Molecular Cell

Review
Posttranscriptional Regulationof MicroRNA Biogenesis in Animals
Haruhiko Siomi1,* and Mikiko C. Siomi1,*
1Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan*Correspondence: awa403@sc.itc.keio.ac.jp (H.S.), siomim@sc.itc.keio.ac.jp (M.C.S.)
DOI 10.1016/j.molcel.2010.03.013

MicroRNAs (miRNAs) control gene expression in animals, plants, and unicellular eukaryotes by promoting
degradation or repressing translation of target mRNAs. miRNA expression is often tissue specific and developmentally regulated, and regulation occurs both transcriptionally and posttranscriptionally. This regulationis crucial, as alteration of miRNA expression has been linked to human diseases, including several cancers.
Here, we discuss recent studies that shed light on how multiple steps in the miRNA biogenesis pathway are
regulated to modulate miRNA function in animals.
Introduction
The lin-4 miRNA was identified in C. elegans in 1993 (Lee et al.,1993). At the time, lin-4 was thought to be a worm-specific curiosity, but with the subsequent identification of the let-7 miRNA,
which is phylogenetically conserved (Pasquinelli et al., 2000;
Reinhart et al., 2000), researchers took greater notice and began
looking for other similar small RNAs. Since then, thousands ofmiRNAs have been found in animals, plants, and unicellular
eukaryotes, and it is becoming clear that miRNAs have a big
impact on shaping transcriptomes and proteomes of eukaryoticorganisms (Baek et al., 2008; Lim et al., 2005; Selbach et al.,2008).
Conventional wisdom holds that miRNAs control the expression of specific genes, typically by base pairing to the 30 untranslated regions (30 UTRs) of target messenger RNAs (mRNAs) tomediate repression of that target message, either by transcript
destabilization, translational inhibition, or both (Filipowicz et al.,2008). However, recent studies have shown that translational
inhibition alone is the exception rather than the rule, at least for
mammalian miRNAs (Hausser et al., 2009; Hendrickson et al.,2009), and that miRNAs can also bind to other regions of agene including the protein-coding exons (Rigoutsos, 2009).
Computational and experimental approaches indicate thata single miRNA may target, on average, more than a hundred
mRNAs. Furthermore, over 60% of human protein-coding genes
are predicted to contain miRNA-binding sites within their 30 UTRs
(Friedman et al., 2009). More than 700 miRNA genes havealready been identified in the human genome alone, whichapproaches about 3% of the number of all human proteincoding genes. Thus, miRNAs constitute one of the most abundant classes of gene-regulatory molecules in animals.
miRNAs are involved in a broad range of developmental andphysiological processes (Bushati and Cohen 2007); thus, theirderegulation is closely linked to human diseases, such as cancer(Chang and Mendell, 2007; Garzon et al., 2009). A large fractionof miRNAs exhibit strict developmental stage-specific andtissue-specific expression patterns, and the levels of many ofthese miRNAs are altered during disease. Although the final
synthesis rate of a miRNA can, in principle, be controlled atany step of miRNA biogenesis, from transcription to mature

miRNA turn over, recent findings have uncovered a significant
role for posttranscriptional mechanisms in the regulation of
miRNA biogenesis and activity (Carthew and Sontheimer,
2009; Davis and Hata, 2009). Here, we review recent progressin our understanding of the posttranscriptional mechanisms ofmiRNA biogenesis in animals.
General miRNA Biogenesis
Most mammalian miRNAs are transcribed by RNA rolymerase II(pol II), which generates a primary miRNA (pri-miRNA) transcriptthat consists of one or more hairpin structures, each composedof a stem and a terminal loop. Pri-miRNAs are 50 -capped,
spliced, and polyadenylated, and they often produce morethan one functional miRNA (Carthew and Sontheimer, 2009;
Kim et al., 2009). Two subsequent, sequential processing reactions—one in the nucleus and one in the cytoplasm—trim thepri-miRNA transcript and generate the mature miRNA.
In the nucleus, the pri-miRNA is ‘‘cropped’’ into a 70 nucleotide (nt) hairpin-structured precursor (pre-miRNA) by a multiprotein complex called the Microprocessor. The two core components of the Microprocessor are Drosha, an RNase III enzyme,
and DGCR8/Pasha, a double-stranded RNA-binding domain(dsRBD) protein. The Microprocessor complex also contains
a variety of cofactors including the DEAD box RNA helicasesp68 (DDX5) and p72 (DDX17), as well as heterogeneous nuclear
ribonucleoproteins (hnRNPs) (Gregory et al., 2004). These auxiliary factors may function to promote the fidelity, specificity, and/or activity of Drosha cleavage. DGCR8/Pasha functions at leastin part by binding to the junction between single-stranded (ss)and double-stranded (ds) region of the pri-miRNA stem anddirecting Drosha to cleave 11 bp away from the junction (Hanet al., 2006). Drosha cleavage occurs cotranscriptionally, before
splicing of the host RNA (Kim and Kim, 2007; Morlando et al.,2008), and generates a product with a 2 nt 30 overhang, characteristic of RNase III-mediated cleavage. The overhang is recognized by Exportin-5, which transports the pre-miRNA into thecytoplasm via a Ran-GTP-dependent mechanism (Kim et al.,2009; Okada et al., 2009). In the cytoplasm, the pre-miRNA
gets further ‘‘diced’’ into a 22 nt long miRNA duplex by Dicer,
another RNase III enzyme, in collaboration with the dsRBD

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Reviewproteins TRBP/PACT (Chendrimada et al., 2005; Haase et al.,2005; Lee et al., 2006). The two miRNA strands are then separated and one of the strands associates with an Argonaute(AGO) protein within the RNA-induced silencing complex (RISCor miRISC) where it acts as a guide to repress target messages.
The miRNA guides RISC to complementary sites within thetarget mRNAs to mediate repression of that target message.
While most miRNAs are generated by this pathway, someanimal miRNAs are not. ‘‘Mirtrons,’’ for example, are premiRNA-like hairpins that are made by splicing and debranchingof short hairpin introns (Okamura et al., 2007; Ruby et al., 2007).
Some small nucleolar RNAs (snoRNAs), transfer RNAs (tRNAs),and endogenous short hairpin RNAs (shRNAs) are also processed into miRNA-like molecules independently of the Microprocessor complex (Babiarz et al., 2008; Ender et al., 2008;
Saraiya and Wang 2008; Cole et al., 2009). Deep sequencing
efforts also reveal that endo-siRNA loci produce a distinct fraction of small RNAs that seem to load onto AGO1, and therefore
might constitute functional miRNAs (Czech et al., 2009). Indeed,
terminal hairpins of endogenous siRNA (endo-siRNA) long-stemloop precursors are another source of miRNAs that bypass the
Drosha processing step (Miyoshi et al., 2010).
The two strands of the miRNA duplex are not equal, andthe inherent features of the duplex help to determine which
strand is incorporated into RISC (Carthew and Sontheimer,
2009; Kim et al., 2009; Siomi and Siomi, 2009). One importantparameter is the thermodynamic asymmetry of the miRNA
duplex (Khvorova et al., 2003; Schwarz et al., 2003). Typically,
the miRNA strand whose 50 end is less stably base-paired will
be more frequently chosen as the guide. In contrast, the miRNA
strand whose 50 end is more stably base-paired, also known as
the passenger strand or the miRNA* strand, is excluded fromthe miRISC and generally thought to be degraded. In Drosophila,
miRNAs and siRNAs are actively sorted into AGO1- and AGO2RISC complexes, respectively, and AGO1 sorting involves
Dicer-1/Loquacious (Loqs) whereas AGO2 sorting involves
Dicer2/R2D2 (Siomi and Siomi, 2009). Central mismatches
reduce binding of small RNA duplexes by the Dicer-2/R2D2 heterodimer, antagonizing AGO2 loading and promoting loading into
AGO1 (Tomari et al., 2007). Recent deep-sequencing efforts,
however, indicate that a large number of miRNA*s are notdegraded, but rather associate with AGO1 or AGO2 (Czech
et al., 2009; Ghildiyal et al., 2010; Okamura et al., 2008, 2009;
Seitz et al., 2008). In such cases both miRNA and miRNA* strands
are functional (Czech et al., 2009; Okamura et al., 2008, 2009).
Differential sorting of these miRNA/miRNA* duplexes correlateswith specific mismatches: Watson-Crick base-pairing at positions 9 and 10 promotes miRNA* sorting to AGO2 (Czech et al.,2009; Okamura et al., 2009). The identity of the 50 nucleotide
also affects strand selection, as observed in plants (Chen 2009;
Czech et al., 2009; Okamura et al., 2009). Together, these findings suggest that miRNA precursors can be bifunctional, withindividual strands adopting different fates within small RNA
pathways.
In humans, all four AGO proteins show remarkably similar preferences for the structures of small RNA duplexes; central
mismatches promote RISC loading, and thus their features arereminiscent of Drosophila AGO1 (Yoda et al., 2010). Although

324 Molecular Cell 38, May 14, 2010 ª2010 Elsevier Inc.

strand selection based on 50 thermodynamic stability still
contributes greatly to the sorting process, relative expression
levels of the miRNA/miRNA* strands vary widely among tissues(Landgraf et al., 2007; Hu et al., 2009), indicating that strand
selection factors other than sequence features might exist andthat miRNA processing pathways are more complex thancurrently recognized.
Regulation of miRNA Maturation: Global and Individual
miRNA biogenesis is stringently controlled and is often subjected to feedback regulation. For example, some miRNAs regulate transcription factors that, in turn, regulate expression of themiRNA, forming a double-negative feedback loop (Li et al., 2009;
Xu et al., 2009; for reviews, see Carthew and Sontheimer, 2009;
Davis and Hata, 2009). This coordinated expression might serve
to prevent misexpression of cell-type-specific miRNAs and toconfer robustness to the underlying gene expression programs
during development.
Interestingly, multiple steps of miRNA biogenesis are specifically regulated during processes like differentiation and tumor
progression. For example, during early development, numerous
pri-miRNAs are expressed but are not efficiently converted into
mature miRNAs (Thomson et al., 2006). Similarly, reduced
processing contributes to widespread downregulation of miRNAs in many human cancers (Garzon et al., 2009; Lu et al.,2005; Thomson et al., 2006). These findings have uncovereda significant role for posttranscriptional mechanisms of miRNAbiogenesis.
Processing of individual miRNAs can also be independently
regulated. For instance, many miRNAs are encoded in the
genome as clusters and can be transcribed as long polycistronic
primary transcripts; however, there is an example of one particular miRNA located in a cluster that is regulated independently
from the other miRNAs in the cluster (Guil and Cáceres, 2007).
This indicates that individual miRNAs can be posttranscriptionally regulated by factors that confer the specificity and function
at various points during the biogenesis pathway. How is miRNAprocessing controlled at the posttranscriptional level? Proteinsof known function or activity in posttranscriptional mechanismsof miRNA biogenesis are summarized in Table 1.
Regulation of miRNA Maturation at the Levelof ‘‘Cropping’’
Recent studies have shown that activity and specificity of Microprocessor-mediated pri-miRNA cleavage is subject to intense
regulation. Adenosine-to-inosine (A-to-I) RNA editing of specificpri-miRNAs by ADAR (adenosine deaminase acting on RNA)
enzymes prevents their effective processing by Drosha and, insome cases, A-to-I editing directs RNA molecules to Tudor-SN(Tudor staphylococcal nuclease homolog) for degradation(Yang et al., 2006). Editing in the miRNA ‘‘seed’’ region canalso lead to the production of modified mature miRNAs thatcan potentially recognize a different set of targets comparedwith the unmodified miRNAs (Kawahara et al., 2007). Thus,
RNA editing is an effective mechanism for modifying the biogenesis and activity of miRNAs.
Depletion of either of the RNA helicases p68 or p72, cofactorsof the Microprocessor complex, results in a reduction in theMolecular Cell

Review
Table 1. Posttranscriptional Regulators of miRNA Biogenesis in Animals
Protein

MotifsKnown Activity

Mechanisms

Target miRNA

References

ADARs

dsRBD

A-I RNA editing

Inhibition of Drosha
and Dicer processing

A subset

Yang et al. 2006;
Kawahara et al. 2007

p68/p72

DEAD-box

Components ofMicroprocessor

Promoting Drosha
cleavage

A subset

Fukuda et al. 2007p53

DNA binding

Tumor suppressor

Binding to p68 andPromoting Drosha cleavage

miR-16-1,
miR-143

Suzuki et al. 2009

SMADs

DNA binding

Signal transducersof TGFb

Binding to p68 andPromoting Drosha cleavage

miR-21,
miR-199a

Davis et al. 2008

ERa

DNA binding

Nuclear estrogen
receptor

Binding to p68/p72 andinhibiting Drosha cleavage

A subset

Yamagata et al. 2009hnRNP A1

RRM, M9

Pre-mRNA splicing

Chaperone forDrosha/DGCR8 binding

miR-18a

Guil and Cáceres, 2007

KSRP

KH

mRNA decay

Promoting Drosha
and Dicer processing

A subset

Trabucchi et al. 2009

ARS2

Plant SERRATE
homolog

Nuclear Cap-binding

Enhancing Drosha
processing

GlobalGruber et al. 2009;
Sabin et al. 2009

DGCR8

dsRBD

Binding to Drosha

Stabilizing Drosha

GlobalHan et al, 2009

Exportin-5

RanBP

Binding to tRNAs
and pre-miRNAs

Nuclear transportof pre-miRNA

GlobalGrimm et al., 2006;
Diederichs et al., 2008

LIN-28CCHC-typezinc finger

Promoting pluripotency

Inhibition of Drosha
and Dicer processing,and Recruiting TUT4

let-7

Heo et al. 2008; Newman
et al 2008; Rybak et al. 2008;
Viswanathan et al, 2008

TUT4

Poly(A) polymerase,
CCHC-type zincfingerTerminal uridylation

Binding to LIN-28 andinhibiting Dicer processing

let-7

Hagan et al. 2009;
Heo et al. 2009

TRBP

dsRBD

Binding to Dicer, MKK
phosphorylation site

Stabilizing Dicer

GlobalParoo et al., 2009

XRN-2

50 to 30
exoribonuclease

exoribonuclease

Degrading miRNA

GlobalChatterjee and
Grosshans, 2009

GLD2

Poly(A) polymerase

Terminal adenylation

Stabilizing miRNA

miR-122

Katoh et al., 2009

mLin41

TRIM-NHL(RING finger)

Ubiquitinylation

Binding to Ago2 andtargeting it for degradation

Let-7 andothers in
ES cells

Rybak et al., 2009

TRIM32

TRIM-NHL(RING finger)

ubiquitinylation

Binding to miRISC andenhancing miRNA activity

A subset

Hammell et al., 2009

NHL-2

TRIM-NHL(RING finger)

ubiquitinylation

Binding to miRISC andenhancing miRNA activity

A subset

Schwamborn et al., 2009

Mei-P26

TRIM-NHL(RING finger)

ubiquitinylation

Binding to miRISC andinhibiting miRNA activity

A subset

Neumüller et al., 2008

Argonautes

PAZ, PIWI

Components of RISC

Stabilizing associated miRNAs

GlobalDiederichs et al., 2008

ADARs, adenosine deaminase acting on RNA enzymes; dsRBD, double-stranded RNA-binding domain; DGCR8, DiGeorge syndrome critical region
gene 8; TGFb, transforming growth factor b; ERa, estrogen receptor a; hnRNP A1, heterogeneous nuclear ribonucleoprotein A1; RRM, RNA recognition motifs; M9, nucleocytoplasmic shuttling signal sequence; KSRP, KH-type splicing regulatory protein; KH, hnRNP K homology domains; RanBP,
ran-binding protein; ARS2, arsenic resistance protein 2; TUT4, terminal uridylyl transferase 4; TRIM-NHL, tripartite motif (consisting zinc fingers of both
RING type and B Box type and a coiled-coil domain)-NHL repeats.

levels of a particular set of miRNAs (Fukuda et al., 2007), suggesting a role for p68 and p72 in promoting Drosha cleavageof a subset of miRNAs. p68 and p72 interact with a large numberof proteins (Davis and Hata., 2009). The tumor suppressor
protein p53, under conditions of DNA damage, is present in
a complex with both Drosha and p68, and enhances Drosha processing of a subset of miRNAs, including miR-16-1 and miR-143(Suzuki et al., 2009). Treatment of human vascular smoothmuscle cells with either bone morphogenetic protein 4 (BMP4)
or transforming growth factor beta (TGF-b) stimulates SMAD
binding to p68 and increases Drosha processing of pri-miR-21
and pri-miR-199a (Davis et al., 2008). Estradiol (E2) treatmentof ovariectomized female mice inhibits the production of a subsetof miRNAs in the uterus through binding to its nuclear estrogen
receptor a (ERa), which interacts with p68 and p72. ERa boundto E2 interacts with Drosha in a p68-/p72-dependent manner

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Figure 1. Posttranscriptional Controls of miRNA Biogenesis Mediated by RNA-Binding Proteins
The binding of RNA-binding proteins to miRNA precursors, particularly to the stem-loop structures within them, either blocks or enhances further processing bycompeting with or recruiting miRNA processing complexes. In (A), specific binding of RNA-binding proteins (X in the figure) to miRNA precursors can induce either
a conformational change (chaperone activity; e.g., hnRNP A1) or a displacement of repressive RNA-binding proteins (Y in the figure).
(B) RNA-binding proteins (X in the figure) can also recruit auxiliary factors (Y in the figure) on specific miRNA precursors, thereby regulating processing, stability
and subcellular location of miRNAs. For example, Lin28 competes with Dicer for pre-let-7. Upon binding by recognizing a specific sequence motif in the terminal
loop, Lin28 recruits TUT4 to pre-let-7, leading to the 30 terminal uridylation and the degradation of pre-let-7.

and appears to induce the dissociation of the Microprocessor
complex from the pri-miRNAs or a conformational change ofthe Microprocessor with the consequence of reduced association prior to interaction with the pri-miRNAs, thereby blocking
Drosha-mediated processing (Yamagata et al., 2009).
These observations indicate that signaling cascades regulate
biogenesis of individual miRNAs and suggest that the interactionof p68 and/or p72 with accessory factors, such as p53 andSMAD proteins, can modulate specificity and activity of Drosha/
DGCR8 processing, probably through a conformational changeof the Microprocessor or pri-miRNAs or by a displacement ofrepressive RNA-binding proteins (Figure 1A). It will be important
to identify factors that supply specificity to the Microprocessor
complex for recognizing to a particular subset of pri-miRNAs
and factors that supply specificity for selecting the Microprocessor complex on a particular subset of pri-miRNAs among
all other Microprocessor complexes. However, an importantrecurring theme is that different signaling pathways can regulate
the Microprocessor complex by modulating the activity of p68-/
p72-interacting proteins.

326 Molecular Cell 38, May 14, 2010 ª2010 Elsevier Inc.

Regulation at Cropping: The Importance of the miRNA
Loop
miR-18a is processed from a polycistronic pri-miR-17-92 transcript, which harbors five additional miRNAs. hnRNP A1, anmRNA splicing regulator, exclusively stimulates maturation of
miR18a from the miR-17-92 cluster. Pri-miR-18a, but not othermembers of the cluster, contains two regions of similarity to
the consensus hnRNP A1-binding site, UAGGGA/U, within its
terminal loop and stem (Guil and Cáceres, 2007; Michlewski
et al., 2008). Direct and specific interaction of hnRNP A1 withthe terminal loop and stem of the pri-miR-18a hairpin induces
a structural rearrangement of the hairpin to generate a morefavorable Drosha/DGCR8 binding and cleavage site (Figure 1A).
Thus, hnRNP A1 acts as a chaperone for recognition and cropping of specific pri-miRNAs by Drosha/DGCR8. Interestingly,
approximately 14% of human miRNAs contain highly conserved
loop sequences, suggesting that processing regulation by
hnRNPs and other nuclear RNA-binding proteins might extend
well beyond miR-18a (Michlewski et al., 2008). Indeed, the KHtype splicing regulator protein (KSRP, also known as KHSRP)

Molecular Cell

Review
recognizes G-rich regions, including a GGG triplet, present
within the terminal loops of a subset of pri-miRNAs, to promote
Drosha/DGCR8 mediated processing (Trabucchi et al., 2009).
Upon binding, KSRP could optimize the positioning and/or
recruitment of the miRNA precursor processing complexes
through protein-protein interactions. KSRP is associated withthe terminal loop of the target miRNA precursors during nucleocytoplasmic transit and also promotes the processing of thetarget pre-miRNAs by Dicer in the cytoplasm. This further
suggests that specific recognition of the terminal loop by RNAbinding proteins is an important means to extend regulation of
miRNA processing down to the level of individual miRNAs.
Binding of RNA-binding proteins to specific sequences
present in the terminal loop regions of miRNA precursors potentially either competes for binding or promotes recruitment of processing factors. Many signaling pathways operate by modifying
the activity of specific RNA-binding proteins, in which RNAbinding motifs are combined with other conserved domains,such as protein-protein interaction domains and consensus
phosphorylation motifs (Davis and Hata, 2009). Changes of
expression, RNA-binding activity, interacting protein partners,
and subcellular localization of RNA-binding proteins in response
to extracellular signals might serve to regulate biogenesis of individual miRNAs.
Global Regulation: Possible Links to Other Nuclear RNA
Processing Events
Accumulation of global miRNA with increasing cell density has
been observed and is associated with enhanced Drosha processing activity without changing protein levels of Drosha or
DGCR8 (Hwang et al., 2009). This suggests that miRNA biogenesis is linked to the proliferative state of the cell and thatprocessing activity can be regulated for the same stoichiometoric amount of the complex by altering turnover rate of thesingle Microprocessor complexes. Arsenate resistance protein
2 (ARS2), whose expression is low during cell quiescence andhigh during proliferation, is a component of the nuclear RNA
cap-binding complex (CBC) that binds and stimulates theactivity and fidelity of the Microprocessor complex (Gruber
et al., 2009; Sabin et al., 2009). Interestingly, the plant homologof ARS2, SERRATE (SE), is an essential factor in miRNA biogenesis that interacts with the nuclear CBC and RNase III enzyme
Dicer-like 1 (DCL1), the main miRNA-processing enzyme inplants (Chen, 2009). The nuclear CBC containing ARS2 mayfunction to link pri-miRNA processing to other RNA processing
events. For example, CBC/ARS2 may recruit the Microprocessor
to 50 -capped nascent pri-miRNA transcripts, as seen for SE inplants, and might also either stabilize pri-miRNAs or enhance
the cleavage activity and specificity of Drosha/DGCR8. Rapid
cytoplasmic transport of pri-miRNA transcripts may competewith Drosha/DGCR8 recognition and cleavage in the nucleus.
Together, CBC and ARS2 may counteract this transport by
repressin nuclear export of pri-miRNA transcripts, thereby
increasing the opportunity for Microprocessor recognition andprocessing of target hairpins. Furthermore, approximately 40%
of miRNAs are encoded in the introns of genes (Kim et al.,2009) and therefore some Pol II transcripts are simultaneously
pre-mRNAs and pri-miRNAs. This suggests that there may bephysical connections between the Microprocessor and thepre-mRNA splicing machinery as SE, like hnRNP A1 and
KSRP, also acts in pre-mRNA splicing. The CBC not only recruits
export factors to nascent pre-mRNAs, but also interacts directlywith splicing factors (Pawlicki and Steitz, 2010) and may, therefore, play an integral role in the coordination of pre-mRNA andpri-miRNA processing. Once processed, pre-miRNAs are transported to the cytoplasm by Exportin-5, which appears to belimiting (Grimm et al., 2006; Diederichs et al., 2008). Thus premiRNA transport can also be regulated.
Autoregulation of the miRNA Cropping Machinery
Drosha and DGCR8 are also tightly regulated in an intricate
feedback circuit, which might contribute to the homeostatic
control of miRNA biogenesis (Han et al., 2009). DGCR8 stabilizes
Drosha through a direct interaction. In turn, Drosha cleaves
two hairpin structures in the 50 UTR and the coding sequencesof the DGCR8 mRNA, whose folds are similar to pri-miRNA
structure, thus leading to degradation of the mRNA. This results
in a double-negative feedback loop, ensuring tight coupling of
the core Microprocessor components. A similar feedback mechanism regulates pri-miRNA processing in plants (Rajagopalan
et al., 2006). DICER-LIKE1 (DCL1) pre-mRNA harbors the premiR838 hairpin. DCL-mediated processing of DCL1 pre-mRNA
releases pre-miR838, generating nonproductive fragments of
DCL1 pre-mRNA. It is likely that animal cells may (de)stabilize
Drosha by modifying its interacting partner DGCR8 or by masking pri-miRNA-like hairpins in the DGCR8 mRNA by recruiting an
RNA-binding protein. This would alter global miRNA biogenesis
in response to the proliferative state of the cell or to extracellular
stimuli. Although analysis of deep-sequencing data of small
RNAs in mouse cells suggests that the Microprocessor’s rolein directly destabilizing coding mRNAs may be only targeted to
DGCR8 mRNA itself (Shenoy and Blelloch, 2009), it is still
tempting to speculate that some mRNAs with pri-miRNA-likehairpins could be involved in a network that balances global
miRNA biogenesis by acting as attenuators that compete withauthentic pri-miRNAs for Microprocessor binding.
Regulation of Dicer Activity
Pre-miRNA processing by Dicer appears to function quite efficiently since pre-miRNA levels are low relative to the levels ofpri- and mature miRNA, suggesting that dicing is a mechanismwith rapid substrate turnover and very little regulation. However,recent studies have shown that the expression and activity ofDicer might serve as an important regulatory point in global
and individual miRNA biogenesis. Dicer activity is repressed by
the nuclear retention of pre-miRNAs that prevents access bycytoplasmic Dicer (Lee et al., 2008). Dicer activity might also
be repressed by the presence of a specific inhibitor that cancompete with Dicer for pre-miRNA binding, though such an
inhibitor has not yet been identified (e.g., pre-miR-138 in mousebrain) (Obernosterer et al., 2006). The levels of Dicer can be
regulated by its products; the identification of let-7 targetingsites within Dicer coding sequence suggests that let-7 miRNAdirectly targets Dicer, thus establishing a miRNA/Dicer autoregulatory negative feedback loop that might regulate the total rateof miRNA biogenesis (Forman et al., 2008). In C. elegans, Dicer

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activity is inhibited by rncs-1, a highly base-paired 800 nt noncoding RNA, presumably by binding to Dicer or accessory
dsRBD proteins to compete with endogenous dsRNAs involved
in silencing (Hellwig and Bass, 2008).
Regulation at Dicing: The Importance of the miRNA Loop
The human let-7 miRNA family is derived from 12 precursor transcripts that contain nine mature miRNA sequences. While mostlet-7 pri-miRNA transcripts are highly expressed throughout
development, the mature let-7 is detectable only in highly differentiated cells, suggesting a posttranscriptional regulation ofbiogenesis (Roush and Slack, 2008). Levels of LIN-28, an RNAbinding protein that promotes pluripotency, inversely correlateswith mature let-7 expression during development (Wu andBelasco, 2005). Although LIN-28 was then found to block accumulation of mature let-7 by repression of both Drosha and Dicer
activities (Heo et al., 2008; Newman et al., 2008; Rybak et al.,2008; Viswanathan et al., 2008), recent studies demonstratethat LIN-28 can induce 30 terminal uridylation of pre-let-7 miRNAin mouse embryonic stem (ES) cells to block Dicer cleavage andlet-7 maturation (Heo et al., 2008). LIN-28 recruits a noncanonicalpoly (A) polymerase (PAP), terminal uridylyl transferase 4 (TUT4,
also known as ZCCHC11 in mammals and PUP-2 in C. elegans)to pre-let-7 by recognizing a tetra-nucleotide sequence motif(GGAG) in the terminal loop (Hagan et al., 2009; Heo et al.,2009) (Figure 1B). TUT4 then adds a uridine (U) tail to the 30
end of pre-miRNA in a template-independent manner. Dicer isunable to cleave hairpin RNAs with such long 30 extensions.
Uridylyl groups are known to recruit 30 -to-50 exonucleases, suggesting that 30 terminal uridylation may also facilitate the degradation of pre-let-7 with a U tail. Several additional miRNAs that
contain the GGAG motif in the terminal loop also undergo uridylation in a LIN-28-dependent manner (Heo et al., 2009). Thissuggests that the ability of LIN-28 and TUT4 to repress premiRNA processing by inducing uridylation might not be specificto let-7. Besides LIN28, other specificity factors may also assist
TUT4 (and other TUTases) to recognize distinct pre-miRNAs.
These findings suggest that the terminal loop is a pivotal structure or landing pad where miRNA processing ‘‘activators’’ (forexample, hnRNP A1 and KSRP) as well as ‘‘repressors’’’(forexample, LIN-28) function in a coordinated way to transformproliferating, apoptotic, or differentiating cues into changes ofmiRNA expression (Figure 1B). In C. elegans, LIN-28 stimulates
uridylation of let-7 pre-miRNA by PUP-2, indicating that LIN-28
and let-7 form an ancient regulatory switch, conserved fromnematodes to humans (Lehrbach et al., 2009). However, theterminal loop of pre-let-7 is not conserved in C. elegans. Thus,
the exact mechanistic details might differ between organisms.
Signal-Mediated Regulation of Dicer Activity
Dicer protein levels can be regulated by TRBP through a directinteraction. Truncation mutations of TRBP that cause diminished
TRBP protein expression are found in sporadic and hereditarycarcinomas, and are associated with both a defect in theprocessing of miRNAs and in destabilization of the Dicer protein(Melo et al., 2009). The mitogen-activated protein kinase (MAPK)
extracellular signal-regulated kinase (ERK) pathway, which promotes cell growth, phosphorylates TRBP and enhances miRNA

328 Molecular Cell 38, May 14, 2010 ª2010 Elsevier Inc.

production by stabilizing the Dicer-TRBP complex (Paroo et al.,2009). The ERK signaling pathway involves a cascade of phosphorylation events from a MAPK kinase kinase, to a MAPK
kinase (MKK), to MAPK ERK. TRBP interacts with phosphorylated ERK1/2 and its phosphorylation requires MKK1 and
ERK2. Interestingly, phosphorylation of TRBP is blocked by the
MKK inhibitor U0126, and concomitantly, the level of growthpromoting miRNA decreases and that of growth-suppressing
miRNAs increases in cancer cell lines treated with U0126. Thesefindings indicate a concerted miRNA regulatory program capable of responding positively to mitogenic signals and negatively
following inhibition of these signals. This also implies that thelevel of the Dicer-TRBP complex determines preferential substrates for pre-miRNA processing, which might account for theobserved decrease of growth-suppressing miRNAs in somecancer cells. How the level of the Dicer-TRBP complex determines processing of preferential target pre-miRNAs remains tobe elucidated; however, these findings also imply therapeuticsignificance.
Regulation of miRNA Accumulation at the Levelof miRNA Turnover
Maintaining proper steady-state levels of miRNAs is crucial fornormal development and overexpression or underexpressionof miRNAs are often linked to various human diseases. The
steady-state levels of miRNAs are presumably determinedby the opposing activities of miRNA biogenesis (transcription
and processing) and degradation. In plants, a family of 30 to 50
exoribonucleases encoded by the SMALL RNA DEGRADINGNUCLEASE (SDN) genes degrades mature miRNAs to limit theiraccumulation (Ramachandran and Chen, 2008). In C. elegans,degradation of mature miRNAs is mediated by the 50 to 30 exoribonuclease XRN-2, a tRNA quality control factor that selectively
removes incompletely modified tRNAs in yeast (Chatterjee and
Grosshans., 2009). Although miRNAs in the cell are present as
miRISC complexes, in which both the 50 and 30 ends are thought
to be bound directly by Argonaute (Wang et al., 2008) and therefore resistant to exonucleases, cells apparently have some wayof dislodging miRNAs from Argonaute to expose the miRNAs to
XRN-2 for degradation. miRNA degradation by XRN-2 can beblocked by the addition of miRNA target RNA, suggesting thattargets can modulate the stability of individual mature miRNAs.
These findings demonstrate that miRNA degradation affects
functional miRNA homeostasis, helping to prevent detrimental
overexpression of miRNAs. A coordination of miRNA and target
levels could permit miRISC reprogramming or turnover whentarget abundance is low.
The stability of individual mature miRNAs can also be regulated. For example, the cytoplasmic poly(A) polymerase GLD2
(also known as TUTase2) specifically monoadenylates and stabilizes miR-122 in mammalian liver cells (Katoh et al., 2009). On theother hand, the addition of nontemplated adenines has been
detected on many different animal miRNAs (Landgraf et al.,2007; Azuma-Mukai et al., 2008). This suggests that despite theirsmall size, specific miRNAs contain additional sequence elements that control their behavior, probably through specificrecognition by RNA-binding protein cofactors, thereby affecting
miRNA turnover (Ambros, 2008; Hwang et al., 2007).

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Review
The protein factors governing the abundance of mature
miRNAs also include Argonaute proteins. Increased levels of
Argonaute proteins in mammalian cells correlate with increased
levels of mature miRNAs (Diederichs et al., 2008). This effectdepends on direct binding of the Argonaute proteins to themiRNA, suggesting that Argonaute proteins are limiting andserve to stabilize miRNAs. This coordinated regulation could
provide a feedback mechanism to titrate the expression of
mature miRNAs with the availability of Argonaute proteins toform functional, stoichiometric miRISCs. Therefore, regulationof the total levels of the Argonaute proteins within the cell
contributes to global miRNA turnover. One such regulator is
the type I collagen prolyl-4-hydroxylase (C-P4H[I]). Human
AGO2 interacts with and is hydroxylated by the C-P4H(I), which
enhances the stability of AGO2 (Qi et al., 2008). C-P4H(I) is upregulated by a variety of factors, including hypoxia and TGF-b,factors which also induce many miRNAs. Another example isthe let-7 target gene mouse Lin41 which encodes a member of
the TRIM protein family that contains a RING finger domain
with E3 ubiquitin ligase activity. mLin41 facilitates polyubiquitination of Ago2 in ES cells and targets it for degradation, suggesting that mLin41 is a negative regulator of cellular Ago2 levels.
This provides evidence of an additional negative feedback loopthat acts in addition to LIN-28 to regulate let-7 activity in mouse
ES cells (Rybak et al., 2009). Similarly, other TRIM family proteinssuch as fly Mei-P26, mouse TRIM32, and worm NHL-2 wererecently found to enhance or suppress RISC loading and RISC
activity by binding to core miRNP components, including Agoproteins (Neumüller et al., 2008; Hammell et al., 2009; Schwamborn et al., 2009). Finally, formation and turnover of miRISC
appears to depend on the endosomal pathway, where factors
required for endosome formation also regulate miRNA loading
into RISC (Gibbings et al., 2009; Lee et al., 2009). Therefore, turnover of the endosome might also affect the net accumulation of
mature miRNAs.
Perspectives
As described above, final mature miRNA expression will dependon the cooperation of multiple mechanisms and their net effect. It
is also becoming apparent that miRNA strand selection can beseen as a form of posttranscriptional regulation. The identification of factors that regulate both global and individual miRNAbiogenesis and turnover represents an important challenge for
future studies. It is important to bear in mind that miRNAs arepart of a large multimolecular complex, miRISC, and theirprecursors associate with multiple RNA-binding proteins. Thus,
one major future challenge will be to understand in more detail
how RNA-binding proteins influence the final outcome of miRNAbiogenesis and how they impose specificity on the expression
and function of individual miRNAs. Thus, it will be critical todevelop novel biochemical approaches to identify complexes
containing miRNA, their precursors, and associated proteinsin vivo. The HITS-CLIP (high-throughput sequencing of RNAs
isolated by crosslinking immunoprecipitation) method recently
developed to decode miRNA-mRNA interactions (Chi et al.,2009) can be applied to the identification of factors that regulate
miRNA biogenesis. The budding yeast model for RNAi (Drinnenberg et al., 2009) could also be used as a tool to study and genet-

ically screen the pathways that regulate miRNAs. For this, it isprobably worth determining if miRNA biogenesis can be reconstituted in S. cerevisiae by expressing human Dicer, TRBP, andAGO2 or their homologs of the unicellular green alga Chlamydomonas reinhardtii, which encodes many miRNAs (Molnár et al.,2007; Zhao et al., 2007). The study of posttranslational modification of both miRNA processing factors and their accessory
factors might allow the identification of upstream signaling pathways that regulate and fine-tune the miRNA machinery. Further
investigation should unravel multiple mechanisms and the interactions occurring among them that regulate and coordinate theprecise expression of many miRNAs at posttranscriptional
levels. Such knowledge will not only expand our understandingof how they are altered in pathological conditions but will present
exciting opportunities for screening of compounds to manipulate
aspects of the miRNA machinery for therapeutic application.
ACKNOWLEDGMENTS
The authors would like to thank members of the Siomi laboratory for discussions and three anonymous referees for their useful comments. We apologize
to colleagues whose relevant primary publications were not cited because of
space constraints. This work was supported by MEXT grants to H.S. andNEDO (New Energy and Industrial Technology Development Organization)
grants to M.C.S. M.C.S. is supported by CREST from the Japanese Scienceand Technology Agency.
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