REVIEW ARTICLE
MicroRNAs – micro in size but macro in function
Sunit K. Singh
1,2
, Manika Pal Bhadra
3
, Hermann J. Girschick
2
and Utpal Bhadra
4
1 Section of Infectious Diseases and Immunobiology, Centre for Cellular and Molecular Biology, Hyderabad, India
2 Section of Infectious Diseases, Immunology and Pediatric Rheumatology, Children’s Hospital, University of Wuerzburg, Germany
3 Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad, India
4 Functional Genomics and Gene Silencing Group, Centre for Cellular and Molecular Biology, Hyderabad, India
Introduction
Small RNAs exhibit a wide spectrum of biological
functions. There are many classes of small RNAs, such
as microRNAs (miRNAs), small interfering RNAs
(siRNAs), repeat associated small interfering RNAs
[1], small nuclear RNA, small nucleolar RNA, Piwi-
interacting RNA [2] and transacting short interfering
RNA [3].
miRNAs are single-stranded RNAs of 19–25 nucleo-
tides in length originating from endogenous hairpin-
shaped transcripts [4]. These miRNAs interact with
their target mRNAs by base pairing, which could lead
to translational repression; decapping, deadenylation
and ⁄or cleavage of target mRNA. The first known
miRNA, lin-4, was discovered in 1993 by Ambros and
coworkers in the nematode Caenorhabditis elegans
[5,6]. The lin-4 gene plays a role in the developmental
timing of stage-specific cell lineages in C. elegans.
Later on, lin-4 was found to encode a 22-nucleotide
noncoding RNA that negatively regulates the transla-
tion of lin-14. A few years later, another small RNA,
Keywords
Dicer; microRNA; miRNA and cancer;
miRNA and disease; miRNA and
therapeutics; miRNA biogenesis; miRNA
function; miRNA inhibitors; small RNA
Correspondence
S. K. Singh, Section of Infectious Diseases
and Immunobiology, Centre for Cellular and
Molecular Biology, Uppal Road,
Hyderabad 500007, India
Fax: +91 40 27160311
Tel: +91 40 27192523
E-mail: sunitsingh@ccmb.res.in
(Received 30 June 2008, revised 30 July
2008, accepted 1 August 2008)
doi:10.1111/j.1742-4658.2008.06624.x
MicroRNAs (miRNAs) are endogenous small RNAs that can regulate
target mRNAs by binding to their 3¢-UTRs. A single miRNA can regulate
many mRNA targets, and several miRNAs can regulate a single mRNA.
These have been reported to be involved in a variety of functions, including
developmental transitions, neuronal patterning, apoptosis, adipogenesis
metabolism and hematopoiesis in different organisms. Many oncogenes
and tumor suppressor genes are regulated by miRNAs. Studies conducted
in the past few years have demonstrated the possible association between
miRNAs and several human malignancies and infectious diseases. In this
article, we have focused on the mechanism of miRNA biogenesis and the
role of miRNAs in human health and disease.
Abbreviations
AD, Alzheimer’s disease; AGO, argonaute; Ab, amyloid b-peptide; Dcp, decapping enzyme; DCR, Dicer; DGCR8, DiGeorge syndrome critical
region gene 8; dsRBD, double-stranded RNA-binding domain; eIF, eukaryotic translation initiation factor; ES, embryonic stem; Exp-5,
exportin-5; IRES, internal ribosome entry site; KSHV, Kaposi sarcoma herpes virus; LNA, lock nucleic acid; miRISC, microRNA-containing
RNA-induced silencing complex; miRLC, microRNA-containing RNA-induced silencing complex loading complex; miRNA, microRNA; miRNP,
microRNA ribonucleoprotein; P-body, processing body; Pol II, RNA polymerase II; Pol III, RNA polymerase III; pre-miRNA, precursor
microRNA; pri-miRNA, primary microRNA; RISC, RNA-induced silencing complex; RLC, RNA-induced silencing complex loading complex;
RNAi, RNA interference; siRISC, small interfering RNA-containing RNA-induced silencing complex; siRNA, small interfering RNA.
FEBS Journal 275 (2008) 4929–4944 ª2008 The Authors Journal compilation ª2008 FEBS 4929
let-7, was reported as an additional regulator of devel-
opmental timing in C. elegans [7]. Similar to lin-4, let-7
also functions by binding the 3¢-UTR of lin-41 and
lin-57 to inhibit their translation.
To date, 678 human miRNAs have been character-
ized in the Sanger miRBase sequence database [8], and
many more are still to be identified. Approximately
50% of known human miRNAs are found in clusters
[9,10]. The clustered miRNAs are often related to each
other, but can also be unrelated. Clustered miRNAs
may be functionally related in terms of targeting the
same gene or different genes in the same biochemical
pathway. Most mammalian miRNA genes have been
reported to be located in defined transcription units
Fig. 1. MicroRNA biogenesis and function. The miRNA gene is transcribed by Pol II into a pri-miRNA in the nucleus. The pri-miRNA is pro-
cessed into pre-miRNA by the RNase III enzyme Drosha. The pre-miRNA is exported to the cytoplasm with the help of Ran-GTP cofactor
and Exp-5. The miRNA duplex is cleaved from the pre-miRNA by the RNase III enzyme Dicer and TRBP. Helicase unwinds the mature
miRNA duplex. Either each strand of the miRNA pair or only one strand of mature miRNA can be incorporated into miRISC. miRNAs bound
to miRISC mediate the degradation or translational inhibition of their target mRNAs.
MicroRNAs and their roles S. K. Singh et al.
4930 FEBS Journal 275 (2008) 4929–4944 ª2008 The Authors Journal compilation ª2008 FEBS
and intergenic regions [11]. Studies have revealed that
miRNAs have key roles in diverse processes such as
developmental control, hematopoietic cell differentia-
tion, neural development, apoptosis, cell proliferation
and organ development. In this review, we discuss the
mechanism of miRNA biogenesis and the roles of
miRNA during development and different pathological
states.
miRNA biogenesis
miRNA biogenesis includes miRNA transcription in
the nucleus, the export of miRNAs from the nucleus
to the cytoplasm, and subsequent processing and mat-
uration in the cytoplasm (Fig. 1). In most cases, the
transcription of miRNA genes is mediated by RNA
polymerase II (Pol II), resulting in long primary miR-
NA (pri-miRNA) transcripts with a fold-back structure
comprising a stem loop along with flanking segments
[12]. A few recent reports have shown the involvement
of RNA polymerase III (Pol III) in miRNA transcrip-
tion [13]. The sequence of the miRNA remains
embedded in the arms of the stem loop. The pri-miR-
NA contains the 7-methylguanosine cap and a poly(A)
tail, which is unique for Pol II transcripts, similar to
mRNAs [12,14]. However, the cap and poly(A) tail are
removed during miRNA processing. miRNA promot-
ers have been identified in many studies [9,15,16], and
reported to have typical Pol II elements such as a
TATA box [17], although the recent report of Borchert
et al. [13] suggests that members of the human chro-
mosome 19 miRNA cluster (miR-515-1, miR-517a,
miR-517c and miR-519a-1) are interspersed among
Alu repeats and expressed through Pol III. The pro-
cessing of pri-miRNAs into final mature miRNAs
occurs in a stepwise fashion, which is discussed in
detail in subsequent sections of this article.
Enzymatic machinery involved in
miRNA biogenesis and maturation
Drosha
In humans, the generation of precursor miRNA
(pre-miRNA) from the pri-miRNA transcript takes
place exclusively in the nucleus, through the action
of the microprocessor complex, composed of the
RNase III enzyme Drosha and the double-stranded
RNA-binding domain (dsRBD) protein DiGeorge
syndrome critical region gene 8 (DGCR8), into 70–
80 nucleotide pre-miRNAs [18,19]; this is followed
by maturation of miRNA in the cytosol. The stem
loop structure of pri-miRNAs is cleaved in the
nucleus by Drosha during the generation of pre-miR-
NA. This process is known as cropping. Drosha
forms a large microprocessor complex of 650 kDa
along with the dsRBD protein DGCR8 in humans
[20] and a 500 kDa complex along with the
dsRBD protein Pasha in flies (Drosophila melanogas-
ter) [18,21,22]. In contrast to the siRNA pathway,
the miRNA pathway is initiated in the nucleus [23].
The cleavage by Drosha generates a pre-miRNA
hairpin bearing two nucleotide 3¢-overhangs. Precur-
sor miRNAs are exported to the cytoplasm from the
nucleus by exportin-5 (Exp-5) in the presence of
Ran-GTP as a cofactor (Fig. 1) [22,24,25].
It is important to realize that many human miRNA
genes remain located in intronic regions of coding
genes, so their biogenesis remains coupled with mRNA
splicing [12,26]. Drosha releases the pre-miRNA from
the intron shortly before splicing, allowing the genera-
tion of both RNA species at the same time. The preci-
sion of Drosha–DGCR8 cleavage is very important for
miRNA maturation. Any shift in the position of the
Drosha cut, even by a single nucleotide on the pri-
miRNA, will affect the position of Dicer cleavage. A
shift in the Dicer cleavage site could result in different
5¢-ends and 3¢-ends in the mature miRNA. This type
of nucleotide shift may invert the relative stability of
the 5¢-end of the miRNA strand and of the other asso-
ciated strand, which is opposite to the miRNA strand.
Such a shift could result in the selection of the wrong
strand as the mature miRNA. Even if the stability
remains unchanged and the correct strand is loaded
into the RNA-induced silencing complex (RISC), then
the shift in the 5¢-end of the miRNA will change the
position of the seed sequence (2–8 nucleotides of
miRNA, which often match the target mRNA very
closely), which could lead to a change in its target
mRNA [27]. The RISC is a multiprotein complex that
cleaves specific mRNAs, and that is targeted for degra-
dation by homologous dsRNAs during the process of
RNA interference. This complex plays a very impor-
tant role in gene regulation by miRNAs and siRNAs.
There is an interesting mechanism that determines the
precision of cleavage by the Drosha–DGCR8 complex
to generate pre-miRNA transcripts from pri-miRNA
transcripts. Some structural features of the RNA have
been shown to be involved in determination of the
Drosha cleavage site [27].
The ssRNA segments flanking the base of the stem
loop are crucial for Drosha cleavage [28]. The deletion
of single-stranded regions or their conversion to
dsRNA greatly impairs the conversion of pri-miRNA
to pre-miRNA [28]. In a recent report, Davis et al. [29]
have shown the role of SMAD protein in Drosha-
S. K. Singh et al. MicroRNAs and their roles
FEBS Journal 275 (2008) 4929–4944 ª2008 The Authors Journal compilation ª2008 FEBS 4931
mediated miRNA maturation. Transforming growth
factor-band bone morphogenetic protein signaling
have been reported to promote the rapid increase in
expression of mature miR-21 by promoting the pro-
cessing of primary transcripts of miR-21 (pri-miR-21)
into precursor miR-21 (pre-miR-21) by the Drosha
(also known as RNASEN) complex [29]. Transforming
growth factor-b-specific and bone morphogenetic pro-
tein-specific SMAD signal transducers are recruited to
pri-miR-21 in a complex with RNA helicase p68 (also
known as DDX5), a component of the Drosha micro-
processor complex [29]. Thus, SMAD protein plays an
important role in Drosha-mediated miRNA matura-
tion [29].
Export and import of miRNAs between the
nucleus and cytoplasm
Exp-5 is a member of the karyopherin family of nucle-
ocytoplasmic transport factors, and plays a role in the
export of miRNAs from the nucleus to the cytoplasm
[30]. The function of Exp-5 is dependent on the GTP-
bound form of Ran cofactor for specific binding to its
export substrate in the cell nucleus. This process
involves the hydrolysis of Ran-GTP to Ran-GDP by
the cytoplasmic Ran GTPase-activating protein [31].
The role of Exp-5 in nucleocytoplasmic transport was
verified by using RNA interference (RNAi). In the
event of Exp-5 depletion by RNAi, the level of mature
miRNAs goes down but pre-miRNA does not accumu-
late in the nucleus. The lack of accumulation could be
due to instability of pre-miRNA. This suggests the
possibility that interaction of pre-miRNA with Exp-5
is required for the stability of pre-miRNA [32]. Exp-5
has been reported to recognize the ‘minihelix motif’ of
pre-miRNA, which consists of a > 14 bp stem and a
short 3¢-overhang.
Hwang et al. recently reported that a hexanucleo-
tide element directs the process of nuclear import
rather than export of miR-29b. In contrast to most
of the animal miRNAs, miR-29b has been reported
to be predominantly localized in the nucleus [33].
The special hexanucleotide terminal motif (AGU-
GUU) acts as a transferable nuclear localization ele-
ment of miR-29b, and is responsible for the nuclear
enrichment of miR-29b. These RNAs may prove to
be useful tools for manipulation of gene expression
in the nucleus. It is supposed that miR-29b could
have a role in regulation of the transcription or
splicing events of target transcripts. This role of
miR-29b is quite unique and is different from the
routine translational regulatory functions performed
by other miRNAs [33].
Role of Dicer in miRNA maturation
Dicer is an ATP-dependent multidomain enzyme of
the RNase III family, and has been reported to be
involved in cleavage of double-stranded siRNA and
miRNA. Dicer was initially identified in Drosophila
[34] and has been subsequently reported in humans,
plants and fungi. The mechanism of recognition of the
pre-miRNA by cytoplasmic Dicer is not known [35].
In the cytoplasm, the pre-miRNAs are processed into
22-nucleotide duplex miRNAs by the RNase III
enzyme Dicer (Fig. 1). Some organisms have a single
Dicer gene [36–39], whereas others have many [40,41].
In species with several Dicers, different homologs are
required for different functions [40,42,43]. Two Dicer
homologs (DCR1 and DCR2) have been reported in
Drosophila. DCR1 processes pre-miRNA, whereas
DCR2 processes long dsRNA in Drosophila [43–45].
The only Dicer gene in C. elegans,DCR1, is required
for the processing of both the long dsRNA and
pre-miRNAs.
Dicer cleavage results in the release of a duplex with
mature miRNA in one of the strands of the stem loop.
Both arms of the pre-miRNA stem loop structures are
imperfectly paired, containing G:U wobble pairs and
single nucleotide insertions. These imperfections cause
one strand of the duplex to be less stably paired at its
5¢-end [27]. The conversion from dsRNAs to ssRNAs
is a complex process, involving several RNA–protein
and protein–protein interactions. RISC loading com-
plex (RLC) is an RNA–protein complex that initiates
the formation of the RISC. The RLC puts a small
RNA duplex in the correct orientation for subsequent
RISC assembly [35]. The small RNAs (siRNAs and
miRNAs) in the RLC remain ready to be unwound
for functional RISC assembly. The siRISC loading
complex (siRLC) of Drosophila contains a DCR2–
R2D2 heterodimer and an siRNA duplex. R2D2 has
been reported to be a DCR2 stabilizer as well as the
asymmetric sensor for setting the siRNA orientation
for RISC assembly [35]. Detailed information on miR-
ISC loading complexes (miRLCs) is not available. In a
recent report, MacRae et al. [46] have demonstrated
the assembly of human RLC in vitro from purified
components without any cofactors or chaperones.
They demonstrated that reconstituted RLC maintains
the endogenous RLC functional activities of dicing,
slicing, guide-strand selection and argonaute (AGO)2
loading [46].
Dicer interacts with the dsRNA-binding protein part-
ner, the TAR RNA-binding protein (TRBP), in humans
[RDE4 in C. elegans and Loquacious (Loqs)inDrosoph-
ila], which probably bridges the initiation and effector
MicroRNAs and their roles S. K. Singh et al.
4932 FEBS Journal 275 (2008) 4929–4944 ª2008 The Authors Journal compilation ª2008 FEBS
steps of miRNA action [47–49]. DCR binds with high
affinity to the ends of dsRNAs bearing two-nucleotide
3¢-overhangs, which results in unwinding of duplexes.
The thermodynamic properties of siRNA–miRNA
duplexes play a critical role in determining molecular
function and longevity [50]. The unwinding of the
duplex strands starts at the ends with the lowest thermo-
dynamic stability. The relative stabilities of the base
pairs at the 5¢-ends of two strands determine the fate of
the strand, which has to participate in the RNAi path-
way [51]. Along with the thermodynamic stabilities, a
role of proteins such as R2D2 has also been reported
during the strand selection process. The orientation of
the DCR2–R2D2 protein heterodimer on the siRNA
duplex determines the siRNA strand, which has to asso-
ciate with the core RISC protein AGO2 in Drosophila
[52]. The exact mechanism by which R2D2 guides the
asymmetric assembly of the RISC in Drosophila is not
known. Dicer has an RNA helicase domain to cleave the
dsRNA.
In general, the miRNA strand, which has its 5¢-ter-
minus at the lowest thermodynamic stability, acts as
the mature miRNA (guide strand), and the other
strand (passenger strand) is degraded. However, a
recent report has shown that both strands could be
coaccumulated as miRNA pairs in some tissues, and
subjected to strand selection in other tissues [53].
Ro et al. [53] also reported that both strands of the
miRNA pair can target equal numbers of genes, and
were able to suppress the expression of their target
genes. This study provided evidence for a novel mecha-
nism involved in tissue-dependent miRNA biogenesis
and miRNA target selection [53]. Mature miRNAs are
incorporated into the effector complexes, known as
miRNP (microRNA ribonucleoprotein), mirgonaute,
or miRISC. The identification of the target by the
RISC is based on the complementarity between mature
miRNA and the mRNA. The degree of complementar-
ity decides whether the complex has to undergo endo-
nucleolytic cleavage of target mRNA or translational
repression.
In contrast to miRNAs, siRNAs are often synthe-
sized in vitro or in vivo from viruses or repetitive
sequences. siRNAs have been reported to be involved
in antiviral defense, and also in protecting the genome
against disruption by transposons. The presence of the
selective AGO protein family is one of the several
common features of siRISC and miRISC.
AGO proteins in the RISC
AGO proteins are well conserved in diverse organisms
[54], and constitute a large family involved in develop-
mental regulation in eukaryotes. Several AGO homo-
logs have been reported in eukaryotic organisms, such
as eight in humans [55], five in Drosophila [54], 27 in
C. elegans [56] and only one in fission yeast [36,56].
These homologs are characterized by the presence of
two domains, PAZ (Piwi ⁄Argonaute ⁄Zwille) and
PIWI. The PAZ domain of AGO proteins binds to
the 3¢-end of the ssRNA, possibly by recognizing the
3¢-overhangs [57,58].
AGO proteins are the core components of the RISC
in different organisms. Different AGO proteins specify
distinct RISC functions. Cofractionation studies in
Drosophila have shown that AGO2 cofractionates and
remains functionally associated with DCR2, whereas
AGO1 remains functionally associated with DCR1
[44,59]. These observations verify that DCR1 is
involved in miRNA maturation, whereas DCR2 is
involved in initiation of RNAi in Drosophila [43,44].
Although miRNAs and siRNAs have distinct biogene-
sis pathways in Drosophila, they have a common
sorting pathway, which partitions them into AGO1-
containing or AGO2-containing effector complexes
[60].
In contrast to Drosophila, humans and C. elegans
contain only one Dicer, which initiates the formation
of both siRISCs and miRISCs. In the case of humans,
different AGO proteins (AGO1 to AGO4) have been
reported to be involved during RISC assembly, but
only AGO2-associated RISCs have been reported to
be involved in the cleavage of target mRNA. There-
fore, AGO2 is also called slicer argonaute [61,62].
Slicer activity has been reported in the PIWI domain
of AGO proteins, on the basis of mutagenesis studies
[61]. Specific amino acid residues of the PIWI domain
of AGO2 are essential for slicer activity in AGO2
proteins of human and Drosophila [35].
Processing bodies (P-bodies) and their
biological function
It was thought that once mRNAs finish their job,
enzymes in the cytoplasm simply break them down.
Several groups reported that most of this degradation
occurs in P-bodies (processing bodies) or glycine-tryp-
tophan or decapping enzyme (Dcp) bodies. P-bodies
are found as discrete cytoplasmic bodies in yeast and
mammals. The conservation of P-bodies from yeast to
mammals suggests their important role in the cytoplas-
mic function of eukaryotic mRNA. P-bodies include
the Dcp1p ⁄Dcp2p, activators of decapping, Dhh1p
(referred to as RCK in mammals), Pat1p, Lsm1-7p,
Edc3p and the 5¢–3¢-exonuclease Xrn1p [63–66].
P-bodies have been reported as the sites for decapping
S. K. Singh et al. MicroRNAs and their roles
FEBS Journal 275 (2008) 4929–4944 ª2008 The Authors Journal compilation ª2008 FEBS 4933