Review
A global view of genomic information –
moving beyond the gene and the
master regulator
John S. Mattick1, Ryan J. Taft1 and Geoffrey J. Faulkner2
1
2
Institute for Molecular Bioscience, The University of Queensland, St Lucia, 4072, QLD, Australia
Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Roslin, EH25 9PS, UK
The current view of gene regulation in complex organisms holds that gene expression is largely controlled by
the combinatoric actions of transcription factors and
other regulatory proteins, some of which powerfully
influence cell type. Recent large-scale studies have confirmed that cellular differentiation involves many different regulatory factors. However, other studies indicate
that the genome is pervasively transcribed to produce a
variety of short and long non-protein-coding RNAs, including those derived from retrotransposed sequences,
which also play important roles in the epigenetic regulation of gene expression. The evidence suggests that
ontogenesis requires interplay between state-specific
regulatory proteins, multitasked effector complexes
and target-specific RNAs that recruit these complexes
to their sites of action. Moreover, the semi-continuous
nature of the transcriptome prompts the reassessment
of ‘genes’ as discrete entities and indicates that the
mammalian genome might be more accurately viewed
as islands of protein-coding information in a sea of cisand trans-acting regulatory sequences.
Regulatory paradigms in metazoa
Perhaps the biggest surprises of the genome sequencing
projects are that the number of protein-coding genes in
animals does not change appreciably with increasing
developmental complexity (known as the ‘G-value paradox’) [1] and that, notwithstanding clade-specific expansions and innovations (e.g. RNA editing enzymes in
vertebrates [2,3]), most proteins are orthologous [4]. It is
generally accepted that phenotypic divergence in animals
is based largely on the variation of the regulatory information that controls the expression of these proteins and
their isoforms [5]. In addition, it is generally assumed that
most regulatory transactions are conveyed by sequencespecific regulatory proteins that bind to enhancers, promoters and transcripts to modulate mRNA expression and
processing, with the vast differences in developmental and
cognitive complexity between nematodes and humans
ascribed to an expanded suite of cis-regulatory elements
and the presumed explosive power of the combinatoric
interactions between the regulatory proteins that recognize these elements [6].
Corresponding authors: Mattick, J.S.
(geoff.faulkner@roslin.ed.ac.uk).
(j.mattick@uq.edu.au); Faulkner, G.J.
Here, we discuss recent advances in our understanding
of the nature and regulation of gene expression in mammals, particularly in relation to the complexity of the
hierarchical networks of regulatory factors involved, the
unfolding discovery of previously hidden layers of regulatory RNAs (including many derived from retrotransposon
sequences and pseudogenes) and the emerging realization
that the genome might not be constructed as a discrete set
of protein-coding genes with associated regulatory
sequences, but as an interleaved continuum of both coding
and cis- and trans-acting regulatory information.
‘Transcription factors’ and regulatory networks
The term ‘transcription factor’ loosely describes proteins
with different modes of action that operate at various levels
to facilitate and control the production of RNA. Transcription factors often act generically, in that they are expressed
in multiple cell types and can regulate the expression of
Glossary
Long non-protein-coding RNAs (lncRNAs): RNAs of little protein-coding
potential, >200 nt in length, some of which can be >100 Kb [59,60]. The 200 nt
lower limit is an arbitrary figure based on a convenient practical cut-off in RNA
purification protocols [32] that excludes most known classes of small RNAs.
MicroRNAs (miRNAs): 22 nt small RNAs that regulate gene expression by
partial complementary base pairing to specific mRNAs. This annealing inhibits
protein translation and can also facilitate the degradation of the target mRNA.
Piwi-interacting RNAs (piRNAs): Dicer-independent 26–30 nt small RNAs
principally restricted to the germline and somatic cells bordering the germline.
They associate with Piwi-clade Argonaute proteins and regulate transposon
activity and chromatin state [27,28].
Promoter-associated short RNAs (PASRs): generally 20–200 nt long with 50
ends that coincide with the transcription start sites of protein-coding and noncoding genes [32].
Pseudogene: a supposedly non-functional paralog of a protein-coding gene
generated by gene duplication or retrotransposition. The vast majority of
pseudogenes are computationally defined by looking for features such as
premature stop codons that prevent the translation of a viable protein.
Small interfering RNAs (siRNAs): 21 nt small RNAs produced by Dicer
cleavage of perfectly complementary dsRNA duplexes. They form complexes
with Argonaute proteins and are involved in gene regulation, transposon
control and viral defense [27,28].
Small nucleolar RNAs (snoRNAs): two classes: C/D box snoRNAs (70–120 nt)
guide the methylation of target RNAs and H/ACA box snoRNAs (100–200 nt)
guide pseudouridylation [121]. Recent evidence suggests that they might also
be precursors for at least two classes of small RNAs that might have miRNAlike activity [30,31].
Transcription initiation RNAs (tiRNAs): 18 nt tiny RNAs derived from
sequences just downstream of transcription initiation sites, which seem to be
linked to the position of the first nucleosome and might be derived from RNA
Pol II backtracking and TFIIS-mediated cleavage [33,34].
Transposed element (TE): repetitive element generated by the activity of
retrotransposons and transposons. Almost always incapable of further
transposition because of mutations and truncations.
0168-9525/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2009.11.002 Available online 26 November 2009
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Review
many genes by recognizing cis-acting binding sites with
relatively relaxed consensus sequences that occur in many
places around the genome, suggesting that another layer of
specificity is required. What determines which subset of
these sites is addressed in a given cell or developmental
context is unknown but is presumed to be influenced by
chromatin accessibility [7].
Some transcription factors are expressed in a cellrestricted manner and can have a powerful influence on
cell fate. Good examples are the transcription factors
Pou5f1 (also known as Oct4), Sox2 and Nanog, which
are considered ‘master regulators’ of stem cell pluripotency
with the ability to revert somatic cells to undifferentiated
states capable of elaborating various developmental trajectories [8], and Hox proteins, which control cellular patterning in many contexts, including the segmental
organization and neural circuitry of the hindbrain [9].
Other proteins that seem to function as master regulators
of subsequent cell differentiation (of which there are many)
include the helix–loop–helix transcription factor Myod1,
which can convert differentiated cells into muscle cells [10],
and the zinc finger protein Egr2 (also known as Krox-20),
which is expressed in and required for the development of
specific rhombomeres (segmented compartments) in the
embryonic hindbrain [11]. However, such proteins are only
parts of larger networks that influence muscle or hindbrain
development in vivo, respectively [9,12], and do not fully
explain the diversity and fine structure of organs and
tissues. Similarly, chromatin-modifying proteins have a
profound impact on developmental processes [13] because
they lie at the functional centre of epigenetic regulatory
networks, not because they themselves make locus-specific
regulatory decisions but because they act on other information that does.
The complex interplay of regulatory factors in cellular
differentiation was recently illustrated by examining the
effects of the systematic small interfering RNA (siRNA)mediated knockdown of 52 transcription factors during the
PMA-induced differentiation of the human monocytic cell
line THP-1, which showed that cellular states are determined by complex networks involving both positive and
negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is necessary and sufficient to drive the
differentiation process [14]. A similar approach using
the short hairpin RNA (shRNA)-mediated perturbation
of 125 transcription factors, chromatin modifiers and
RNA-binding proteins found that the transcriptional
response of mouse dendritic cells to pathogens involves
(at least) 24 ‘core regulators’ and 76 ‘fine-tuners’ [15]. The
layered complexity of regulatory networks is also illustrated by the interplay between microRNAs (miRNAs)
and transcription factors in cell pluripotency and differentiation [16–18].
The regulatory challenge and regulatory hierarchies
This complexity is no surprise. Beyond cells in culture, the
enormous and underappreciated challenge for genetic
programming is not simply to define the phenotypic state
of a cell, but rather to organize the 4-dimensional growth
and differentiation of cells into a myriad of precisely
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Trends in Genetics Vol.26 No.1
sculpted organs and tissues. In humans, these include
the lungs, kidneys, heart, liver, pancreas, intestine and so
on, a wide range of skeletal muscles including those in the
face and many bones such as the vertebrae, each of which
has a specific and unique architecture, as well as the
dizzyingly complex organization of the brain with some
1011 neurons and 1014 synapses. Organogenesis, which
involves directional cell division, cell movement, cell
differentiation and programmed apoptosis, requires networked interactions between many hierarchical levels of
gene regulation, including the modulation of chromatin
architecture, transcription initiation and elongation,
alternative splicing, RNA editing and other forms of
post-transcriptional modifications, translation, posttranslational modifications, RNA half-life and RNA and
protein trafficking and signaling. This is an extensive list,
elements of which are often studied in isolation from
others, with respect to both the gene(s) and level(s) of
regulation under scrutiny. Each can have important if not
profound effects on the cellular phenotype, as exemplified
by the pleiotropic effects of protein phosphorylation by the
serine/threonine kinases Akt1–3 on cell survival, growth,
division, migration and metastasis, depending on the
phosphorylated substrate and crosstalk with other pathways [19], although the complex interactions between
different gene products and different levels of gene
regulation in these networks are as yet only poorly
understood.
Guide RNAs in regulatory networks
Although it is presumed that differentiation and development are mainly controlled by regulatory proteins, it is
becoming increasingly apparent that there exists an
additional, potentially vast, layer of regulatory RNAs that
interact with some of these proteins and provide specificity
to them (Figure 1). A well-documented, although by no
means fully explored, example is that of miRNAs, which
are generated by the RNA interference (RNAi) pathway
and have unexpectedly emerged over the past decade as
major players in global and specific gene regulation.
Indeed, two recent reports have shown that a single
miRNA miR-302, like the transcription factors Pou5f1,
Sox2 and Nanog, is capable of reprogramming cells into
an embryonal stem cell-like pluripotent state, including
the induction of these transcription factors [16,17], which
are then repressed by other miRNAs during differentiation
[18]. miRNAs have no intrinsic catalytic function but act as
guides to recruit a relatively generic protein complex (the
RNA-induced silencing complex, or RISC, which contains
‘regulatory’ proteins of the Argonaute family [20]) to
regulate the translation and half-life of specific target
mRNAs through binding sites in both their coding
[18,21] and 3’ untranslated regions, whose length and
miRNA-recognition repertoire is modulated during differentiation [22,23].
Altogether, miRNAs regulate almost every known
developmental process [24] and are perturbed in pathological processes such as cancer [25]. Although these
examples illustrate the regulatory interplay between regulatory factors themselves, the key general point is that in
the case of miRNAs, and other classes of regulatory RNAs,
Review
Trends in Genetics
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Figure 1. Transcriptional complexity at a single locus. Recent research indicates that most of the eukaryotic genome is transcribed into interweaving nests of both sense
and antisense RNA species, whose expression is regulated to some extent by transcription factor activity, and also by local chromatin modifications, boundary elements
and TEs, and other regulatory RNA species. Examples of these phenomena are depicted in this figure. (a) Transcription can initiate at multiple sites in a single locus,
including from the 50 ends of annotated genes (left-hand edge of the red blocks), which frequently show evidence of antisense transcription in addition to a single dominant
and many minor sense-oriented TSSs. Transcription can also initiate at transposed elements upstream of a canonical protein-coding gene or at sense- or antisense-oriented
sites in introns. Such noncanonical transcriptional activity can also have a direct regulatory function – blocking the activity and accessibility of downstream promoters. Both
protein-coding and non-coding RNA TSSs are regulated by transcription factors, and transcription factor targeting itself can be regulated by small ncRNAs. Tethered long
ncRNAs (represented by light blue wavy line) can recruit chromatin or other DNA modifying complexes to regulate TSS accessibility to transcription factors and RNA
polymerase II. (b) Transcripts generated from the TSSs described in panel (a) are depicted. The first three large transcripts shown below the arrow depict a canonical mRNA,
an mRNA-like ncRNA and an alternate mRNA product with an exon extension derived from an alternative TSS in a repetitive element. The long ncRNAs shown in yellow are
derived from the bidirectional transcription of the protein-coding gene and transcription factor-regulated intronic TSSs. Like protein-coding mRNAs, long ncRNAs can be
spliced and capped. Small TSS-proximal RNAs (e.g. tiRNAs and PASRs) are derived from both protein-coding and non-coding transcription and might regulate
transcriptional activity. Other small RNAs, such as snoRNAs and pre-miRNAs, can be processed from the introns of protein-coding or non-coding transcripts, and further
cleaved into sdRNAs and mature miRNAs. Indeed, miRNAs bound to RISC complexes (top right) add an additional layer of regulation by targeting transcripts for
degradation or inhibiting translation. Thin blocks represent short (green) or long (yellow) non-coding RNAs, TEs (purple) or 50 and 30 untranslated regions (red). Thick blocks
indicate protein-coding exons (red). Blocks connected by thin lines indicate spliced transcripts and their respective splicing patterns. Double-stranded structures are
representative of the genome. The size of the arrow indicates the relative abundance of transcripts derived from the TSS. Abbreviations: transcription initiation RNA
(tiRNA); promoter-associated small RNA (PASR); small nucleolar RNA (snoRNA); sno-derived RNA (sdRNA); microRNA (miRNA); RNA-induced silencing complex (RISC);
transcription factor (TF); 7-methylguanosine cap (7mG).
the regulatory signal has been de-coupled from the consequent analog action, which provides enormous efficiency
and flexibility in the evolution and deployment of such
regulatory signals and networks. The number of known
miRNAs in mammals is of the order of 103, but might be
much higher given the indications of deep sequencing and
evidence of cell-specific miRNAs [26].
In recent years, a number of other classes of small RNAs
have emerged, including (i) Piwi-interacting small RNAs
(piRNAs) that interact with other members of the Argonaute family and seem to have a role in silencing transposon activity in the germline [27,28], (ii) siRNAs derived
from sense–antisense duplexes that play a role in the
epigenetic regulation of adjacent loci [27,28] or are imputed
to do so by Argonaute-dependent processes [29], (iii) at
least two classes of small RNAs derived from small nucleolar RNAs (snoRNAs) that might also have miRNA-like
activity [30,31], (iv) promoter-associated small RNAs
(PASRs) of unknown function [32] and (v) transcription
initiation RNAs (tiRNAs) linked to transcription start sites
and nucleosome positioning [33,34]. Interestingly, it has
recently been shown that exons are preferentially positioned in nucleosomes in both somatic [35–38] and sperm
cells [38], which might provide a mechanistic basis for the
23
Review
observed coupling of chromatin structure, transcription
and splicing, and a potential basis for exon selection
through various histone modifications within these nucleosomes that report the status of particular exons during
differentiation and development, a process that itself
might be RNA-directed.
Hidden layers of RNA
It is now evident that the genomes of mammals are almost
entirely transcribed (as are, as far as one can tell, the
genomes of all other organisms), apparently in a developmentally regulated manner. That is, the expansion in
the extent of non-coding sequences with increased complexity is paralleled by a corresponding increase in the
extent of transcription [4]. This has been shown by whole
chromosome tiling arrays [39–42] and deep sequencing
normalized cDNA libraries, which has revealed tens of
thousands of intergenic, antisense, overlapping and intronic long non-protein-coding RNAs (long ncRNAs or
lncRNAs) that are dynamically expressed from the mammalian genome [43–46], yielding a picture of extraordinary
transcriptional complexity at individual loci (Figure 1).
Although initially suspected to be transcriptional
‘noise’, a view that has been entertained if not favored
because it does not disturb the orthodox view of the informational structure of the genome, there is now substantial
genome-wide evidence pointing to the intrinsic functionality of these transcripts (for a review, see [47]). In
addition, although the mechanisms are not yet well understood, there is increasing evidence that these RNAs play
important roles in the regulation of differentiation and
development, including the involvement of lncRNAs in the
regulation of the expression of homeotic genes [48,49] and
oncogenes [50], and in the regulation of skeletal development, eye development and epithelial-to-mesenchymal
transition among many others (for reviews, additional
examples and references, see [51,52]).
The range of functions of the large number of documented lncRNAs has barely been explored, and there is as yet
little information that would allow their structural parsing
and functional classification. Nonetheless, lncRNAs might
be expected to perform a wide range of functions in eukaryotic cell and developmental biology, given the capacity of
RNA to form sophisticated structures that can be allosterically altered by interactions with other molecules
[53,54], as well as to embrace highly sequence-specific
recognition of other RNAs, DNA and proteins. Preliminary
evidence suggests that many if not most lncRNAs are trafficked to specific subcellular locations [55], and there are
well-documented examples of lncRNAs that are required for
the formation and structural integrity of nuclear paraspeckles (which seem to regulate mRNA export) in differentiated cells [56,57] or are specifically associated with a
novel subnuclear domain in a subset of neurons [58]. These
observations demonstrate that RNA molecules themselves
are crucial for proper cellular function.
There are even more subterranean layers of the transcriptome. Most identified lncRNAs seem to be polyadenylated and produced by RNA polymerase II, although
many are derived from internal oligo(dT) priming of
internal A-rich sequences during cDNA cloning, often from
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Trends in Genetics Vol.26 No.1
much larger precursors (‘macroRNAs’) that can extend
over 100 Kb in length [59,60]. However, tiling array
analyses, which do not depend on oligo(dT)-based capture
and priming to reduce the background of rRNA and other
infrastructural RNAs, have shown that almost half of all
transcripts are not polyadenylated and that this fraction is
largely distinct in sequence composition from polyadenylated RNAs [41], indicating that a large proportion of the
dynamic transcriptome has remained hidden from view for
unexpected technical reasons. Many of these RNAs, including those derived from transposed elements (see
below), might be produced by RNA polymerase III (RNAPIII), which also transcribes various types of known or
putative regulatory RNAs, although the full extent of the
RNAPIII transcriptome is unknown [61].
Large-scale screening of ncRNAs, using siRNA- or
shRNA-mediated knockdown (which is surprisingly effective [47]) or ectopic expression strategies, in parallel with
existing efforts to deconstruct the roles of protein-coding
genes, would undoubtedly reveal their role in many cellular and developmental processes. Indeed, given the phenotypic differences between species that retain a similar
complement of proteins, it is possible that regulatory
RNAs, including those that are not well conserved [62–
64], underlie many adaptations.
RNA regulation of the epigenome
Dynamic and mitotically heritable changes to chromatin
architecture are a hallmark of differentiation and development and are central to the 4-dimensional control of
these processes. These changes include DNA methylation
as well as a myriad of different modifications to different
residues in the tails of histones that form the nucleosome
[65], and that are imposed by a relatively generic set of
enzymes and chromatin-modifying complexes that, like
RISC, lack inherent sequence specificity. To date, the field
of epigenomics has largely been concerned with cataloging
the changes in these modifications during differentiation
(including cancer) and their association with particular
features such as promoters or exons [66], and it seems
to have been widely assumed, although not often articulated, that the positional specificity of these modifications
is regulated by proteins that recruit the appropriate chromatin-modifying enzymes and/or complexes to their various sites of action at different loci in different cell types at
different stages of growth and differentiation [67]. There is
a degree of circularity in this assumption because it is also
thought that changes to chromatin structure can also
facilitate or restrict access to the transcription factors
[7] that regulate the next level of specificity in the gene
expression hierarchy (i.e. transcription itself).
Recent evidence strongly suggests that both short and
long ncRNAs are involved in, and perhaps central to, the
control of chromatin architecture [68,69], as predicted earlier [70]. Indeed, ncRNA-directed regulatory circuits
underpin most if not all complex epigenetic phenomena
in eukaryotes, including transcriptional and post-transcriptional gene silencing, position effect variegation,
hybrid dysgenesis, chromosome dosage compensation,
parental imprinting and allelic exclusion, and possibly
transvection and transinduction [47]. More specifically,
Review
it has been shown that many long ncRNAs are spatially
and temporarily expressed from homeotic loci during development [71], and are associated with either chromatin
repressor (Polycomb group) complexes [49,72] or chromatin activator (Trithorax group) complexes and activated
forms of histones [73,74], suggesting that these RNAs
function to guide infrastructural chromatin-modifying
complexes to specific genomic loci to regulate gene expression [67–69]. Interestingly, and significantly for conceptions of evolution [75], RNA-directed epigenetic
changes can also be meiotically inherited in animals and
plants [76,77].
Enhancers are long distance and often very highly conserved regulatory elements that drive tissue-specific patterns of gene expression during development, a process
that is not well understood but is thought to involve the
recruitment of transcription factors and chromosomal looping to bring the resultant complexes into contact with
specific promoters [67,78]. Intriguingly, the available evidence suggests that enhancers are themselves transcribed
in the tissues in which they are active [79,80]. The resulting ncRNAs have been thought not to be functional but
rather to be a passive byproduct of a transcriptional event
that is required to open up the enhancer (or the promoter)
to protein binding [81], but there are also documented
examples of trans-acting functions for ncRNAs in regulating the expression of developmental genes [48,49,73].
There is evidence that both the act of transcription and
the RNA itself are crucial factors in establishing cellular
identity, and it remains an open question whether ncRNAs
have an integral role in enhancer function [71]. There is
also evidence that some classes of transcription factors and
chromatin-modifying complexes have RNA-binding
domains or high affinity for RNA:DNA structures ([82]
and references therein).
Retrotransposon sequences and pseudogenes
Sequences derived from transposable elements (TEs) comprise at least half of the mammalian genome. They are
thought to be largely non-functional and have consequently been used to assess the rate of ‘neutral evolution’, despite McClintock’s originally derided but later
celebrated depiction of them as ‘controlling elements’ in
maize [83], and Britten and Davidson’s subsequent suggestion that they form gene regulatory networks in
animals [84]. Indeed, there is increasing evidence that
these sequences might not only play a key role in genome
evolution [85,86] but also in genome biology and the control of gene expression [87–91], including the potentially
dynamic remodeling of the somatic genome in neurogenesis and/or neuronal function [92]. Moreover, it has been
shown that the transcription of ncRNAs from a SINE B2
element controls chromatin structure in the mouse growth
hormone locus [81] and that human Alu RNA acts as a
modular transacting repressor of mRNA transcription
during heat shock [93]. More recently, it has been found
that thousands of TEs are transcribed in a tissue-specific
manner, are enriched and typically coincide with the
expression of nearby protein-coding genes and can comprise up to 30% of the total capped RNA present in a cell
[91].
Trends in Genetics
Vol.26 No.1
The central message here is that TEs contribute an
integral – and underestimated – fraction to the mammalian transcriptome and regulome. Owing to their enrichment near protein-coding genes, TEs can harness nearby
exons to generate a large number of protein-coding transcripts, or produce ncRNAs that overlap or regulate
protein-coding genes [94]. Moreover, the dramatic variation in TE composition among mammals might reflect
lineage-specific functional exaptation [95], as well as
examples of convergent evolution [96], calling into question
the relevance of conservation-based metrics of TE function
[97]. Because TEs contribute a substantial proportion of
human polymorphisms [98], they might also be responsible
for many phenotypic differences between individuals, a
particularly important perspective for genome resequencing projects given genome-wide association studies
indicate that the vast majority of variations affecting
complex traits and complex diseases lie within non-coding
regions of the genome [99].
Another related class of sequences that might have a
regulatory role is pseudogenes, which are presumed nonfunctional paralogs of functional protein-coding genes
generated by gene duplication or retrotransposition. Computational analyses of genome sequence data currently
indicate a cohort of 20 000 pseudogenes per mammalian
genome [100]. Numerous pseudogenes have been discovered in recent years that contain active promoters and
generate either sense or antisense RNAs distinct to their
ancestral paralogs [100–104], including key markers of
embryonic stem cell pluripotency (Oct4 and Nanog have
at least six and 10 pseudogenes, respectively) [105]. The
functional consequences of pseudogene transcription are
unclear, but it has been proposed that ncRNAs generated
by pseudogenes might silence paralogous mRNAs in trans
directly by forming RNA–RNA duplexes [102] or generating siRNAs from such duplexes [106,107]. Taken together
these data suggest that the term ‘pseudogene’ might ultimately prove to be a misnomer.
It should be noted that the repetitive nature of both TEs
and pseudogenes hinders, but does not prevent, the
reliable identification of their associated RNAs. Massively
parallel sequencing technologies can achieve a degree of
resolution impossible with hybridization-based strategies,
which can only reliably target the non-repetitive half of the
human genome [41]. If deep sequencing reads of sufficient
quality and length are produced, particularly with the use
of paired-end protocols and sophisticated bioinformatic
methods [108,109], it is possible to discriminate the expression of individual repetitive elements and investigate these
species with conventional laboratory techniques [91] including RNAi, despite challenges in the use of exogenous
siRNA molecules against repetitive regions.
An information continuum?
All these observations suggest that the extent of regulatory
information in mammalian genomes is far greater than
previously thought. They also call into question the longstanding assumption that most genes encode proteins
(with their cis-acting regulatory elements) and that
proteins transact most genetic information. We suggest
that a new paradigm needs to emerge that expands the
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Review
definition of genetic information to include large numbers
of regulatory RNAs, and recognizes the possibility that
many ‘regulatory’ proteins, including those that might be
state-specific, are provided with an additional layer of
target specificity by guide RNAs. The most parsimonious
explanation for the G-value paradox is that we have constrained ourselves to a limited and incomplete definition of
the gene, with respect not only to the fact that many
different product isoforms can be produced by post-transcriptional and post-translational modifications, but also
the increasing likelihood that many functional genes do not
encode proteins.
Moreover, even the conception of a discrete ‘gene’ as a
basic organizational unit might be inappropriate to
describe the true nature of genetic information in higher
organisms. Not only is there the pervasive transcription of
the genome, but also a complex mix of overlapping, interleaved and bidirectional coding and non-coding, sense and
‘antisense’ transcripts expressed from most loci
[32,110,111]. Many transcripts contain distal 50 exons
(an average of 20 Kb in Drosophila and almost 200 Kb
in human) that are only used in particular developmental
contexts and traverse large genomic regions including
other genes [112,113], contain internal initiation sites
for alternative transcripts [46,114] and are processed by
alternative splicing and other pathways to produce a
variety of long and short RNAs [32,110,111]. Thus, the
boundaries of ‘genes’ are blurred and become indefinable.
In addition, transcriptomic studies have revealed interesting and unexpected species, such as chimeric transcripts,
that might indicate a higher order network organization
[115,116], consistent with the cell type-specific organization of chromosomes into territories and transcription
factories [117]. For this reason it might be difficult to parse
the genome in only one dimension or associate a genomic
locus with a single developmental process, physiological
function or role in disease progression. Rather, the genome
might be better viewed as a highly organized, information
dense and heavily transcribed structure wherein each
region variably responds to feed–forward regulatory signals and environmental stimuli through a myriad of RNA
and protein products [53,82]. This creates challenges for
resolving the complex genetic bases for common human
diseases, where gene networks rather than master genes
drive specific phenotypes [118], and modeling genetic networks based on the limited conventional descriptions of a
gene.
This conceptual upheaval requires a fresh look at how
the genomes of complex organisms, and perhaps all organisms, might be described and parsed to best reflect their
biological information content. It has been suggested that
genes might be redefined as ‘fuzzy transcription clusters
with multiple products’ [119] and more recently as a ‘union
of genomic sequences encoding a coherent set of potentially
overlapping functional products’ [120], which explicitly
includes ncRNAs, moves away from a protein-centric
model and recapitulates some of the earliest definitions
of genes as genetic loci. However, this does not deal with
transcripts containing distal exons that traverse apparently unrelated loci. A not mutually exclusive alternative
would be to invert the functional genomics paradigm of
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Trends in Genetics Vol.26 No.1
annotating ‘genes’ as discrete entities by the product(s)
they produce. Instead, RNAs could be annotated by their
genomic origin, genomic environment and what is known of
their function, including open reading frame content and
interactions with other molecules. In effect, this would
circumvent the issue of gene definition (including its
boundaries) by consigning it to obsolescence.
Acknowledgements
We thank Alistair Forrest, Piero Carninci and the reviewers for their
constructive and helpful comments. JSM and RJT are supported by a
Federation Fellowship grant (FF0561986) and a Discovery Project grant
(DP0988851) from the Australian Research Council. GJF is supported by
an Overseas Based Biomedical Fellowship (CJ Martin Award) from the
Australian National Health and Medical Research Council (ID 575585)
and a UK BBSRC Institutional Strategic Programme Grant.
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