Small regulatory RNAs in mammals
John S. Mattick*and Igor V. Makunin
ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience,
University of Queensland, Brisbane QLD 4072, Queensland, Australia
Received January 3, 2005; Revised and Accepted February 23, 2005
Mammalian cells harbor numerous small non-protein-coding RNAs, including small nucleolar RNAs
(snoRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs) and small double-stranded RNAs,
which regulate gene expression at many levels including chromatin architecture, RNA editing, RNA stability,
translation, and quite possibly transcription and splicing. These RNAs are processed by multistep pathways
from the introns and exons of longer primary transcripts, including protein-coding transcripts. Most show
distinctive temporal- and tissue-specific expression patterns in different tissues, including embryonal
stem cells and the brain, and some are imprinted. Small RNAs control a wide range of developmental and
physiological pathways in animals, including hematopoietic differentiation, adipocyte differentiation and
insulin secretion in mammals, and have been shown to be perturbed in cancer and other diseases. The
extent of transcription of non-coding sequences and the abundance of small RNAs suggests the existence
of an extensive regulatory network on the basis of RNA signaling which may underpin the development
and much of the phenotypic variation in mammals and other complex organisms and which may have differ-
ent genetic signatures from sequences encoding proteins.
Although only 1.2% of the human genome encodes protein, a
large fraction of it is transcribed. Indeed, ?98% of the tran-
scriptional output in humans and other mammals consists
of non-protein-coding RNAs (ncRNA) from the introns of
protein-coding genes and the exons and introns of non-
protein-coding genes (1,2), including many that are anti-
sense to or overlapping protein-coding genes (3–5).
Until recently, the non-coding RNA fraction was considered
mainly useless with the exception of the common infrastruc-
tural RNAs involved in protein synthesis, transport and spli-
cing. Introns have long been regarded as evolutionary debris
with intronic RNA assumed to be simply degraded after spli-
cing excision, and the increasing number of non-protein-
coding transcripts being detected in mammalian cells has
been suggested, at least by some, to be largely ‘transcriptional
noise’(6). However, a significant proportion of ncRNAs
appears to be stable in eukaryotic cells. For example, some
excised introns have half-lives comparable with mRNA and
are even exported from the nucleus to the cytoplasm (7,8).
Whole chromosome tiling chip arrays have shown that the
range of detectable ncRNAs in human cells is much greater
than can be accounted for by mRNAs (9) and that there
appear to be roughly equal numbers of protein-coding and
non-coding transcripts regulated by common transcription
factors in the human genome (4,10). Similar data have been
reported in Drosophila (11).
All intensively studied gene loci, including those that are
imprinted and conventional loci such as beta-globin have
been shown to contain a majority of non-coding transcripts
(12–15). The number of known functional ncRNA genes has
risen dramatically in recent years and over 800 ncRNAs [exclu-
ding, transfer RNAs (tRNAs), ribosomal RNAs (rRNAs)
and small nuclear RNAs (snRNAs)] have been catalogued in
mammals, at least some of which are alternatively spliced
(16,17), along with almost 20 000 putative ncRNA transcripts
identified in cDNA libraries (16,18). ncRNAs have been
implicated in diseases including various cancers and neuro-
logical diseases (2,16), and at least some are processed into
smaller functional molecules (19,20).
Apart from tRNAs and spliceosomal snRNAs, which are
housekeeping RNAs involved in mRNA splicing and trans-
lation, there are several functionally and structurally distinct
classes of short RNAs in eukaryotic cells. In most, if not all,
cases, their function is based on recognition of RNA or
DNA target sequences by specific base pairing, analogous to
digital signaling (21). Because of this feature, even short
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Human Molecular Genetics, 2005, Vol. 14, Review Issue 1
RNAs contain sufficient information to specify individual
targets in the genome and the transcriptome, in a much
more compact and energy-efficient manner than proteins,
which may have been a necessary adaptation to address the
accelerating regulatory requirements of more complex organ-
isms (21,22) and have been crucial to their evolution and
development (23,24). These small RNAs and their role in
mammalian cell and developmental biology are the subjects
of the current review.
SMALL NUCLEOLAR RNAS
Small nucleolar RNAs (snoRNAs) guide the site-specific
modification of nucleotides in target RNAs. Two types of
modification occur, 20-O-ribose methylation and pseudouridy-
lation, directed by two large families of snoRNAs termed box
C/D and box H/ACA snoRNAs, respectively (reviewed in
25,26). SnoRNAs recognize target sequences by formation
of a canonical guide RNA duplex and recruit associated pro-
teins to perform the corresponding modification at the target
site. Generally, snoRNAs range between 60 and 300 nucleo-
tides in length, but only short sequences participate in target
recognition via antisense interactions. Initially, it was
thought that the role of snoRNAs was restricted to rRNA
modification in ribosome biogenesis, but it is now evident
that they can target other RNAs including snRNAs and poss-
ibly mRNAs (25,27). Interestingly, a pseudouridine synthase
and pseudouridinylation of the steroid receptor RNA activator
SRA have recently been shown to be involved in retinoic acid
and other nuclear receptor-dependent transactivation (28).
Over 300 different snoRNAs are known in humans and
almost 200 in mouse (see databases at http://noncode.bioinfo.
org.cn and http://www.sanger.ac.uk/Software/Rfam) (17,29),
many of which occur in polycistronic clusters and at least
some of which show tissue-specific expression, suggesting
that they are specifically regulated and in turn have specific
regulatory roles in the differential modification of selected
target RNAs in different tissues including brain. Interestingly,
a number of brain-specific snoRNAs come from imprinted
regions and some of them represent orphan snoRNAs with
unknown targets (30,31). One of these snoRNAs has an 18
nucleotide phylogenetically conserved sequence complemen-
tary to a critical alternative splice site and adenosine-to-
inosine (A–I) RNA editing site in the serotonin 2C receptor
mRNA whose gene also encodes another snoRNA that itself
has an unknown target (30). Moreover, at least some brain-
specific snoRNAs appear to have evolved recently and to be
restricted in their phylogenetic distribution (32), suggesting
their importance in the epigenetic control of behavior.
Mammalian snoRNAs are derived from introns of pre-
mRNA transcripts, and are produced by processing of the
excised intron (debranched lariat) as well as by endonucleoly-
tic cleavage of unspliced primary transcripts, by a complex
pathway involving endonucleases, exonucleases and helicases
(25,26). In many cases, the snoRNA-containing introns occur
within protein-coding transcripts, such as those encoding ribo-
somal proteins and others involved in ribosomal biogenesis/
nucleolar function, a clear example of a parallel genetic
output. However, in many other cases, snoRNAs are derived
from the introns of transcripts that do not have any protein-
coding capacity (25–27,33), the function of whose exons
(if any) is unknown.
No mutations with phenotypic consequences have been
recorded in snoRNA sequences, which suggest that they are
lethal, functionally redundant or (most likely) cause subtle
effects, for example on growth or brain function. It has been
reported that certain snoRNAs, present as multicopy genes
(designated as HBII-52 and HBII-85 snoRNAs, respectively),
are absent from the cortex of a patient with Prader–Willi Syn-
drome (PWS) and from a PWS mouse model, demonstrating
their paternal imprinting and pointing to their potential role
in the etiology of PWS (30). However, a recent study of a
genomic deletion of the HBII-52 snoRNA gene cluster in
humans indicates that these snoRNAs do not play a major
role in PWS on their own, or do so only in connection with
the HBII-85 snoRNA cluster (34).
In relation to RNA regulation, it should be noted that
another form of RNA editing, A–I conversion, catalyzed by
adenosine deaminases that act on RNA (ADARs), is also
common in the brain, and aberrant editing has been associated
with certain cancers and a range of abnormal behaviors includ-
ing epilepsy and depression (35). In humans, A–I editing
has recently been shown to be much more widespread than
was previously thought and to occur primarily in Alu elements
(which are primate-specific) in non-coding RNA sequences in
both protein-coding and non-coding transcripts (36–38). A–I
editing also appears to modulate RNA interference (RNAi)
(described subsequently) by altering the sequence of intro-
duced (39,40) and naturally occurring (41) double-stranded
MIRNAS AND SIRNAS
The other broad class of small regulatory RNAs in mammals
(and indeed in animals and plants generally) are the tiny, gene-
rally 21–25 nt long, molecules named microRNAs (miRNAs)
and short interfering RNAs (siRNA), which have been the
subject of intense recent interest (42–48). miRNAs come
from endogenous short hairpin precursor structures (described
subsequently) and usually target other loci with similar but not
identical sequences for translational repression. siRNAs are
produced from longer double-stranded (bimolecular) RNAs
or long hairpins, often of exogenous origin, and usually
target homologous sequences at the same locus or elsewhere
in the genome for destruction (gene silencing) (43–45), the
phenomenon termed RNAi (49). However, the distinction
between miRNAs and siRNAs is becoming blurred as both
are produced by similar pathways and have similar mechan-
isms of action (43–45). Recent observations have shown
that both miRNAs and siRNAs can suppress translation of
mRNAs (in the case of an imperfect match) and can cleave
target RNAs (in the case of a perfect match) (44,50,51).
miRNAs have been shown to be involved in a variety of devel-
opmental processes in animals and plants (43,46) (described
subsequently). In contrast, siRNAs were originally proposed
to act mainly as an antiviral defense and transposon repression
system via the phenomenon of RNAi (52), but recent findings
R122Human Molecular Genetics, 2005, Vol. 14, Review Issue 1
indicate that such RNAs may play a much broader role in gene
and genome regulation (described subsequently).
A database of known and predicted endogenous miRNAs in
various animal and plant species is available at http://
www.sanger.ac.uk/Software/Rfam/mirna (29,53). The data-
base (release 6.1) currently lists 222 human and 224 mouse
miRNAs (as well as 186 rat miRNAs), many of which are
orthologous. Roughly half are conserved in fishes and a
quarter are conserved in invertebrates (54), which presumably
have evolutionarily conserved functions in vertebrate and
metazoan development. However, new human and rodent
miRNAs are constantly being identified (55). Very recently,
976 candidate miRNAs were identified by scanning whole-
genome human/mouse and human/rat alignments, most of
which are also conserved in other vertebrates, and around
20% of which have experimental support (56).
Two databases of siRNAs directed against human genes
have also recently been published (see http://www.human-
These databases contain several hundred siRNAs that have
been experimentally verified to be active against human
genes (57,58) and thousands of siRNA sequences designed
computationally to be active against the RefSeq curated
human gene set (58).
Biogenesis of miRNAs and siRNAs
miRNAs are processed by the RNAi machinery in a two-step
cleavage process (59) from longer primary transcripts that
have been termed ‘pri-miRNAs’ (60) but that in reality appear
to be conventional pre-mRNAs and ncRNAs (15,20,55,
61–66), including antisense transcripts (47,55), at least some
of which are polycistronic (15,60,61,66–68). miRNA-contain-
polymerase II and to be polyadenylated and capped (62,65).
Many human and mouse miRNAs are derived from the
introns of protein-coding genes and the remainder from the
introns and the exons of mRNA-like ncRNA genes (63,66).
These transcripts, presumably after splicing (64), are pro-
cessed by the RNase III endonuclease Drosha (69). Drosha
cleaves RNA hairpins that contain a large (?10 nt) terminal
loop approximately two helical turns into the stem, to excise
65–75 nt precursors called ‘pre-miRNAs’ (69,70). Drosha
appears to occur in two complexes in the nucleus. The
larger of these complexes includes a variety of RNA-associ-
ated proteins including RNA helicases, proteins that bind
double-stranded RNA, novel heterogeneous nuclear ribonu-
cleoproteins and the Ewing’s sarcoma family of proteins
(71), whereas the smaller complex is composed of Drosha
and the double-stranded-RNA-binding protein, DGCR8 (also
called Pasha), the product of a gene deleted in DiGeorge syn-
drome (71–74). The pre-miRNAs are then exported from the
nucleus by Exportin 5 (75,76) and processed by the cyto-
plasmic RNase III endonuclease Dicer (77) into ?22 bp
(imperfect) duplexes with a 2 nt overhang at their 30ends
siRNAs are also processed by Dicer from double-stranded
RNA precursors but do not require Drosha (reviewed in
43,46). These precursors may be produced endogenously,
for example, from sense–antisense transcripts. They may
also be supplied exogenously, as occurred in the initial discov-
ery of RNAi, whereby such RNAs can act catalytically to
destroy endogenous RNAs with matching sequence (78),
now a widespread tool for probing gene function by siRNA-
induced target knockdown (57,58).
The Dicer-processed short duplex RNAs are incorporated
into the RISC ribonucleoprotein complex, which contains a
member of the Argonaute family. There are many Argonaute
homologs in animals, plants and fungi, implying that
there may be many forms of such complexes that may recog-
nize different RNA substrates (reviewed in 79). Recent
evidence suggests that different RISC complexes containing
different Argonaute proteins may be involved in miRNA-
and siRNA-mediated RNAi (80), although there are conflic-
ting reports (81). Argonaute proteins intersect with the
Wingless/Wnt and Hedgehog pathways that control cell
fate and developmental patterning (82,83). Mutations in
Argonaute family members affect a variety of developmental
processes including germ cell development and stem cell
fate, as well as being implicated in various human cancers
and developmental abnormalities (79). The fragile X mental
retardation protein (FMRP), an RNA-binding protein which
associates with hundreds of mRNAs in neurons via a
G-quartet structure and/or U-rich sequences (84–88), is also
associated with the RISC complex, as well as with Dicer
itself (89–92), and is involved with the control of behavior
via a process involving Argonaute2 (93). There is a strong
enrichment of predicted miRNA targets in mRNAs associated
with FMRP in mammals (94), and it has also recently been
reported that FMRP is phosphorylated by casein kinase II
(95), hinting at the enormous complexity in these RNA pro-
cessing and signaling pathways and their regulation.
In general, only one strand from the processed duplex is
retained in the RISC complex, the selection of which
appears to be determined by the relative stability of the two
ends of the duplex, favoring the one whose 50-end is less
tightly paired (96,97). The RISC complex then forms a
complex with a target RNA and either leads to its translational
repression by an, as yet, unknown mechanism, but which may
involve interaction with polyribosomes (98), or in the case of
(near) perfect identity cleaves the target RNA approximately
in the middle of the paired region. Recent evidence suggests
that the endonuclease activity within the RISC complex,
dubbed ‘slicer’, is in fact mediated by an RNase H-like
domain (piwi) in Argonaute (99–102).
The roles of miRNAs and siRNAs in development
Some mammalian miRNAs appear to be ubiquitously
expressed, but most have been found to exhibit developmen-
tally regulated expression patterns in a variety of cells and
tissues, including brain, lung, liver, spleen, heart and skeletal
muscle, using northern blots, PCR, microarray chips and
sensor transgenes (15,46,68,103–112). Many miRNAs are
specifically expressed during embryonal stem cell differen-
tiation (68,105) and embryogenesis (112), as well as during
brain development (104,108,113,114), neuronal differentiation
(103,115) and hematopoietic lineage differentiation (106,111).
Human Molecular Genetics, 2005, Vol. 14, Review Issue 1R123
Studies in model organisms have shown that miRNAs are
involved in the control of developmental timing, cell prolifer-
ation, left–right patterning, neuronal cell fate, apoptosis and
fat metabolism in invertebrates (as well as in a variety of
developmental processes in plants) (reviewed in 43,45–
47,116), and there is every reason to expect a similar range
of functions in vertebrates. Indeed the archetypal miRNAs,
lin-4 and let-7, which were first discovered by genetic
screens to control developmental timing in Caenorhabditis
elegans, have been shown to have close homologs in other
species, including mammals (117–119), as do many other
miRNAs (54,67,118–120). The target of lin-4 and let-7,
lin-28, is also conserved in mammals (121). siRNAs targeted
against let-7 cause developmental abnormalities in fish and
frogs (122), and human let-7 paralogs are able to suppress a
sensor transgene containing the human homolog of lin-28
(123). It has also been shown that let-7b miRNA associates
together with lin-28 mRNA in polyribosomes in human
cells, indicating a possible physical interaction between this
miRNA and its target mRNA (98). Reduced expression of
let-7 is observed in certain human lung cancers in association
with shortened postoperative survival, and over-expression of
let-7 in a lung adenocarcinoma cell line inhibited lung cancer
cell growth in vitro (124).
Knockout of the miRNA-producing enzyme Dicer1 in mice
leads to lethality early in development, with Dicer1-null
embryos depleted of stem cells (125). These observations
and the apparent inability to generate viable Dicer1-null
embryonic stem cells in vitro suggest a role for Dicer, and,
by implication, miRNAs, in maintaining stem cell populations
during early mouse development (125). Dicer-defective ES
cells also exhibit severe defects in differentiation in vitro as
well as in centromeric silencing (126). Inactivation of Dicer
also causes developmental arrest in zebrafish embryos (127).
In mammals, miRNAs have been shown to regulate B-cell
differentiation (106), adipocyte differentiation (128) and
insulin secretion (129). Chen et al. have shown that three
miRNAs are differentially expressed during mouse hemato-
poiesis and that ectopic over-expression of one of them
(miR-181) in hematopoietic stem/progenitor cells increases
the fraction of B-lineage cells both in vitro and in vivo
(106). Reduction in the level of miR-143, one of whose
predicted targets is the MAP kinase BMK1/ERK5 mRNA
(130), resulted in an increase in the level of BMK1/ERK5
and inhibited adipocyte differentiation in culture (128). In pan-
creatic endocrine cells, inhibition of miR-375 enhanced
glucose-induced insulin secretion and conversely, over-
expression of miR-375 suppressed insulin secretion, an effect
that could be mimicked by siRNAs directed against Myotro-
phin, the putative target of miR-375 (129). The authors
suggest that many of 67 miRNA sequences cloned from pan-
creatic cells (11 of which had not been previously identified)
may regulate endocrine pancreas development (129).
Some miRNAs are embedded in Hox clusters and exhibit
expression patterns that are reminiscent of Hox genes
(51,112). At least one of these miRNAs (miR-196) has exten-
sive, evolutionarily conserved complementarity to HoxB8,
HoxC8 and HoxD8 sequences and has been shown to nega-
tively regulate HoxB8 and other Hox genes, suggesting a
miRNA-mediated mechanism for the posttranscriptional
restriction of Hox gene expression during vertebrate develop-
It has also been shown that some mammalian miRNAs are
imprinted (15,61). The imprinting process clearly involves
transactions with non-coding RNAs (131), although the mech-
anisms remain unknown. Mouse miR-127 and miR-136 are
transcribed antisense to a reciprocally imprinted retrotranspo-
son-like gene, which are expressed from maternal and paternal
chromosomes, respectively. In addition, the neighboring
region contains maternally expressed clusters of snoRNA
and miRNAs, and it has been suggested that these miRNAs
may play a role in the imprinting process, either by directing
allele-specific chromatin modification or by targeting particu-
lar transcripts (15,61).
These observations all indicate that miRNAs are part of the
molecular circuitry and complex regulatory networks that
control cell fate during mammalian development, a conclusion
supported by a variety of studies in other animals. For example,
the miRNAs, miR-7 and miR-2a/b, have been shown to regu-
late the Notch pathway and proapoptotic genes reaper, grim
and sickle in Drosophila (132).
Consistent with their role in developmental processes,
perturbations of miRNA expression are observed in aberrant
developmental states, i.e. oncogenesis (133), in human
B-cell chronic lymphocytic leukemia (109,134), Burkitt
lymphoma (20), colorectal cancer (135), lung cancer (124)
and in a number of cancer cell lines (110). A high proportion
of known miRNAs are located at fragile sites or in cancer-
associated genomic regions (minimal regions of loss of hetero-
zygosity, minimal regions of amplification or common
breakpoint regions) (136). Interestingly, some miRNAs from
the same genomic cluster show different expression patterns
in cancer cells, indicating that the regulation of some
miRNAs might occur post transcriptionally (109), perhaps,
themselves regulated by other miRNAs as part of more
complex regulatory networks (described subsequently).
It has been reported that some siRNAs can induce sequence-
dependent off-target effects on proteins such as p53 and p21
that are sensitive markers of cell state (137), which suggest
care in the interpretation of such experiments. These effects
may occur either because of partial complementary sequence
matches to other genes (137) or because perturbation
of miRNA-regulated pathways can have pleiotropic effects
on wider networks of gene expression.
Target prediction in mammals
Thus far, only a few miRNA targets have been identified in
mammals and the rules of interaction are largely unknown.
The majority of identified mammalian miRNAs has non-
perfect matches to target mRNAs but the rules are complex
(138). Because of the very short target sequences and the pre-
sence of mismatches, the bioinformatic prediction of miRNA
targets, especially in the complex genomes of mammals, is a
very challenging task. Most studies predict miRNA targets
on the basis of an evolutionarily conserved sequence comple-
mentarity and low free energy of interaction, usually focused
on 30-UTRs of known genes (94,123,130,132), on the pre-
sumption that most miRNAs are involved in translational
R124 Human Molecular Genetics, 2005, Vol. 14, Review Issue 1
repression via UTRs, by extension from the well-studied
examples of lin-4 and let-7.
Such approaches potentially minimize the numbers of false
positives but may well seriously underestimate the actual
numbers of such RNAs (described subsequently). On the
basis of minimal binding energies, Kiriakidou et al. (123) pre-
dicted 5031 target sequences for 94 miRNAs. More than 400
targets were predicted by Lewis et al. (130) and 11 out 15
were confirmed experimentally. Predictions from a bigger
miRNA dataset (218 known mammalian miRNAs) identified
2273 target genes with one or more target sites showing
90% sequence conservation between human, mouse and rat
in aligned UTRs (94). The predicted target genes had
diverse functions, but were enriched for genes encoding
mRNAs coding for transcription factors, components of
the miRNA machinery, other proteins involved in translational
regulation and components
degradation machinery (94,130), many of which are known
to play important roles in developmental regulation and
some of which are involved in the molecular etiology of
cancer. Very recently, sophisticated bioinformatics analyses
based on the overabundance of conserved adenosines flanking
the complementary sites in mRNAs have implicated more than
5300 human genes (.20% of all known or predicted human
protein-coding genes) as potential miRNA targets, most of
which occur in 30-UTRs, but some of which occur in coding
sequences (139). The fact that many miRNAs are predicted
to have multiple cognate mRNAs, and vice versa, suggests
that the regulatory networks in which they participate are
very complex indeed.
Small RNAs regulate chromosome dynamics and
As described previously, miRNAs and siRNAs target mRNAs
for either translational inhibition or destruction by RISC-
mediated cleavage (43,45,46). However, there is also con-
siderable evidence that small RNAs also regulate chromosome
dynamics, chromatin modification and epigenetic memory,
including imprinting, DNA methylation and transcriptional
gene silencing (2).
The RNAi pathway and non-coding RNAs have been shown
to be central to the formation of silenced chromatin and chro-
mosomal dynamics in animals, plants, fungi and protozoa
(reviewed in 140–142). In the fission yeast, Schizosaccharo-
myces pombe, the RNAi pathway has been shown to be
involved in heterochromatin formation, as well as in centro-
mere function in meiosis and mitosis, via the methylation of
histone H3 on lysine-9 and the RITS (RNA-induced initiation
of transcriptional gene silencing) complex which contains
Argonaute, the Chp1 chromodomain protein (among others)
target DNA in heterochromatic regions (143–148). Meiosis
in S. pombe has also been reported to require a number of
specific non-coding RNAs (149). Similar observations have
been made in Drosophila where mutations in components of
the RNAi machinery affect silencing and heterochromatin
formation, accompanied by reduction in histone H3 lysine-9
methylation and delocalization of the heterochromatin pro-
teins HP1 and HP2 (150). Short 25–27 nt RNAs, derived
RNAs homologousto the
from dsRNA of Drosophila Su(Ste) repeats from the Y
chromosome, suppress the Stellate gene on the X chromosome,
and complementarity between the Stellate transcript and the
Su(Ste) repeats is essential for silencing (151). Knockout of
Dicer has been recently reported to affect centromeric hetero-
chromatin formation in mouse (126).
These observations suggest that small RNAs may be central
to chromatin regulation in all eukaryotes including mammals.
Indeed, it has been shown that the localization of mammalian
HP1 to heterochromatin involves its co-ordinate binding to
methylated histone H3 and RNA, involving interactions in
the hinge region between its chromodomains (152,153). This
is consistent with previous reports that chromodomains
(which are present in many different types of chromatin-
binding and chromatin remodeling proteins, including the
polycomb family, the histone methyltransferase and histone
acetyltransferase families, the retinoblastoma binding protein
1 family, the CHD family and the SWI3 family) bind RNA
as well as modified histones (154–156). RNA-interacting
proteins are also components of the mammalian DNA methyl-
ation system (157).
Moreover, it has recently been shown that synthetic siRNAs
targeted to CpG islands in the E-cadherin promoter reduced
the expression of the gene and induced significant DNA
methylation and histone H3 lysine-9 methylation in human
cultured cells (158). Similar results were obtained with
siRNAs directed against the erbB2/HER2 promoter (158)
and the elongation factor 1alpha promoter (159), providing
strong support for the notion that endogenous small RNAs
may perform similar functions in vivo. However, these
results remain to be confirmed, and there is a recent report
that over-expression of fragments of a non-coding antisense
RNA Khps1 results in demethylation of CG sites and methyl-
ation of CC(A/T)GG sites in a region of the Sphk1 gene
promoter that is subjected to tissue-specific differential
methylation (160), suggesting that RNA signaling may
control epigenetic modifications in more than one way. RNA
signaling may also play a key role in the control of transcrip-
tion and splicing (see below), although such RNAs have yet to
Many other small RNAs appear to exist in animal and plant
cells. Ambros and colleagues (116) recently described 33
small non-coding RNAs in C. elegans, which are similar
in size to miRNAs and are developmentally regulated but
which are not derived from hairpin precursors and are not
evolutionarily conserved. They also described over 700 small
RNAs that are antisense to known protein-coding sequences
(compared with only 49 from sense strands), some of which
are detectable as ?22 nt species in northern blots, which are
potential endogenous siRNAs (116). Endogenous siRNAs
derived from the sense and antisense strands of non-coding
RNA have been described in Arabidopsis (161). A number
of small RNAs, including new snoRNAs and 22 others
ranging from 70 to 450 bp derived from intergenic and genic
regions, including splice junctions, were detected and con-
firmed by northern blot analyses in Drosophila (162).
Human Molecular Genetics, 2005, Vol. 14, Review Issue 1R125
It seems that other small RNAs also exist in mammals. Only
30% of 179 small RNA sequences cloned from mouse ES cells
appear to come from hairpin precursors (68). Approximately
20% show similarity to tRNA or rRNA, which leaves close
to half that may act as small regulatory RNAs in some other
capacity. Of 733 non-redundant sequences isolated from
human ES cells, only 36 could have been derived from
hairpin precursors (105). More than 50 unknown short non-
coding RNAs were cloned from neural stem cells (163). One
of these sequences is present in more than 60 copies in the
mouse genome and has similarity to the NRSE/RE1 sequence,
which is preferentially localized in promoter regions of
neuron-specific genes. This RNA, which occurs in the
nucleus as a small ?20 nt dsRNA, controls the differentiation
of adult neural stem cells and activates the transcription of
genes containing NRSE/RE1 sequence, apparently mediated
through dsRNA–protein interactions, rather than through
siRNA or miRNA (163).
PARALLEL OUTPUTS AND
Approximately two-thirds of annotated mammalian miRNAs
are encoded within known genes and (like snoRNAs) mainly
occur within introns of protein-coding and non-coding
genes, with some residing within exons of non-coding genes
(63). Some of these transcripts may be very long and
contain multiple miRNAs (61,67). The situation appears to
be even more complicated as some miRNAs from intronic
regions are derived from anti-sense transcripts (54). The
most common molecular function for mammalian protein-
coding host genes are those annotated as ‘purine nucleotide
binding’, ‘DNA binding’ (63) and those containing homeobox
and RNA-binding domains (94), all of which point to the par-
allel output of proteins and regulatory RNA sequences as part
of complex networks which underpin mammalian biology
(1,24) (Fig. 1). Moreover, it has also been suggested, on the
basis of sequence homologies, that some miRNAs may regu-
late other miRNAs rather than mRNAs via a network of regu-
latory interactions at the RNA level (164).
THE TIP OF THE ICEBERG?
There are only limited numbers of known miRNAs in
mammals. The fact that some of these miRNAs show up
repeatedly in cloning experiments had led some to suggest
that the set of miRNAs in mammals and other organisms
is small. However, there are good reasons to think that this
is not the case and that there are in fact tens or even hundreds
of thousands of RNA signals which constitute a hitherto
hidden control network that regulates chromatin architecture
and gene expression during mammalian ontogeny (24).
Some miRNAs are present in large amounts (54), but it is
clear that at least some miRNAs and other small regulatory
RNAs are present at very low levels (45,165), and it is poss-
ible, indeed likely, that most will exhibit very restricted
expression patterns in specific cell types such as observed in
hematopoietic and pancreatic
miRNAs, such as that encoded by the lys-6 locus, which
controls the asymmetry of chemosensory neurons in C.
elegans (166), and that encoded by the bantam locus in
Drosophila (167), were only discovered by sensitive genetic
screens, which are difficult if not impossible to carry out in
mammals. The miRNA encoded by lys-6 is very scarce and
cannot be detected by normal biochemical procedures, and
loss-of-function mutations in it result in a very subtle pheno-
The lack of known mutations in miRNAs (and no doubt
other types of regulatory RNAs) in mammals is likely to be
due to a combination of ascertainment bias focused on
exons of protein-coding genes and the difficulty of mutation
screening across large tracts of non-coding sequences
in regions identified by genome scanning for quantitative
trait or disease associations. In this context, it is worth
noting that the mutations underlying the callipyge (‘beautiful
bottom’) phenotype in sheep or the enhanced muscling of
domestic pigs are single base substitutions within non-
coding sequences (a long intergenic sequence of unknown
transcriptional status in the DLK1-GTL2 imprinted region
and the third intron of the IGF2 gene, respectively), the
identification of which involved tour-de-force analyses in
well structured pedigrees (168–170).
The problems of cloning small RNAs (171) and the con-
tamination of cDNA libraries with rRNAs and other
common RNA sequences have led to the conclusion that not
many more miRNAs will be identified by this approach
(54). Bioinformatic predictions (at least to date) have been
limited by the tight constraints on the search parameters,
Figure 1. Regulatory networks involving small non-coding RNAs. Small
non-coding RNAs regulate genome structure and gene expression at many
levels. miRNAs, siRNAs, snoRNAs and other small RNAs are involved in
the regulation of translation, mRNA stability and chromatin structure, as
well as self-regulation (dashed lines) and possibly also the control of transcrip-
tion and splicing (question marks).
R126 Human Molecular Genetics, 2005, Vol. 14, Review Issue 1
including their focus on hairpin precursors, mRNA/UTR
targets and strongevolutionary
improved filters based on the different patterns of miRNA
and flanking sequence conservation in different species have
recently identified almost 1000 candidates (56). Moreover,
new algorithms based on secondary structural parameters are
being developed, which appear to have the potential to ident-
ify other types of non-coding RNAs (172) that presumably
also have regulatory functions.
There is strong evidence that chromatin dynamics and
heterochromatin formation are controlled by small regulatory
RNAs and that local chromatin architecture (in promoter
regions) can also be directed by small RNAs. Indeed, this
would make a lot of sense. It is well established that chromatin
modification occurs at many different loci in different cells
and that this is central to developmental ontogeny. There
must either be an army of sequence-specific DNA binding
proteins that carry out these modifications, which is not the
case—there are only a limited number of DNA and histone
modifying enzymes (methylases, acetylases and deacetylases
etc.) (173)—or these enzymes must be directed to their sites
of action by some other signal, most logically sequence-
specific RNAs. Such signals would also potentially solve the
conundrum of how to select from the huge number of tran-
scription factor binding sites that exist in the genome. In this
context, it is interesting to note that triplexes, which may
contain RNA, are very common in human chromosomes
(174) and many transcription factors have high affinity for
Trans-acting guide RNAs may also regulate alternative spli-
cing (177), which is currently mainly thought to be controlled
by the combinatorial effects of protein ‘splicing factors’ but is
not at all well understood in these terms (2,178,179). Consist-
ent with the possibility that site-specific trans-acting RNAs are
involved, the nucleotide sequences around alternative splice
sites are often highly conserved between species (180,181),
and it has been shown by many studies that splicing patterns
may be easily altered in cultured cells and in whole animals
by introducing small antisense RNAs, an approach which is
showing considerable promise for gene therapy of splice site
mutations in muscular dystrophy and other human genetic
diseases (182–186). It is not a big leap of faith to conclude
that RNA control of splice site selection is also likely to
happen naturally and that the reason that it has not yet been
demonstrated to be the case is because of the sheer complexity
of the numbers and variety of such signals in regulatory net-
works in different cells. If cells are awash with small RNA
signals processed from longer precursors, which (as such)
have short half-lives, identification of these signals will be dif-
ficult, although bioinformatics using appropriate search algor-
ithms may provide a means to do so.
The known miRNAs tend to be highly conserved (54,56),
presumably because their sequence is constrained by func-
tional interactions with multiple targets (94,187), which is
possibly also the case for the ultra-conserved elements that
are far more conserved than protein-coding sequences in the
mammalian genome (188). In contrast, endogenous siRNAs
are less conserved presumably because these RNAs and their
homologous targets can easily co-vary and still maintain
specificity (43), which would make them difficult to identify
bioinformatically, at least on the basis of evolutionary conser-
vation. The level of selection pressure on such sequences (as
signaling molecules largely dependent on primary sequence
recognition and secondary structure) will be a function of
the number of interactions that must be maintained, rather
than the precise sequence itself. Those with one or few inter-
acting partners will be able to evolve relatively freely and also
explore new connections in regulatory networks, which them-
selves can evolve to explore new developmental space and
which (given a relatively stable proteome) may be the major
route to higher complexity and phenotypic variation.
Thus, many small regulatory RNAs, including possibly the
majority of miRNAs, may not show strong evidence of
sequence conservation over significant evolutionary distances.
In this context, it is worth pointing out that known non-coding
RNAs with conserved functions, such as Xist, are not highly
conserved at the primary sequence level among mammals
(189,190). This will, of course, also contribute to the percep-
tion that genomic sequences encoding such ncRNAs are
(in general) drifting neutrally and that the majority of the
genome which is transcribed is non-functional, which may
not be the case at all (21). Indeed, a recent analysis suggests
that the proportion of the human genome which is under
purifying selection for functions held in common with other
mammals is (at least) an order of magnitude higher in non-
coding than in protein-coding sequences (191), an observation
which is hard to reconcile with protein-based models of regu-
lation of gene expression.
We have argued elsewhere that the majority of the genome
of humans and other complex organisms is in fact devoted to
extensive, but hitherto largely hidden, regulatory networks that
are trans-acted by non-coding RNAs and that were essential to
the evolution of complex organisms (1,2,21). We predicted
that regulatory ncRNAs would be derived from the vast
tracts of transcribed introns (23,24), which has now been
confirmed in principle, as well as from non-protein-coding
transcripts, which also appear to be the case. Indeed, the
evidence is accumulating that the major advance in the
evolution of complex organisms was the co-option of RNA
as a digital signaling network, which was required to over-
come the limitations of an analog (protein) based regulatory
system (21,22,24). These RNA networks are likely to be
intrinsically robust and their perturbation, particularly in the
case of single nucleotide polymorphisms, may lead to a
range of subtle phenotypes (in contrast to the generally
severe effects of non-synonymous mutations in protein-
coding sequences) (2). These may underpin much of the vari-
ation observed between species and individuals, including
differences in quantitative and behavioral traits, and variation
in susceptibility to complex diseases. If this is correct, most of
our conceptions of gene regulation and approaches to molecu-
lar genetic analysis will have to be revised.
We have barely begun to investigate RNA regulatory networks
in mammals. The majority of the mammalian genome is tran-
scribed into non-protein-coding RNA. Numerous short RNAs
are processed from longer transcripts and possess various
Human Molecular Genetics, 2005, Vol. 14, Review Issue 1R127
expression patterns, but the biological functions and targets
are known for very few of them. The mechanism of RNAi
is relatively well studied but our knowledge of chromatin
modification by siRNAs is incomplete to say the least. It
also seems likely that other short non-coding RNAs exist in
the cell that may regulate many other processes and utilize
other mechanisms, which remain to be discovered. Our under-
standing of the mammalian genome is undergoing major
change. We used to consider most non-coding regions to be
junk, but the extent of non-coding RNA transcription, the
rapidly emerging evidence of regulatory networks controlled
by RNA and a new logic about the genetic structure of
complex organisms suggest that most of the mammalian
genome may in fact be functional or at least that this possi-
bility should be more seriously considered.
The authors thank the Australian Research Council and the
Queensland State Government for financial support and their
colleagues for many stimulating discussions. They also
thank Alex Hu ¨ttenhofer and two anonymous reviewers for
alerting us to some important omissions and for some very
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