Theme Issue Article
Regulation of microRNA biogenesis and function
Thomas Treiber; Nora Treiber; Gunter Meister
Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany
Summary
MicroRNAs (miRNAs) are considered as key regulators of literally all
cellular pathways. Therefore, miRNA biosynthesis and their individual
cellular functions must be tightly regulated as well. MiRNAs are tran-
scribed as primary transcripts, which are processed to mature miRNAs
in two consecutive maturation steps. Finally, the mature miRNA is incor-
porated into a miRNA-protein complex, where it directly interacts with
a member of the Argonaute (Ago) protein family. The miRNA guides
such protein complexes to partial complementary target sites, which
Correspondence to:
Gunter Meister
Biochemistry Center Regensburg (BZR)
Laboratory for RNA Biology, University of Regensburg
Universitätsstrasse 31, 93053 Regensburg, Germany
Tel.: +49 941 943 2847, Fax: +49 941 943 2936
E-mail: gunter.meister@vkl.uni-regensburg.de
Introduction
MicroRNAs (miRNAs) are short non-coding RNAs that are viewed
as fundamental regulators of cell function (1, 2). MiRNAs are ap-
proximately 20–25 nucleotides (nt) long and are generated from
double stranded RNA precursors (see below). One strand is se-
lected and interacts with a member of the Ago protein family to
form a miRNA-induced silencing complex (miRISC), also referred
to as micro-ribonucleoprotein (miRNP). The miRNA guides such
complexes to partially complementary target RNAs. Most of the
target sites are located within the 3’ untranslated region (UTR) of
mRNAs. However, functional miRNA binding sites in the 5’ UTR
as well as in the open reading frame have also been reported (3, 4).
Nucleotides 2–8 of the miRNA are particularly important for pair-
ing with the target mRNA. This sequence motif is referred to as the
miRNA seed sequence (1). Depending on the recognition site,
binding of miRISC to the cognate target can have different out-
comes (5). In case the target site is perfectly complementary to the
miRNA, the miRNA functions like short interfering RNAs (siR-
NAs) and the target is sequence-specifically cleaved by miRISC.
This is very rare in mammals but more frequent in plants. Binding
to partially complementary target sites is the rule in mammals and
leads to repression of translation or degradation of the target tran-
script (6). This degradation process, however, is different to RNAi-
like mechanisms and involves the recruitment of deadenylase
complexes such as the CCR4-NOT complex to the mRNA to re-
move or shorten the poly(A) tail. Poly(A) tail shortening induces
the removal of the 5’ cap of the mRNA, a process referred to as de-
capping. Uncapped mRNAs are rapidly removed from the cell by 5’
to 3’ exoribonucleases such as Xrn1 (7). Ribosome profiling ex-
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© Schattauer 2012 605
MicroRNAs in vascular biology
are typically located in the 3’ untranslated region (UTR) of mRNAs lead-
ing to inhibition of gene expression. MiRNA activity and abundance is
regulated on various levels ranging from transcription and processing
to target site binding and miRNA stability. Recent advances in our
understanding of how miRNA activity is regulated in mammalian cells
are summarised and discussed in this review article.
Keywords
microRNAs, Dicer, Drosha, Argonaute, gene silencing
Received: December 15, 2011
Accepted after minor revision: January 11, 2012
Prepublished online: February 8, 2012
doi:10.1160/TH11-12-0836
Thromb Haemost 2012; 107: 605–610
periments in conjunction with mRNA level measurements reveal-
ed that mRNA decay accounts for approximately 85%, while trans-
lational repression contributes only about 15% to miRNA-guided
gene silencing effects in mammals (8).
MiRNA-guided gene silencing is mediated by Ago proteins,
which are the direct binding partners of mature miRNAs and
therefore central components of miRISC. In mammals, four differ-
ent Ago proteins exist (Ago1-Ago4) and they are characterised by
PAZ (PIWI-Argonaute-Zwille), MID and PIWI (P-element-in-
duced wimpy testes) domains. The PAZ domain anchors the 3’ end
of the miRNA, while the MID domain contains a highly specific
binding pocket for the 5’ end of the miRNA (9). In addition, the
MID domain discriminates between the four different bases at the
5’ terminal nucleotide and prefers binding to uridines and, with a
slightly lower affinity, to adenosines explaining the U bias at the 5’
end of miRNAs (10). The PIWI domain is structurally similar to
RNase H and indeed some PIWI domains possess endonuclease
activity and mediate target RNA cleavage in RNAi (11, 12). In
mammals, only Ago2 is endonucleolytically active (13, 14).
MiRNA-guided regulation of gene expression has been impli-
cated in literally every cellular pathway. In addition, each cell type
expresses a specific subset of miRNAs to ensure that cell type spe-
cific mRNA profiles are established and maintained. For example,
expression of a neuron-specific miRNA in non-neuronal cells re-
sults in shifting the global gene expression program towards tran-
script profiles typically found in neurons (15). Another prominent
example for the importance of miRNAs is the effect of a number of
miRNAs on the reprogramming of somatic cells to induced pluri-
potent stem cells (iPS) (16).
Thrombosis and Haemostasis 107.4/2012
606 Treiber et al. microRNA-guided gene silencing and its regulation
Given their fundamental cellular roles, it is not surprising that
miRNAs are affected in many diseases. Almost all types of cancer
MicroRNAs in vascular biology
have been analysed and miRNA missexpression was frequently
noted (17, 18). Indeed, different cancers have different miRNA
profiles, which can even be found in the serum of patients (19).
The functions of such circulating miRNAs have not been ident-
ified, but profiling of serum miRNAs might be a powerful ap-
proach for early cancer diagnosis.
Disease-causing changes in miRNA levels can have many differ-
ent reasons (6, 20). i) Similar to other genes, transcription can be
positively or negatively affected. ii) miRNA processing can be regu-
lated at each individual step (see below). iii) miRNA binding to
Ago proteins as well as the activity of Ago proteins can be altered.
iv) Accessibility of miRNA target sites on mRNAs can be changed.
v) Other RNAs can function as sponges and sequester miRNAs
thereby regulating their activity (21). Recent progress towards the
understanding of miRNA regulation will be summarised in several
chapters of this review.
miRNA biogenesis
MiRNAs are transcribed as parts of longer primary transcripts
(pri-miRNAs) that are generated mainly by RNA polymerase II
(22, 23). MiRNA genes are frequently located in transcripts of pro-
tein-coding genes, where they mainly reside in introns (24, 25).
Other miRNAs are transcribed as part of long non-coding RNAs
and are often arranged as clusters in the genome, which leads to
one pri-miRNA being subsequently processed into several func-
tional miRNAs (24). Like other Pol II transcripts, pri-miRNAs pos-
sess a 5’ cap and a 3’ poly-A-tail (23). Within the primary tran-
scripts, miRNAs form stem-loop structures, which contain the ma-
ture miRNA as part of an imperfectly paired double stranded stem
connected by a short terminal loop. In the canonical miRNA bio-
genesis pathway, these structures are recognised by the micropro-
cessor complex, a multiprotein complex with the two core compo-
nents Drosha and Di George Syndrome critical region gene 8
(DGCR8) (26–29). The double-stranded RNA binding protein
DGCR8 binds to the base of the stem loop structure and thereby
guides the positioning of the RNase III enzyme Drosha, which con-
stitutes the catalytic center of the complex (30). Drosha cleaves the
double-stranded stem about 11 bp from the base and generates a
two nucleotide (nt) overhang at the 3’ end (27, 30). This cleavage
reaction liberates a hairpin-shaped RNA molecule of 70–100 bp
called miRNA precursor or pre-miRNA. The mammalian micro-
processor contains several additional protein factors, most of
which have not been assigned a specific function in miRNA pro-
cessing yet (27). Notably, the two DEAD-box RNA helicases DDX5
and DDX17 (also known as p68 and p72) have been identified as
integral part of the microprocessor and are necessary for the effi-
cient cleavage of a subset of pri-miRNAs (31).
The pre-miRNAs are specifically recognised by the nuclear ex-
port receptor Exportin 5 and exported in a Ran-GTP dependent
manner (32–34). In the cytosol, the pre-miRNA is further pro-
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cessed by the RNase III enzyme Dicer (35). This enzyme binds the
3’ overhang of the pre-miRNA with its PAZ domain and thereby
positions the substrate correctly for the cleavage by the two cata-
lytic domains 22 nt upstream within the double stranded stem (36,
37). A recent study has reported that in metazoa, the 5’ end of the
pre-miRNA is contacted by Dicer proteins as well. This interaction
contributes to pre-miRNA recognition and correct processing
(38). The result of Dicer cleavage is a double stranded RNA of 22 nt
in length with 3’ overhangs of 2nt on both ends (1). One of the
strands (the mature miRNA) is transferred into an Ago protein,
whereas the other strand (the star (*)-strand) is degraded. In
mammals, the strand selection and RISC assembly is accomplished
by a complex that contains Dicer, Ago and the double stranded
RNA binding protein TRBP (39–41). Statistical analyses have dis-
covered that generally the strand with the less stable base pairing at
the 5’ end is chosen as guide strand (42, 43), which requires an
analysis of the thermodynamic properties of the double stranded
miRNA after dicing. Recently, it has been proposed that the sensor
for the thermodynamic asymmetry is the helicase domain of
human Dicer itself. In this model, the double stranded Dicer cleav-
age product is repositioned on Dicer after the cleavage reaction.
During this process, the helicase domain of Dicer senses the
thermodynamic stability of the ends and positions the double
stranded RNA in an orientation that allows for correct guide
strand incorporation into RISC (씰Fig. 1) (44).
Regulation of miRNA biogenesis
Given the tremendous impact of miRNA-guided gene regulation
on almost all aspects of cellular biology, it is not surprising that
miRNA levels are tightly controlled and that deregulation can lead
to various diseases including cancer (6, 17, 20).
The first and one of the most important layers governing
miRNA abundance is the regulation of pri-miRNA transcription.
The transcription of miRNAs encoded within introns of protein
coding genes is mostly driven by the promoter of the host gene
leading to a strong correlation of mRNA and miRNA expression.
However, an analysis of chromatin signatures characteristic for
promoters has identified additional promoters for about one third
of the intronic miRNAs enabling a separate regulation from the
host gene (45). Intergenic miRNAs have dedicated promoters,
which show all features commonly associated with Pol II-mediated
transcription such as histone marks, CpG islands, transcription
factor binding sites etc. (45). Clustered miRNAs share one pro-
moter and are coregulated as part of long pri-miRNAs. Via the pro-
moters, pri-miRNA transcription can be strongly regulated by
binding of transcription factors. The tumour suppressor p53, for
instance, has been shown to upregulate the transcription of
miR-34 family members, which in turn repress important factors
for cell proliferation and survival, such as Bcl2 and Cdk4 and 6
(46–49).
In addition to the transcriptional regulation, it was noted that
also the processing of pri-miRNA transcripts can be the limiting
© Schattauer 2012
 Treiber et al. microRNA-guided gene silencing and its regulation 607

factor for miRNA expression and can be regulated at different steps

leading to the accumulation of specific precursor forms (50, 51).

MicroRNAs in vascular biology

This posttranscriptional regulation can affect a large number of
miRNAs and is especially prominent in cancer samples and cell
lines (50, 52). Since these discoveries, a small but increasing
number of mechanisms that specifically regulate the processing of
miRNA subsets have been reported and will be discussed below.

Regulation of Drosha processing

An emerging group of proteins that can modulate pri-miRNA pro-
cessing by the microprocessor complex in response to diverse sti-
muli are transcription factors. For example, activation of Smad
proteins by stimulation of cells with bone morphogenic protein
(BMP) or tumour growth factor β (TGF-β) can stimulate the
maturation of specific miRNAs. This activity is independent of
Smad phosphorylation and binding of the Co-Smad4 and leads to
increased recruitment of the pri-miRNAs to the microprocessor
complex, enabling a more efficient cleavage by Drosha (53). Analy-
sis of the about 20 miRNAs that are affected by this regulation re-
vealed a consensus sequence that strongly resembles the Smad-
binding element in DNA and is located in the double stranded
stem of the pri-miRNAs. This motif is bound by the DNA-binding
MH1 domain of Smad1, 3 and 5 and establishes the specificity of
the regulated miRNAs (54). Another well-characterised Smad do-
main, the MH2 domain, binds to p68 (DDX5), which is part of the
microprocessor complex and is necessary for the induction of
miRNA processing by the Smad proteins (씰Fig. 1) (53).
Similarly, activation of the p53 pathway through DNA damage
leads to an association of the p53 protein with the microprocessor
complex via the p68 helicase (씰Fig. 1). This recruitment causes
increased processing of a set of pri-miRNAs including mir-145,
Figure 1: MiRNAs are transcribed as primary miRNA transcripts (pri-
which exerts a tumour suppressive function via repression of
miRNAs) in the nucleus of eukaryotic cells. In a first processing step, the
c-myc (55). In contrast to the Smad-regulated miRNAs, no com- microprocessor complex containing the catalytic subunit Drosha, its co-fac-
mon sequence motif could be delineated from the induced miR- tor DGCR8 and a number of other proteins including p68 and p72, liberates
NAs and it remains unclear how the target-miRNAs are selected. a hairpin-structured miRNA precursor (pre-miRNA). Pre-miRNAs are ex-
An alternative mechanism is used by the estrogen receptor ported to the cytoplasm by the export receptor Exportin 5. In the cytoplasm,
alpha (ERα), which associates with the microprocessor complex Dicer cleaves the pre-miRNA molecule and produces a short-lived double
and inhibits the cleavage of specific miRNAs after ligand binding stranded intermediate. One strand is selected and becomes the mature
(씰Fig. 1). Again, the helicase subunits of the microprocessor p68 miRNA whereas the other strand (referred to as miRNA-star) is degraded. The
and p72 are crucial for the recruitment of ERα (56). Interestingly, mature miRNA directly interacts with a member of the Argonaute protein
several estrogen-regulated miRNAs target mRNAs that are tran- family. MiRNA processing is regulated at many different steps. Factors that
scriptionally upregulated during estrogen-response. Thus, the in- positively influence miRNA processing or function are shown in green. Fac-
tors that inhibit miRNA maturation/function are highlighted in red.
hibition of miRNA maturation by ERα synergises with its tran-
scriptional effects by stabilising the target mRNAs (56). However,
how the specificity for the regulated miRNAs is achieved, needs to
be elucidated. stranded GGG-triplet. KHSRP binds to pri-miRNAs including
A second group of factors regulating microprocessor cleavage pri-let-7a and is necessary for microprocessor cleavage. Knock-
of a subset of miRNAs are RNA binding proteins that also have a down of KHSRP leads to a strong decrease in mature let-7a levels.
known role in RNA splicing (씰Fig. 1). The KH-type splicing regu- KHSRP is present in most cells and constitutively promotes pro-
latory protein (KHSRP) binds with high affinity to conserved ter- cessing of so far 14 identified miRNA targets (57). Its activity can
minal loop regions of a specific set of miRNAs. The binding motif be augmented by phosphorylation of three serine residues by the
is similar to binding sites in mRNA and consists of a single ATM kinase, which increases the affinity of KHSRP for the pri-

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608 Treiber et al. microRNA-guided gene silencing and its regulation
miRNA-loops in response to DNA damage (58). The stimulatory
role of KHSRP in miRNA processing is antagonised by hnRNPA1,
MicroRNAs in vascular biology
which binds to the same site in pri-miRNAs and competes with
KHSRP binding (59). In addition to its inhibitory role, hnRNPA1
can also activate microprocessor cleavage of pri-miR-18a by bind-
ing to the double stranded stem resulting in a more relaxed local
helix structure more favourable for Drosha cleavage (60, 61). Inter-
estingly, in spite of being part of the large miR-17∼92 cluster,
miR-18a is the only member that is regulated post-transcription-
ally by hnRNPA1 (60, 61). This observation illustrates how regu-
lation of miRNA processing is used to uncouple the expression lev-
els of individual clustered miRNAs through posttranscriptional
mechanisms.
Similar to the regulation of miR-18 by hnRNPA1, the SR-pro-
tein SF2/ASF can bind to the lower stem of pri-miR-7 and promote
Drosha cleavage. As the translation of SF2/ASF mRNA is repressed
by miR-7, this leads to the formation of a negative feedback loop
that attenuates SF2 expression (62).
Adenosine deaminase acting on RNA (ADAR) enzymes bind
double stranded RNA and deaminate adenosine to inosine. Apart
from their function in mRNA editing they can also accept certain
miRNA precursors as substrates resulting in an I-U wobble base
pair in the modified RNA. For miR-142 it has been shown that
Drosha processing of the edited pri-miRNA is substantially im-
paired. Instead, the pri-miRNA is recognised and degraded by the
I-U specific nuclease Tudor-SN, resulting in a severe decrease in
mature miRNA levels (63).
Regulation of pre-miRNA export
The pre-miRNA export receptor Exportin 5 is, like other importin-
β/karyopherin family proteins, composed of HEAT repeats that
form a solenoid-shaped molecule with a large binding groove for
the cargo. In Exportin 5, this binding region is strongly basic and
can perfectly accomodate the negatively charged RNA double helix
of the pre-miRNA stem. An additional small pocket at the base of
the groove binds the 2 nt 3’ overhang and ensures the specificity for
Drosha cleavage products. The terminal loop is not bound and can
vary in size and shape (64). This mode of binding might allow for
the co-export of proteins bound to the terminal loop but not the
double stranded pre-miRNA stem.
C-terminally truncated mutations of Exportin 5 have been
identified in tumours with microsatellite instability and result in a
global defect of pre-miRNA export. The resulting general decrease
of mature miRNA levels is a hallmark of miRNA profiles of cancer
cells (65). So far there is only indirect evidence for specific regu-
lation of nuclear export of individual miRNA precursors. Several
pri-miRNAs including mir-31 are processed by the microproces-
sor but retained in the nucleus in most cell lines (52). However, the
detailed mechanisms as well as the functional consequences re-
main to be elucidated.
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Regulation of Dicer processing
The best-studied regulator of pre-miR processing by Dicer is the
protein Lin-28 (씰Fig. 1). In an evolutionary highly conserved
mechanism, this protein binds to the terminal loop of almost all
let-7-family miRNAs (66–68). This high affinity interaction is me-
diated by a cold shock domain and two zinc fingers that display a
high homology to the NCp7 protein of HIV-1 (69–71). Lin-28 is
highly expressed in embryonic stem cells and leads to a complete
block in let-7 maturation thus ensuring that the expression of sev-
eral let-7 targets (including c-myc) that are essential for the main-
tenance of the pluripotent state, remain unrepressed. During dif-
ferentiation, the expression of Lin-28 is lost, and mature let-7 miR-
NAs can be produced (66). Repression of Dicer processing involves
the cytoplasmic polyuridylation of the pre-miRNA at its 3’ end
(72), a reaction catalysed by terminal uridyl transferases (TUT-
ases). Recent high-throughput sequencing data of miRNAs has
shown that a considerable amount of pre-miRNAs carry at least a
single U added to their 3’ end (73). Pre-let-7 bound Lin-28 recruits
a specific TUTase, TUT4, and thereby acts as a processivity-factor
mediating the specific polyuridylation of its bound pre-miRNA
(74–76). In addition to the regulation of pre-miRNA processing by
Dicer, it has been proposed that Lin-28 also plays a role in the nu-
cleus by blocking Drosha cleavage. This has been shown in Caenor-
habditis elegans where Lin-28 binds pri-let-7 co-transcriptionally
and inhibits microprocessor activity (77). Very recently, it has been
reported in mammals, that a homolog of Lin-28, Lin-28b, re-
presses Drosha processing by sequestering pri-let-7 to nucleoli,
where they are not accessible for the microprocessor complex (78).
Apart from stem cells, Lin-28 and Lin-28b are expressed in special-
ised tissues and are frequently upregulated in cancer, where they
promote tumour growth by repressing let-7 miRNAs.
Members of the let-7 family are not the only targets of repres-
sion by Lin-28 and Tut4. It has been shown that in cardiac tissue,
pre-miR-1 can be uridylated and degraded in a Lin-28-dependent
manner. Yet, under normal conditions, this process is prevented by
the binding of the MBNL1-protein to the terminal loop of miR-1,
thereby competing with Lin-28 for binding. In myotonic dys-
trophy, however, MBNL1 is sequestered by binding to expanded
CUG or CCUG sequences that cause the disease. Thus, Lin-28 can
bind pre-miR-1 and the expression of the mature miRNA is lost,
which contributes to the cardiac manifestation of the dystrophy
through dysregulation of membrane channels (79).
Regulation of miRISC stability
The final regulatory level of miRNA abundance is the stability of
miRISC. More and more specific mechanisms that can destabilise
miRNAs as well as miRISC protein components are uncovered. In
stem cells, the tripartite motif protein Lin-41 acts as specific ubi-
quitin ligase for Ago proteins. As Ago proteins appear to be limiting
for the activity of the miRNA pathway, this results in the global at-
tenuation of miRNA-guided gene silencing (80). Upon differenti-
© Schattauer 2012

ation of stem cells, Lin-41 expression is downregulated, at least
partially as result of repression by let-7 family members that are re-
leased from the maturation block imposed by Lin-28 and destabi-
lise Lin-41 mRNA (80).
On the miRNA side, it is becoming evident that different miR-
NAs have distinct inherent half-lives that might even be encoded in
their sequence. miR-382, for example, is relatively short lived and
degraded by the exosome. A sequence in the 3’ end of the miRNA
is crucial in this process (81). Moreover, several retinal miRNAs are
rapidly turned over upon light stimulation (82). The detailed
mechanisms of active miRNA turn over in mammals, however, has
not been elucidated. In C. elegans, it has been demonstrated that
miRNAs, which are not bound to target RNAs, are degraded by the
5’ to 3’ exoribonuclease Xrn2 (83). It is therefore very likely that
more, so far unrecognised factors might influence miRNA stability
and thereby regulate gene silencing.
Outlook
It is becoming more and more apparent that miRNA activity is
heavily regulated at all steps of the miRNA pathway. It is likely that
numerous, so far not identified RNA binding proteins are engaged
in the regulation of miRNA function. It might even be possible that
each miRNA family or miRNA cluster possesses its own repertoire
of regulatory proteins. In addition, key protein components of the
miRNA pathway are subject to phosphorylation (84). Currently,
we do not understand how miRNA pathways are embedded into
cellular signalling networks and it is possible that kinases and
phosphatases are involved in the interplay of miRNAs and signal-
ling molecules.
Very recently, pseudogenes have been implicated in the regu-
lation of miRNA activity as well. Numerous conserved pseudo-
genes exist in the human genome and many of them contain 3’
UTRs with conserved miRNA binding sites. Interestingly, these
binding sites are active and function as decoys or sponges seques-
tering miRNAs and preventing them from binding to their cognate
mRNA targets. Such RNAs have been termed competing endogen-
ous RNAs (ceRNAs) (85). These new findings highlight the com-
plexity of regulatory miRNA networks. In order to understand the
impact of miRNAs on disease and the development of miRNA-
based therapeutics, it is essential to characterise such miRNA net-
works in molecular detail.
Acknowledgements
We apologise to those, whose work was not cited due to space con-
straints. Our research is supported in part by grants of the Euro-
pean Research Council (ERC, sRNAs to G.M.), the EU FP7 project
‘ONCOMIRs’, the Bavarian Genome Research Network (BayGene
to G.M.) and the Deutsche Forschungsgemeinschaft (DFG).
Conflicts of interest
None declared.
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Treiber et al. microRNA-guided gene silencing and its regulation 609
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