large surprises:
the large fraction of the genome comprised of sequences derived from transposable elements
and the much lower than expected number of protein-coding genes [11,12]. In fact, the number
of recognized human protein-coding genes (20687) [2] is similar to that in the nematode
worm C. elegans (20517) [108] and that in a basal metazoan, the sponge Amphimedon queenslandica
(18500–30000) [109]. Furthermore, much of the protein-coding ‘toolkit’ that controls
multicellular processes in more complex animals is also present in the sponge [109]. There is
widespread use of alternative splicing in human genes [110], which can diversify the proteome
M.B. Clark and others 9
© 2013 Biochemical Society
without an increase in gene number, suggesting that it is one mechanism to explain differences
in complexity. However, alternative splicing itself requires regulation and hence it has been
hypothesized that increases in gene regulatory complexity underlie much of morphological
complexity [5,8,10].
Moreover, given the relatively stable protein-coding complement, it is clear that most evolutionary
adaptation occurs in regulatory sequences, which are fast evolving and show little
conservation over long evolutionary distances [111–113]. The discovery that most of this
non-coding DNA is dynamically transcribed to generate tens of thousands of ncRNAs
[1,15,16] provides a hitherto unexpected mechanism to explain this increase in regulatory
complexity.
ncRNAs, with their potential to bind DNA, RNA and protein in a sequence- or structurespecific
manner, are versatile and effective regulatory molecules. By providing specificity to
generic protein complexes [3], ncRNAs can act as guides to selectively target effector proteins
to different loci and thereby regulate the transcription or translation of many genes [90,114].
Lastly the pervasive transcription of different ncRNAs in the genome provides a large
dynamic pool of transcripts for selection to act upon, as most ncRNAs are subject to more
flexible structure–function constraints than protein-coding RNAs [17,111,115]. For instance,
many ncRNAs function via the formation of stable secondary and tertiary structures, which
can accommodate compensatory nucleotide substitutions, e.g. A:U base pairs to G:U or G:C,
without disrupting their structural (and thus functional) integrity [116]. Moreover, regulatory
sequences are also subject to positive selection for adaptive radiation [117]. The overarching
conclusion is that regulatory ncRNAs represent a vast hidden layer of evolutionarily plastic cisand
trans-acting regulatory information that directs the epigenetic pathways that underpin
animal development and diversity [8–10].
Conclusions
The last decade has revolutionized our understanding of genomes and what constitutes a gene.
It has become increasingly apparent that many cellular functions are mediated by RNA, a realization
that has far-reaching implications for understanding human biology and treating
human disease. The following chapters outline the state-of-the-art in the characterization of
various types of ncRNAs, although the continuing rapid pace of discovery and unknown function
of so many ncRNAs makes it clear that much remains to be done before this poorly
charted sphere of biology is fully explored.
Summary
• Non-coding RNA genes are abundant in the genome, with similar numbers
of protein-coding and non-coding genes in humans.
• Non-coding RNAs are structurally diverse, ranging from less than 20 nt to
over 100 kb in length.
• The properties of RNA molecules allow them to function through both
sequence complementarity to other RNAs or DNA, as well as forming structures
that can interact with proteins and/or nucleic acids.
10 Essays in Biochemistry volume 54 2013
© The Authors Journal compilation © 2013 Biochemical Society
The authors acknowledge the support of the Australian NHMRC (National Health and
Medical Research Council) [NHMRC Australia Fellowship number 631668 (to J.S.M.)],
the Australian Research Council [DECRA Fellowship (to R.J.T.)] and the University of
Queensland [University of Queensland International Research Tuition Award and
University of Queensland Research Scholarship (to A.C.)].
References
1. Djebali, S., Davis, C.A., Merkel, A., Dobin, A., Lassmann, T., Mortazavi, A., Tanzer, A.,
Lagarde, J., Lin, W., Schlesinger, F. et al. (2012) Landscape of transcription in human cells.
Nature 489, 101–108
2. Harrow, J., Frankish, A., Gonzalez, J.M., Tapanari, E., Diekhans, M., Kokocinski, F., Aken,
B.L., Barrell, D., Zadissa, A., Searle, S. et al. (2012) GENCODE: the reference human genome
annotation for The ENCODE Project. Genome Res. 22, 1760–1774
3. Huttenhofer, A. and Schattner, P. (2006) The principles of guiding by RNA: chimeric RNAprotein
enzymes. Nat. Rev. Genet. 7, 475–482
4. Atkins, J.F., Gesteland, R.F. and Cech, T. (2011) RNA worlds: from life’s origins to diversity in
gene regulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
5. Taft, R.J., Pheasant, M. and Mattick, J.S. (2007) The relationship between non-protein-coding
DNA and eukaryotic complexity. BioEssays 29, 288–299
6. Amaral, P.P., Dinger, M.E., Mercer, T.R. and Mattick, J.S. (2008) The eukaryotic genome as an
RNA machine. Science 319, 1787–1789
7. Brosnan, C.A. and Voinnet, O. (2009) The long and the short of noncoding RNAs. Curr. Opin.
Cell Biol. 21, 416–425
8. Mattick, J.S. and Makunin, I.V. (2006) Non-coding RNA. Hum. Mol. Genet. 15, R17–R29
9. Prasanth, K.V. and Spector, D.L. (2007) Eukaryotic regulatory RNAs: an answer to the
‘genome complexity’ conundrum. Genes Dev. 21, 11–42
10. Mattick, J.S. (2011) The central role of RNA in human development and cognition. FEBS Lett.
585, 1600–1616
11. Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M.C., Baldwin, J., Devon, K.,
Dewar, K., Doyle, M., FitzHugh, W. et al. (2001) Initial sequencing and analysis of the human
genome. Nature 409, 860–921
12. Waterston, R.H., Lindblad-Toh, K., Birney, E., Rogers, J., Abril, J.F., Agarwal, P., Agarwala, R.,
Ainscough, R., Alexandersson, M., An, P. et al. (2002) Initial sequencing and comparative
analysis of the mouse genome. Nature 420, 520–562
13. Stephen, S., Pheasant, M., Makunin, I.V. and Mattick, J.S. (2008) Large-scale appearance of
ultraconserved elements in tetrapod genomes and slowdown of the molecular clock. Mol.
Biol. Evol. 25, 402–408
• Many well-characterized subclasses of small non-coding RNAs are known,
with each member of a subclass having a similar functional mechanism,
whereas the subclasses and mechanism-of-action of long non-coding
RNAs are much less well understood.
• Most functionally characterized non-coding RNAs (whether small or long)
function in the regulation of gene expression.
• Non-coding RNAs play essential roles in many biological processes and are
crucial for development and disease, and perhaps even the evolution of
organisms.
M.B. Clark and others 11
© 2013 Biochemical Society
14. Clark, M.B., Amaral, P.P., Schlesinger, F.J., Dinger, M.E., Taft, R.J., Rinn, J.L., Ponting, C.P.,
Stadler, P.F., Morris, K.V., Morillon, A. et al. (2011) The reality of pervasive transcription. PLoS
Biol. 9, e1000625
15. Carninci, P., Kasukawa, T., Katayama, S., Gough, J., Frith, M.C., Maeda, N., Oyama, R.,
Ravasi, T., Lenhard, B., Wells, C. et al. (2005) The transcriptional landscape of the mammalian
genome. Science 309, 1559–1563
16. Cheng, J., Kapranov, P., Drenkow, J., Dike, S., Brubaker, S., Patel, S., Long, J., Stern, D.,
Tammana, H., Helt, G. et al. (2005) Transcriptional maps of 10 human chromosomes at
5–nucleotide resolution. Science 308, 1149–1154
17. Birney, E., Stamatoyannopoulos, J.A., Dutta, A., Guigo, R., Gingeras, T.R., Margulies,
E.H., Weng, Z., Snyder, M., Dermitzakis, E.T., Thurman, R.E. et al. (2007) Identification and
analysis of functional elements in 1% of the human genome by the ENCODE pilot project.
Nature 447, 799–816
18. Kampa, D., Cheng, J., Kapranov, P., Yamanaka, M., Brubaker, S., Cawley, S., Drenkow, J.,
Piccolboni, A., Bekiranov, S., Helt, G. et al. (2004) Novel RNAs identified from an in-depth
analysis of the transcriptome of human chromosomes 21 and 22. Genome Res. 14, 331–342
19. Kapranov, P., Cheng, J., Dike, S., Nix, D.A., Duttagupta, R., Willingham, A.T., Stadler, P.F.,
Hertel, J., Hackermuller, J., Hofacker, I.L. et al. (2007) RNA maps reveal new RNA classes
and a possible function for pervasive transcription. Science 316, 1484–1488
20. Fejes-Toth, K., Sotirova, V., Sachidanandam, R., Assaf, G., Hannon, G.J., Kapranov, P.,
Foissac, S., Willingham, A.T., Duttagupta, R., Dumais, R. and Gingeras, T.R. (2009) Posttranscriptional
processing generates a diversity of 5′-modified long and short RNAs. Nature
457, 1028–1032
21. Bachellerie, J.P., Cavaille, J. and Huttenhofer, A. (2002) The expanding snoRNA world.
Biochimie 84, 775–790
22. Taft, R.J., Glazov, E.A., Lassmann, T., Hayashizaki, Y., Carninci, P. and Mattick, J.S. (2009)
Small RNAs derived from snoRNAs. RNA 15, 1233–1240
23. Wachtel, C. and Manley, J.L. (2009) Splicing of mRNA precursors: the role of RNAs and
proteins in catalysis. Mol. Biosyst. 5, 311–316
24. Sim, S., Weinberg, D.E., Fuchs, G., Choi, K., Chung, J. and Wolin, S.L. (2009) The subcellular
distribution of an RNA quality control protein, the Ro autoantigen, is regulated by noncoding
Y RNA binding. Mol. Biol. Cell 20, 1555–1564
25. Berger, W., Steiner, E., Grusch, M., Elbling, L. and Micksche, M. (2009) Vaults and the major vault
protein: novel roles in signal pathway regulation and immunity. Cell. Mol. Life Sci. 66, 43–61
26. Ghildiyal, M. and Zamore, P.D. (2009) Small silencing RNAs: an expanding universe. Nat. Rev.
Genet. 10, 94–108
27. Lai, E.C. (2002) Micro RNAs are complementary to 3′ UTR sequence motifs that mediate
negative post-transcriptional regulation. Nat. Genet. 30, 363–364
28. Wightman, B., Ha, I. and Ruvkun, G. (1993) Posttranscriptional regulation of the heterochronic
gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75, 855–862
29. Lee, R.C., Feinbaum, R.L. and Ambros, V. (1993) The C. elegans heterochronic gene lin-4
encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854
30. Kozomara, A. and Griffiths-Jones, S. (2011) miRBase: integrating microRNA annotation and
deep-sequencing data. Nucleic Acids Res 39, D152–D157
31. Lee, Y., Jeon, K., Lee, J.T., Kim, S. and Kim, V.N. (2002) MicroRNA maturation: stepwise
processing and subcellular localization. EMBO J. 21, 4663–4670
32. Landgraf, P., Rusu, M., Sheridan, R., Sewer, A., Iovino, N., Aravin, A., Pfeffer, S., Rice, A.,
Kamphorst, A.O., Landthaler, M. et al. (2007) A mammalian microRNA expression atlas based
on small RNA library sequencing. Cell 129, 1401–1414
33. Friedman, R.C., Farh, K.K., Burge, C.B. and Bartel, D.P. (2009) Most mammalian mRNAs are
conserved targets of microRNAs. Genome Res. 19, 92–105
34. Khraiwesh, B., Arif, M.A., Seumel, G.I., Ossowski, S., Weigel, D., Reski, R. and Frank, W.
(2010) Transcriptional control of gene expression by microRNAs. Cell 140, 111–122
12 Essays in Biochemistry volume 54 2013
© The Authors Journal compilation © 2013 Biochemical Society
35. Hwang, H.W., Wentzel, E.A. and Mendell, J.T. (2007) A hexanucleotide element directs
microRNA nuclear import. Science 315, 97–100
36. Taft, R.J., Simons, C., Nahkuri, S., Oey, H., Korbie, D.J., Mercer, T.R., Holst, J., Ritchie, W.,
Wong, J.J., Rasko, J.E. et al. (2010) Nuclear-localized tiny RNAs are associated with
transcription initiation and splice sites in metazoans. Nat. Struct. Mol. Biol. 17, 1030–1034
37. Watanabe, T., Totoki, Y., Toyoda, A., Kaneda, M., Kuramochi-Miyagawa, S., Obata, Y., Chiba,
H., Kohara, Y., Kono, T., Nakano, T. et al. (2008) Endogenous siRNAs from naturally formed
dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539–543
38. Czech, B., Malone, C.D., Zhou, R., Stark, A., Schlingeheyde, C., Dus, M., Perrimon, N.,
Kellis, M., Wohlschlegel, J.A., Sachidanandam, R. et al. (2008) An endogenous small
interfering RNA pathway in Drosophila. Nature 453, 798–802
39. Okamura, K., Balla, S., Martin, R., Liu, N. and Lai, E.C. (2008) Two distinct mechanisms
generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster.
Nat. Struct. Mol. Biol. 15, 581–590
40. Kim, V.N., Han, J. and Siomi, M.C. (2009) Biogenesis of small RNAs in animals. Nat. Rev. Mol.
Cell Biol. 10, 126–139
41. Chung, W.J., Okamura, K., Martin, R. and Lai, E.C. (2008) Endogenous RNA interference
provides a somatic defense against Drosophila transposons. Curr. Biol. 18, 795–802
42. Li, S.D., Chono, S. and Huang, L. (2008) Efficient oncogene silencing and metastasis
inhibition via systemic delivery of siRNA. Mol. Ther. 16, 942–946
43. Aravin, A., Gaidatzis, D., Pfeffer, S., Lagos-Quintana, M., Landgraf, P., Iovino, N., Morris, P.,
Brownstein, M.J., Kuramochi-Miyagawa, S., Nakano, T. et al. (2006) A novel class of small
RNAs bind to MILI protein in mouse testes. Nature 442, 203–207
44. Thomson, T. and Lin, H. (2009) The biogenesis and function of PIWI proteins and piRNAs:
progress and prospect. Annu. Rev. Cell Dev. Biol. 25, 355–376
45. Brennecke, J., Aravin, A.A., Stark, A., Dus, M., Kellis, M., Sachidanandam, R. and Hannon,
G.J. (2007) Discrete small RNA-generating loci as master regulators of transposon activity in
Drosophila. Cell 128, 1089–1103
46. Houwing, S., Kamminga, L.M., Berezikov, E., Cronembold, D., Girard, A., van den Elst, H.,
Filippov, D.V., Blaser, H., Raz, E., Moens, C.B. et al. (2007) A role for Piwi and piRNAs in germ
cell maintenance and transposon silencing in Zebrafish. Cell 129, 69–82
47. Aravin, A.A., Sachidanandam, R., Bourc’his, D., Schaefer, C., Pezic, D., Toth, K.F., Bestor, T.
and Hannon, G.J. (2008) A piRNA pathway primed by individual transposons is linked to
de novo DNA methylation in mice. Mol. Cell 31, 785–799
48. Rajasethupathy, P., Antonov, I., Sheridan, R., Frey, S., Sander, C., Tuschl, T. and Kandel, E.R.
(2012) A role for neuronal piRNAs in the epigenetic control of memory-related synaptic
plasticity. Cell 149, 693–707
49. Taft, R.J., Glazov, E.A., Cloonan, N., Simons, C., Stephen, S., Faulkner, G.J., Lassmann, T.,
Forrest, A.R., Grimmond, S.M., Schroder, K. et al. (2009) Tiny RNAs associated with
transcription start sites in animals. Nat. Genet. 41, 572–578
50. Taft, R.J., Hawkins, P.G., Mattick, J.S. and Morris, K.V. (2011) The relationship between
transcription initiation RNAs and CCCTC-binding factor (CTCF) localization. Epigenetics
Chromatin 4, 13
51. Preker, P., Nielsen, J., Kammler, S., Lykke-Andersen, S., Christensen, M.S., Mapendano,
C.K., Schierup, M.H. and Jensen, T.H. (2008) RNA exosome depletion reveals transcription
upstream of active human promoters. Science 322, 1851–1854
52. Seila, A.C., Calabrese, J.M., Levine, S.S., Yeo, G.W., Rahl, P.B., Flynn, R.A., Young, R.A.
and Sharp, P.A. (2008) Divergent transcription from active promoters. Science 322,
1849–1851
53. Esteller, M. (2011) Non-coding RNAs in human disease. Nat. Rev. Genet. 12, 861–874
54. Boyd, S.D. (2008) Everything you wanted to know about small RNA but were afraid to ask.
Lab. Invest. 88, 569–578
M.B. Clark and others 13
© 2013 Biochemical Society
55. Sahoo, T., del Gaudio, D., German, J.R., Shinawi, M., Peters, S.U., Person, R.E., Garnica, A.,
Cheung, S.W. and Beaudet, A.L. (2008) Prader–Willi phenotype caused by paternal deficiency
for the HBII-85 C/D box small nucleolar RNA cluster. Nat. Genet. 40, 719–721
56. Nakatani, J., Tamada, K., Hatanaka, F., Ise, S., Ohta, H., Inoue, K., Tomonaga, S., Watanabe,
Y., Chung, Y.J., Banerjee, R. et al. (2009) Abnormal behavior in a chromosome-engineered
mouse model for human 15q11-13 duplication seen in autism. Cell 137, 1235–1246
57. Lyle, R., Watanabe, D., te Vruchte, D., Lerchner, W., Smrzka, O.W., Wutz, A., Schageman, J.,
Hahner, L., Davies, C. and Barlow, D.P. (2000) The imprinted antisense RNA at the Igf2r locus
overlaps but does not imprint Mas1. Nat. Genet. 25, 19–21
58. Furuno, M., Pang, K.C., Ninomiya, N., Fukuda, S., Frith, M.C., Bult, C., Kai, C., Kawai, J.,
Carninci, P., Hayashizaki, Y. et al. (2006) Clusters of internally primed transcripts reveal novel
long noncoding RNAs. PLoS Genet. 2, e37
59. Amaral, P.P., Clark, M.B., Gascoigne, D.K., Dinger, M.E. and Mattick, J.S. (2011) lncRNAdb: a
reference database for long noncoding RNAs. Nucleic Acids Res. 39, D146–D151
60. Brown, C.J., Hendrich, B.D., Rupert, J.L., Lafreniere, R.G., Xing, Y., Lawrence, J. and Willard,
H.F. (1992) The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains
conserved repeats and is highly localized within the nucleus. Cell 71, 527–542
61. Penny, G.D., Kay, G.F., Sheardown, S.A., Rastan, S. and Brockdorff, N. (1996) Requirement
for Xist in X chromosome inactivation. Nature 379, 131–137
62. Erdmann, V.A., Szymanski, M., Hochberg, A., de Groot, N. and Barciszewski, J. (1999)
Collection of mRNA-like non-coding RNAs. Nucleic Acids Res. 27, 192–195
63. Ravasi, T., Suzuki, H., Pang, K.C., Katayama, S., Furuno, M., Okunishi, R., Fukuda, S.,
Ru, K., Frith, M.C., Gongora, M.M. et al. (2006) Experimental validation of the regulated
expression of large numbers of non-coding RNAs from the mouse genome. Genome Res
16, 11–19
64. Guttman, M., Amit, I., Garber, M., French, C., Lin, M.F., Feldser, D., Huarte, M., Zuk, O.,
Carey, B.W., Cassady, J.P. et al. (2009) Chromatin signature reveals over a thousand highly
conserved large non-coding RNAs in mammals. Nature 458, 223–227
65. Sleutels, F., Zwart, R. and Barlow, D.P. (2002) The non-coding Air RNA is required for
silencing autosomal imprinted genes. Nature 415, 810–813
66. Ji, P., Diederichs, S., Wang, W., Boing, S., Metzger, R., Schneider, P.M., Tidow, N., Brandt,
B., Buerger, H., Bulk, E. et al. (2003) MALAT–1, a novel noncoding RNA, and thymosin β4
predict metastasis and survival in early–stage non–small cell lung cancer. Oncogene 22,
8031–8041
67. Rinn, J.L., Kertesz, M., Wang, J.K., Squazzo, S.L., Xu, X., Brugmann, S.A., Goodnough,
L.H., Helms, J.A., Farnham, P.J., Segal, E. and Chang, H.Y. (2007) Functional demarcation
of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129,
1311–1323
68. Engstrom, P.G., Suzuki, H., Ninomiya, N., Akalin, A., Sessa, L., Lavorgna, G., Brozzi, A., Luzi,
L., Tan, S.L., Yang, L. et al. (2006) Complex loci in human and mouse genomes. PLoS Genet.
2, e47
69. Nakaya, H.I., Amaral, P.P., Louro, R., Lopes, A., Fachel, A.A., Moreira, Y.B., El–Jundi, T.A.,
da Silva, A.M., Reis, E.M. and Verjovski-Almeida, S. (2007) Genome mapping and expression
analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in
genes related to regulation of transcription. Genome Biol. 8, R43
70. Kim, T.K., Hemberg, M., Gray, J.M., Costa, A.M., Bear, D.M., Wu, J., Harmin, D.A.,
Laptewicz, M., Barbara-Haley, K., Kuersten, S. et al. (2010) Widespread transcription at
neuronal activity-regulated enhancers. Nature 465, 182–187
71. Guttman, M., Garber, M., Levin, J.Z., Donaghey, J., Robinson, J., Adiconis, X., Fan, L., Koziol,
M.J., Gnirke, A., Nusbaum, C. et al. (2010) Ab initio reconstruction of cell type-specific
transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat.
Biotechnol. 28, 503–510
14 Essays in Biochemistry volume 54 2013
© The Authors Journal compilation © 2013 Biochemical Society
72. Cabili, M.N., Trapnell, C., Goff, L., Koziol, M., Tazon-Vega, B., Regev, A. and Rinn, J.L. (2011)
Integrative annotation of human large intergenic noncoding RNAs reveals global properties
and specific subclasses. Genes Dev. 25, 1915–1927
73. Derrien, T., Johnson, R., Bussotti, G., Tanzer, A., Djebali, S., Tilgner, H., Guernec, G., Martin, D.,
Merkel, A., Knowles, D.G. et al. (2012) The GENCODE v7 catalog of human long noncoding
RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 22, 1775–1789
74. Marques, A.C. and Ponting, C.P. (2009) Catalogues of mammalian long noncoding RNAs:
modest conservation and incompleteness. Genome Biol. 10, R124
75. Chodroff, R.A., Goodstadt, L., Sirey, T.M., Oliver, P.L., Davies, K.E., Green, E.D., Molnar, Z.
and Ponting, C.P. (2010) Long noncoding RNA genes: conservation of sequence and brain
expression among diverse amniotes. Genome Biol. 11, R72
76. Dinger, M.E., Amaral, P.P., Mercer, T.R. and Mattick, J.S. (2009) Pervasive transcription of the
eukaryotic genome: functional indices and conceptual implications. Briefings Funct.
Genomics Proteomics 8, 407–423
77. Rinn, J.L. and Chang, H.Y. (2012) Genome regulation by long noncoding RNAs. Annu. Rev.
Biochem. 81, 145–166
78. Nagano, T., Mitchell, J.A., Sanz, L.A., Pauler, F.M., Ferguson–Smith, A.C., Feil, R. and Fraser,
P. (2008) The Air noncoding RNA epigenetically silences transcription by targeting G9a to
chromatin. Science 322, 1717–1720
79. Zhao, J., Sun, B.K., Erwin, J.A., Song, J.J. and Lee, J.T. (2008) Polycomb proteins targeted
by a short repeat RNA to the mouse X chromosome. Science 322, 750–756
80. Huarte, M., Guttman, M., Feldser, D., Garber, M., Koziol, M.J., Kenzelmann-Broz, D., Khalil,
A.M., Zuk, O., Amit, I., Rabani, M. et al. (2010) A large intergenic noncoding RNA induced by
p53 mediates global gene repression in the p53 response. Cell 142, 409–419
81. Kino, T., Hurt, D.E., Ichijo, T., Nader, N. and Chrousos, G.P. (2010) Noncoding RNA gas5
is a growth arrest- and starvation-associated repressor of the glucocorticoid receptor. Sci.
Signaling 3, ra8
82. Guttman, M., Donaghey, J., Carey, B.W., Garber, M., Grenier, J.K., Munson, G., Young, G.,
Lucas, A.B., Ach, R., Bruhn, L. et al. (2011) lincRNAs act in the circuitry controlling
pluripotency and differentiation. Nature 477, 295–300
83. Lanz, R.B., McKenna, N.J., Onate, S.A., Albrecht, U., Wong, J., Tsai, S.Y., Tsai, M.J. and
O’Malley, B.W. (1999) A steroid receptor coactivator, SRA, functions as an RNA and is
present in an SRC–1 complex. Cell 97, 17–27
84. Clemson, C.M., Hutchinson, J.N., Sara, S.A., Ensminger, A.W., Fox, A.H., Chess, A. and
Lawrence, J.B. (2009) An architectural role for a nuclear noncoding RNA: NEAT1 RNA is
essential for the structure of paraspeckles. Mol. Cell 33, 717–726
85. Sunwoo, H., Dinger, M.E., Wilusz, J.E., Amaral, P.P., Mattick, J.S. and Spector, D.L. (2009)
MENε/β nuclear-retained non-coding RNAs are up-regulated upon muscle differentiation and
are essential components of paraspeckles. Genome Res. 19, 347–359
86. Shevtsov, S.P. and Dundr, M. (2011) Nucleation of nuclear bodies by RNA. Nat. Cell Biol. 13,
167–173
87. Poliseno, L., Salmena, L., Zhang, J., Carver, B., Haveman, W.J. and Pandolfi, P.P. (2010) A
coding-independent function of gene and pseudogene mRNAs regulates tumour biology.
Nature 465, 1033–1038
88. Cesana, M., Cacchiarelli, D., Legnini, I., Santini, T., Sthandier, O., Chinappi, M., Tramontano,
A. and Bozzoni, I. (2011) A long noncoding RNA controls muscle differentiation by functioning
as a competing endogenous RNA. Cell 147, 358–369
89. Tsai, M.C., Manor, O., Wan, Y., Mosammaparast, N., Wang, J.K., Lan, F., Shi, Y., Segal, E.
and Chang, H.Y. (2010) Long noncoding RNA as modular scaffold of histone modification
complexes. Science 329, 689–693
90. Gupta, R.A., Shah, N., Wang, K.C., Kim, J., Horlings, H.M., Wong, D.J., Tsai, M.C., Hung, T.,
Argani, P., Rinn, J.L. et al. (2010) Long non-coding RNA HOTAIR reprograms chromatin state
to promote cancer metastasis. Nature 464, 1071–1076
M.B. Clark and others 15
© 2013 Biochemical Society
91. Zhang, X., Gejman, R., Mahta, A., Zhong, Y., Rice, K.A., Zhou, Y., Cheunsuchon, P., Louis,
D.N. and Klibanski, A. (2010) Maternally expressed gene 3, an imprinted noncoding RNA
gene, is associated with meningioma pathogenesis and progression. Cancer Res. 70,
2350–2358
92. Ishii, N., Ozaki, K., Sato, H., Mizuno, H., Saito, S., Takahashi, A., Miyamoto, Y., Ikegawa, S.,
Kamatani, N., Hori, M. et al. (2006) Identification of a novel non-coding RNA, MIAT, that
confers risk of myocardial infarction. J. Hum. Genet. 51, 1087–1099
93. Mus, E., Hof, P.R. and Tiedge, H. (2007) Dendritic BC200 RNA in aging and in Alzheimer’s
disease. Proc. Natl. Acad. Sci. U.S.A. 104, 10679–10684
94. Waters, L.S. and Storz, G. (2009) Regulatory RNAs in bacteria. Cell 136, 615–628
95. Omer, A.D., Lowe, T.M., Russell, A.G., Ebhardt, H., Eddy, S.R. and Dennis, P.P. (2000)
Homologs of small nucleolar RNAs in Archaea. Science 288, 517–522
96. Jager, D., Sharma, C.M., Thomsen, J., Ehlers, C., Vogel, J. and Schmitz, R.A. (2009) Deep
sequencing analysis of the Methanosarcina mazei Go1 transcriptome in response to nitrogen
availability. Proc. Natl. Acad. Sci. U.S.A. 106, 21878–21882
97. Toledo-Arana, A., Dussurget, O., Nikitas, G., Sesto, N., Guet-Revillet, H., Balestrino, D., Loh,
E., Gripenland, J., Tiensuu, T., Vaitkevicius, K. et al. (2009) The Listeria transcriptional
landscape from saprophytism to virulence. Nature 459, 950–956
98. Wurtzel, O., Yoder-Himes, D.R., Han, K., Dandekar, A.A., Edelheit, S., Greenberg, E.P., Sorek,
R. and Lory, S. (2012) The single-nucleotide resolution transcriptome of Pseudomonas
aeruginosa grown in body temperature. PLoS Pathog. 8, e1002945
99. Sharma, C.M., Hoffmann, S., Darfeuille, F., Reignier, J., Findeiss, S., Sittka, A., Chabas, S.,
Reiche, K., Hackermuller, J., Reinhardt, R. et al. (2010) The primary transcriptome of the
major human pathogen Helicobacter pylori. Nature 464, 250–255
100. Thomason, M.K. and Storz, G. (2010) Bacterial antisense RNAs: how many are there, and
what are they doing? Annu. Rev. Genet. 44, 167–188
101. Tomizawa, J. and Itoh, T. (1981) Plasmid ColE1 incompatibility determined by interaction of
RNA I with primer transcript. Proc. Natl. Acad. Sci. U.S.A. 78, 6096–6100
102. Kawano, M., Aravind, L. and Storz, G. (2007) An antisense RNA controls synthesis of an
SOS-induced toxin evolved from an antitoxin. Mol. Microbiol. 64, 738–754
103. Lenz, D.H., Mok, K.C., Lilley, B.N., Kulkarni, R.V., Wingreen, N.S. and Bassler, B.L. (2004) The
small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi
and Vibrio cholerae. Cell 118, 69–82
104. Wassarman, K.M. and Storz, G. (2000) 6S RNA regulates E. coli RNA polymerase activity.
Cell 101, 613–623
105. Ishino, Y., Shinagawa, H., Makino, K., Amemura, M. and Nakata, A. (1987) Nucleotide
sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in
Escherichia coli, and identification of the gene product. J. Bacteriol. 169, 5429–5433
106. Jore, M.M., Brouns, S.J. and van der Oost, J. (2012) RNA in defense: CRISPRs protect
prokaryotes against mobile genetic elements. Cold Spring Harbor Perspect. Biol. 4, a003657
107. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero,
D.A. and Horvath, P. (2007) CRISPR provides acquired resistance against viruses in
prokaryotes. Science 315, 1709–1712
108. Flicek, P., Amode, M.R., Barrell, D., Beal, K., Brent, S., Chen, Y., Clapham, P., Coates, G.,
Fairley, S., Fitzgerald, S. et al. (2011) Ensembl 2011. Nucleic Acids Res. 39, D800-D806
109. Srivastava, M., Simakov, O., Chapman, J., Fahey, B., Gauthier, M.E., Mitros, T., Richards,
G.S., Conaco, C., Dacre, M., Hellsten, U. et al. (2010) The Amphimedon queenslandica
genome and the evolution of animal complexity. Nature 466, 720–726
110. Wang, E.T., Sandberg, R., Luo, S., Khrebtukova, I., Zhang, L., Mayr, C., Kingsmore, S.F.,
Schroth, G.P. and Burge, C.B. (2008) Alternative isoform regulation in human tissue
transcriptomes. Nature 456, 470–476
111. Meader, S., Ponting, C.P. and Lunter, G. (2010) Massive turnover of functional sequence in
human and other mammalian genomes. Genome Res. 20, 1335–1343
16 Essays in Biochemistry volume 54 2013
© The Authors Journal compilation © 2013 Biochemical Society
112. Kutter, C., Watt, S., Stefflova, K., Wilson, M.D., Goncalves, A., Ponting, C.P., Odom, D.T. and
Marques, A.C. (2012) Rapid turnover of long noncoding RNAs and the evolution of gene
expression. PLoS Genet. 8, e1002841
113. Schmidt, D., Wilson, M.D., Ballester, B., Schwalie, P.C., Brown, G.D., Marshall, A., Kutter, C.,
Watt, S., Martinez-Jimenez, C.P., Mackay, S. et al. (2010) Five-vertebrate ChIP-seq reveals
the evolutionary dynamics of transcription factor binding. Science 328, 1036–1040
114. Guo, H., Ingolia, N.T., Weissman, J.S. and Bartel, D.P. (2010) Mammalian microRNAs
predominantly act to decrease target mRNA levels. Nature 466, 835–840
115. Heinen, T.J., Staubach, F., Haming, D. and Tautz, D. (2009) Emergence of a new gene from
an intergenic region. Curr. Biol. 19, 1527–1531
116. Smit, S., Knight, R. and Heringa, J. (2009) RNA structure prediction from evolutionary
patterns of nucleotide composition. Nucleic Acids Res. 37, 1378–1386
117. Heimberg, A.M., Sempere, L.F., Moy, V.N., Donoghue, P.C. and Peterson, K.J. (2008)
MicroRNAs and the advent of vertebrate morphological complexity. Proc. Natl. Acad. Sci.
U.S.A. 105, 2946–2950
118. Mercer, T.R., Wilhelm, D., Dinger, M.E., Solda, G., Korbie, D.J., Glazov, E.A., Truong, V.,
Schwenke, M., Simons, C., Matthaei, K.I. et al. (2011) Expression of distinct RNAs from
3′ untranslated regions. Nucleic Acids Res. 39, 2393–2403