J. Biochem. 2010;148(4):381–392 doi:10.

1093/jb/mvq096

JB Review
Mechanisms of control of microRNA biogenesis
Received July 29, 2010; accepted August 16, 2010; published online September 9, 2010

Brandi N. Davis-Dusenbery1 and RNA product containing the mature miRNA guide
Akiko Hata1,2,* strand and the passenger (miRNA*) strand (Fig. 1).
The mature miRNA then promotes the association of
1
Molecular Cardiology Research Institute, Tufts Medical Center and a large protein complex, termed the RNA-induced
2
Department of Biochemistry, Tufts University School of Medicine2,
Boston, MA 02111, USA
silencing complex (RISC), with specific regions in
the 30 -untranslated region (30 -UTR) of target genes
*Akiko Hata, PhD, Molecular Cardiology Research Institute, (Fig. 1) (8).
Tufts Medical Center, 800 Washington Street, Box 8486, Boston,
MA 02111, USA. Tel: þ1 617 636 0614, Fax: þ1 617 636 5649, Selection of miRNA targets is mediated by imperfect
email: akiko.hata@tufts.edu base pairing between the miRNA and miRNA-
recognition site present in the 30 -UTR of the target
MicroRNAs (miRNAs) are a class of 22 nt non- mRNA. The imperfect nature of the miRNA:mRNA
coding RNAs that control diverse biological functions interaction means that a single miRNA can potentially
in animals, plants and unicellular eukaryotes by pro- target tens to hundreds of mRNAs (9—11). Association
moting degradation or inhibition of translation of of the miRNA—RISC results in the repression of the
target mRNAs. miRNA expression is often tissue spe- target gene by promoting mRNA degradation and/or
cific and developmentally regulated. Aberrant expres- translational inhibition (12—16). Through the repres-
sion of miRNAs has been linked to developmental sion of targets, miRNAs elicit critical changes in
abnormalities and human diseases, including cancer gene expression programmes which have been reported
and cardiovascular disorders. The recent identification to underlie diverse aspects of biology, including
of mechanisms of miRNA biogenesis regulation un- developmental timing, differentiation, proliferation,
covers that various factors or growth factor signalling cell death, and metabolism (17—19).
pathways control every step of the miRNA biogenesis
pathway. Here, we review the mechanisms that control
the regulation of miRNA biogenesis discovered in Regulation of miRNA Genes
human cells. Further understanding of the mechanisms Transcriptional control of miRNA genes
that control of miRNA biogenesis may allow the devel- Transcription is one of the major regulatory steps in
opment of tools to modulate the expression of specific the biosynthesis of miRNAs. The majority of miRNAs
miRNAs, which is crucial for the development of novel are transcribed by RNA Pol II and bear the 7-methyl-
therapies for human disorders derived from aberrant guanylate cap at the 50 -end and poly (A) tail at the
expression of miRNAs. 30 -end characteristic of mRNAs (2, 20). Large-scale
Keywords: biogenesis/Dicer/Drosha/processing/ mapping of the promoters of 175 human miRNA
miRNA/microRNA. genes through nucleosome positioning and chromatin
immunoprecipitation-on-genomic DNA microarray
Abbreviations: ds, double strand; miR, microRNA; chip (or ChIP-on-chip) analysis suggests that many
nt, nucleotide; Pre-miRNA, precursor miRNA; characteristics of miRNA gene promoters, such as
pri-miRNA, primary microRNA; ss, single strand. the relative frequencies of CpG islands, TATA box,
TFIIB recognition, initiator elements and histone
modifications, are similar to the promoters of protein
Introduction to the Basics of MicroRNA coding genes (21, 22). The DNA-binding factors
that regulate miRNA transcription largely overlap
Biogenesis with those that control protein-coding genes, including
The mechanism of microRNA (miRNA) biosynthesis c-myc and p53, as well as cell-type specific
is evolutionarily conserved and involves sequential transcription factors such as MEF2, PU.1 and REST
endonucleolytic cleavages mediated by two RNase III (23—26). Furthermore, transcription of primary
Featured Article
enzymes, Drosha and Dicer (Fig. 1) (1). Following miRNA transcripts can be dynamically regulated
transcription by RNA polymerase II (RNA Pol II), in response to growth factor stimulation, including
Drosha processes the primary miRNA transcript platelet-derived growth factor (PDGF), transforming
(pri-miRNA) into a 60—100 nt hairpin structure growth factor-b (TGF-b) and bone-derived neuro-
termed the precursor-miRNA (pre-miRNA) in the trophic factor (26—29).
nucleus (Fig. 1) (2—5). Following cleavage by The proto-oncogene c-myc encodes a transcription
Drosha, the pre-miRNA is transported out of the factor which regulates 10—15% of human genes and
nucleus through the interaction with Exportin-5 and plays a central role in control of cell growth and apop-
Ran-GTP. The pre-miRNA then undergoes further tosis (30). It is clear that c-Myc binds to E-boxes and
processing catalysed by Dicer (Fig. 1) (6, 7). This cleav- activates transcription of the miR-17-92 cluster (31).
age event gives rise to a 22 nt double-stranded (ds) Consistent with the prominence of c-myc activation

ß The Authors 2010. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved 381
B.N. Davis-Dusenbery and A. Hata

nucleus
RNA Pol II

Transcription

Drosha Complex

7
pri-miRNA m G AAAAA A

Cropping
pre-miRNA

Exportin 5

cytoplasm

Export

Dicer Complex

Dicing

RISC Complex

miRNA:miRNA* Strand Selection

duplex

mature miRNA

Targeting

Translational
Deadenylation &
repression
degradation

AAAAAAAA
ORF

Fig. 1 miRNA biogenesis pathway. miRNAs are initially transcribed as a long, capped and polyadenylated pri-miRNA. The Drosha complex
crops the pri-miRNA into a hairpin-shaped pre-miRNA. Next, Exportin-5 promotes the nuclear translocation of the pre-miRNA which is
further processed by the Dicer complex. Following Dicing, the resulting miRNA : miRNA* is dissociated and the mature miRNA is incorporated
into the RISC where it functions to mediate gene silencing either by translational inhibition or by promoting the degradation of target mRNAs.

in tumours, miR-17-92 is often highly expressed in tu- cells (33). The miR-17-92 cluster contains six
mours (32). Furthermore, amplification of the miRNAs, including miR-17-5p and miR-20, which
13q31-q32 gene locus, which contains the miR-17-92 are known to repress the cell cycle regulator E2F1
cluster, is commonly observed in c-myc-rearranged (31). Interestingly, c-Myc also promotes E2F1 tran-
lymphomas, suggesting that c-myc and the miR-17 scription, suggesting that c-Myc is likely to fine tune
cluster may cooperate to promote transformation of cell cycle progression via the regulation of both

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 Control of miRNA biogenesis

miRNA and mRNA expression (34). In addition, and forms a large complex known as the ‘Drosha
c-Myc also decreases the expression of several microprocessor complex’. The Drosha microprocessor
tumour suppressor miRNA genes, including the complex minimally includes Drosha and a required
miR-15a, -29, -34 and let-7 families (35). Exogenous cofactor, DGCR8, which is able to promote the
expression of c-Myc-repressed miRNAs in lymphoma efficient cleavage of pri-miRNA in vitro (4, 42).
cells reduced cell growth, indicating that down regula- DGCR8 is thought to recognize the region between
tion of a subset of miRNAs is an important mechan- the single-stranded RNA (ssRNA) and the stem in
ism of c-Myc-mediated tumourigenesis (35). Finally, order to direct Drosha cleavage one helical (11 bp)
c-Myc is shown to increase transcription of Lin-28B turn away from the ssRNA—dsRNA junction (42).
which mediates the post-transcriptional repression of Although the cropping of many miRNAs can be
let-7 family members (36). In summary, c-Myc activity mediated by a complex of purified DGCR8 and
contributes to tumourigenesis by both positively and Drosha in vitro, the pri-miRNA to pre-miRNA pro-
negatively regulating the expression of diverse cessing of some miRNA is relatively inefficient (43).
miRNAs. Therefore, the efficient processing of some miRNAs
by the Drosha/DGCR8 complex may require the
Epigenetic control of miRNA genes involvement of accessory factors.
Many of the mechanisms of epigenetic control known
to regulate protein-coding genes, such as DNA methy- Control of Drosha/DGCR8 expression
lation and modifications of histones, seem to be also The total levels of Drosha and DGCR8 in the cell are
applied to miRNA genes. For example, in bladder tightly controlled and may play a role in the regulation
cancer, the expression of miR-127 is reduced due to of pri-miRNA processing. Increased Drosha expres-
promoter hypermethylation (37). Several additional sion is observed in late-stage cervical cancer samples
miRNA loci, such as miR-9-1, -193a, -137, -342, -203 and is associated with poor prognosis in oesophageal
and -34b/c, are also found to be hypermethylated in cancer patients (44, 45). Interestingly, despite a 2- to
multiple human cancers similar to known tumour sup- 7-fold increase in Drosha expression in cervical cancer
pressor gene loci (38, 39). miRNA gene promoters are samples, only miR-31 is increased, while the other
also regulated by histone modifications during devel- differentially expressed miRNAs were decreased (45).
opment and pathogenesis. For example, low expres- DGCR8 was originally identified as a gene located
sion of miR-1 in lung cancer cells can be rescued within a common chromosomal deletion at ch22q11,
by treating with histone deacetylase (HDAC) inhibi- which gives rise to a syndrome characterized by learn-
tors, suggesting that the promoter of miR-1 may be ing disabilities and heart defects termed DiGeorge
aberrantly acetylated in tumours. Restored miR-1 syndrome (46). Mouse models of a homologous
expression reduced cell growth, mobility and tumour chromosomal deletion exhibit a moderate decrease of
formation in vivo. Inhibitors of HDAC have the po- only 59 miRNAs (47). Given the critical role of Drosha
tential to promote downregulation of some miRNAs. and DGCR8 in mediating pri-miRNA processing, it is
For example, in the breast cancer cell line SKBr3, 32 surprising that relatively minor changes are found in
miRNAs are significantly downregulated following miRNA levels upon dramatic alteration of DGCR8/
treatment with HDAC inhibitors (40). Additionally, Drosha levels (47). These findings may be explained, at
miR-27a was strongly repressed by HDAC inhibitors least in part, by the observation that the total levels of
and was found to target genes previously shown to be Drosha and DGCR8 are coupled in an intricate
upregulated by HDAC treatment (40). feed-back circuit. In addition to cleaving pre-miRNA
hairpins, the Drosha microprocessor complex can
Regulation of the First Processing Step also promote cleavage of hairpin structures within
annotated protein coding genes which are not further
from Pri- to Pre-miRNA
processed by Dicer (48, 49). This type of cleavage
miRNA processing by the Drosha complex allows Drosha to modulate protein-coding gene
Long-primary transcripts of miRNA genes expression independent of miRNA production.
(pri-miRNAs), which are generally several thousand Indeed, tiling microarray analysis in Drosophila cells
nucleotides, undergo two sequential cleavages to following depletion of Drosha indicates that many
become the mature 22 nt miRNAs (Fig. 1). The annotated protein-coding genes, including DGCR8,
first step of miRNA processing is catalysed in the are targeted for degradation through this mechanism
nucleus by the RNase III enzyme, Drosha (3, 5, 41). (48). Therefore, Drosha maintains a highly regulated
Pri-miRNAs contain a distinctive stem-loop structure. level of DGCR8 through the Drosha microprocessor-
Drosha cleaves at the base of the stem to generate a mediated cleavage of DGCR8 mRNA. Furthermore,
60—100 nt hairpin pre-miRNA with a characteristic DGCR8 stabilizes Drosha protein levels and ensures
2 nt overhang at the 30 -end (Fig. 1) (3). Although the tight coupling of the core microprocessor proteins.
Drosha contains a highly conserved RNase III This regulatory loop seems to be highly effective in vivo
domain, purified recombinant Drosha fails to as in the DGCR8 heterozygous knockout mouse,
efficiently generate pre-miRNAs in vitro (42). This DGCR8 protein level is only modestly reduced despite
observation suggests that other cofactors might be a 50% decrease in genomically encoded DGCR8 (49).
required to enhance the catalytic activity of Drosha. Thus the relative levels of DGCR8 and Drosha may
Mass-spectrometric analysis indicates that Drosha need to be closely coupled for accurate pri-miRNA
associates with at least 20 distinct polypeptides (4) processing. Interestingly, in vitro pri-miRNA

383
B.N. Davis-Dusenbery and A. Hata

processing experiments using recombinant proteins in- transcription factors, they may serve as a scaffold for
dicate that as little as a 3-fold excess of DGCR8 over the recruitment of other protein factors in the Drosha
Drosha dramatically inhibits processing activity of microprocessor complex (54, 55).
Drosha (4). A role for p68/p72 in miRNA processing is further
In addition to alteration of protein levels, the supported by the regulation of pri-miRNA processing
overall activity of the Drosha/DGCR8 complex mediated by multiple p68-interacting proteins, includ-
may be altered under some circumstances. Cells ing the Smads, p53 and estrogen receptor a (ER-a)
grown to high confluency exhibit increased pre- and (Fig. 2). The Smads are the signal transducers of the
mature-miRNA expression without alteration of TGF-b family of signalling pathways. In the canonical
Drosha or DGCR8 protein level (50). Furthermore, pathway, ligand binding to the types I and II TGF-b
extracts from high-confluency cells promoted the receptors promotes the nuclear accumulation of
more efficient conversion of pri- to pre-miRNA receptor-specific Smads (R-Smads) in complex with
in in vitro processing assays (50). Although the mech- the common-Smad (co-Smad), Smad4. The complex
anism of this increased processing is unknown, it is of R-Smad and co-Smad bind to specific DNA se-
possible that post-transcriptional modifications or quences within the promoter of target genes and regu-
association with accessory factors could alter lates gene transcription either positively or negatively.
the activity of Drosha or DGCR8. For example, it is TGF-b and its family member, bone morphogenetic
interesting to note that DGCR8 is a heme-binding protein 4 (BMP4) are particularly important for the
protein and binding of heme promotes the dimeriza- differentiation of vascular smooth muscle cells
tion of DGCR8 and facilitates pri-miRNA-processing (VSMCs). Treatment with either BMP4 or TGF-b in-
activity (51). creases expression of VSMC-specific genes; this pro-
cess is due, at least in part, to the miR-21-mediated
Control of Drosha processing by the DEAD-box RNA repression of programmed cell death protein-4
helicase-interacting proteins (PDCD4). miR-21 is rapidly induced by BMP4 or
The DEAD-box RNA helicases p68 (DDX5) and TGF-b in VSMC which results in a subsequent de-
p72 (DDX17) were initially identified as crease in PDCD4 and increased VSMC gene expres-
components of the Drosha microprocessor complex sion (56). Interestingly, although knockdown of the
by immunoprecipitation-mass spectrometry analysis R-Smads prevents upregulation of mature and
and subsequently shown to also associate with pre-miR-21 in response to BMP4, no alteration in
DGCR8 (4, 52). Knockout of either p68 or p72 in pri-miR-21 transcription is detected, suggesting that
mouse results in embryonic lethality (E11.5 or P2, R-Smads post-transcriptionally affect the level of
respectively) and animals with double knockout of miR-21. Furthermore, BMP4 could increase the ex-
p68 and p72 show earlier lethality without obvious pression of pre- and mature miR-21 derived from an
specific degeneration of organogenesis (53). expression construct driven by a cytomegarovirus pro-
Microarray analysis indicates that the steady state moter, suggesting that miR-21 is post-transcriptionally
levels of 35% (94 out of 266 surveyed) of mature regulated at the Drosha processing step. The identifi-
miRNAs in p72 null mouse embryonic fibroblasts cation of R-Smads as binding partners of p68 by
(MEFs) are reduced verses control, wild-type MEFs. yeast-two-hybrid system suggests that R-Smads could
Most of the miRNAs which are reduced in p72 knock- associate with the Drosha complex (57). Consistently,
out mice are similarly reduced in p68 knockout mice, co-immunoprecipitation and RNA-ChIP studies indi-
while combined knockout of p68 and p72 does cate that R-Smad is present in a complex with Drosha
not further reduce mature miRNA levels. This obser- and p68 on the pri-miR-21 hairpin following BMP4
vation suggests that the two proteins are largely or TGF-b stimulation (56). An increase in Drosha
functionally redundant or act as a heterodimer in binding to pri-miR-21 is also observed after growth
the Drosha complex. Interestingly, while the level of factor treatment, suggesting that R-Smads may
pre-miRNA was also reduced in p72 or p68 knockout promote the association of Drosha with miRNA
MEFs, the level of pri-miRNA was unchanged. hairpins (56). These results indicate that in addition
Furthermore, in vitro pri-miRNA-processing assays to the transcriptional regulation mediated by the
indicate that the Drosha processing activity of extracts R-Smads, TGF-b can also regulate gene expression
depleted of p68 and p72 is attenuated compared through miRNA processing. In addition to miR-21,
to control extracts. Additionally, in vivo RNA- several other miRNAs are post-transcriptionally
immunoprecipitation studies show reduced Drosha induced by BMP and TGF-b, suggesting that
association with pri-miR-199a in MEFs derived rapid modulation of miRNA levels plays an import-
from p68 and p72 knockout animals. Together, these ant role in cellular response to cytokine signalling
results strongly support a role of p68 and p72 in (Fig. 2) (56).
promoting the Drosha processing of a sub-set of The tumour suppressor protein p53 was recently
miRNAs (53). The precise mechanism of p68/p72- identified to modulate miRNA processing through
mediated processing is unclear, but it may involve association with p68 and Drosha, similarly
the rearrangement of the pri-miRNA hairpin structure to the R-Smads (58). Under conditions of DNA
which leads to enhanced Drosha recruitment or damage, several miRNAs, such as miR-143 and
association with the pri-miRNA. Alternatively, as miR-16, are post-transcriptionally induced (Fig. 2)
p68 andp72 are known to interact with a variety of (58). p53 is essential for this process as p53-null
proteins, including RNA processing enzymes and HCT116 cells do not induce miRNAs in response

384
 Control of miRNA biogenesis

TGF-β miRNA (64). It is intriguing to speculate that p53

pri-miRNA
Signal
and/or ER a similarly select pri-miRNAs based on a
+
ad specific sequence or structure of pri-miRNAs.
DGCR8
Sm
p68/p72
Control of Drosha processing by other auxiliary
+ DNA proteins
p53 Damage In addition to the mechanisms described above, several
Drosha processing mechanisms independent of the DEAD-box
RNA helicases have been studied. For example,
-
hnRNP A1 is required for the processing of a
E2-ER
α Estrogen
Signal member of the miR-17-92 cluster, miR-18a. Addition
of hnRNP A1 to in vitro pri-miRNA-processing assays
dramatically increases the conversion of pri-miR-18a
to pre-miR-18a (Fig. 3A). Conversely, depletion of
hnRNP A1 lowers pre-miR-18a levels, but does not
alter the expression of the other five clustered
miRNAs (65). Further studies indicate that direct
binding of hnRNP A1 to the loop region of miRNA
pre-miRNA
hairpins causes the structural rearrangement of the
hairpin to generate a more favourable Drosha cleavage
Fig. 2 DEAD box RNA helicase-dependent miRNA processing site (Fig. 3A) (66). The loop region of miR-18a
pathways. The RNA helicases p68 and p72 play a critical role in is well conserved throughout evolution (66), emphas-
the post-transcriptional regulation of numerous miRNAs in izing the importance of the hnRNP A1-loop
response to cellular signals, including TGF-b stimulation, DNA
damage and estrogen stimulation. The downstream mediators of interaction for the processing of pri-miRNA.
TGF-b stimulation and DNA damage, the Smads and p53, act to Interestingly, 14% of human miRNAs contain
promote miRNA processing. Conversely, when bound to E2, ER-a highly conserved loop regions, suggesting that process-
reduces the processing of a subset of miRNAs. For clarity, some ing regulation by hnRNPs may extend well beyond
potential members of the Drosha processing complex are not shown.
miR-18a. Consistently, the processing of two other
pri-miRNAs bearing conserved loops, miR-101 and
let-7a-1 is also prevented by complementary
to DNA damage (58). Co-immunoprecipitation studies looptomiRNAs, suggesting that the loop represents a
indicate that p53 is present in a complex critical regulatory region for the generation of these
with both Drosha and p68, and addition of miRNAs (66).
p53 to in vitro pri-miRNA processing assays could en- The importance of the loop region in the regulation
hance the processing reaction by Drosha (Fig. 2) (58). of pri-miRNA processing is further supported by recent
Interestingly, several mutants of p53 which are linked studies which indicate KH-type splicing regulatory
to oncogenesis show reduced post-transcriptional protein (KSRP) directly interacts with G-rich regions
miRNA expression (58). present within the loop of a sub-set of pri-miRNAs to
More recently, it was reported that estrogen promote both Drosha and Dicer mediated miRNA
receptor-a (ER-a) also associates with p72 and processing (Fig. 3B) (67). Transient knockdown of
Drosha upon estradiol (E2) stimulation (59). Unlike KSRP in HeLa cells strongly reduced the expression
the Smads or p53, however, association of E2-bound of a group of mature miRNAs, including let-7a and
ER-a with the Drosha complex inhibits the association miR-206. Furthermore, knockdown of KSRP altered
of Drosha with a subset of pri-miRNAs (Fig. 2) (59). cellular responses mediated by these miRNAs, includ-
In summary, these results indicate that the association ing proliferation and skeletal muscle differentiation.
of p68/Drosha with transcription factors, such as p53, KSRP was found to be present in both the Drosha
R-Smads and ER-a, is important for the rapid regula- and Dicer complexes, and knockdown of KSRP
tion of miRNA expression in response to extracellular reduced the cropping activity of Drosha and Dicer
stimuli. It is intriguing to speculate that other known (67). The interaction of KSRP with sequential
p68-interacting transcription factors, such as MyoD pri-miRNA and pre-miRNA cleavage steps may
(60), and Runx2 (61), androgen receptor (62) and serve to promote the synergistic activation of process-
b-catenin (63), might also play a role in the Drosha ing for a subset of miRNAs (Fig. 3B). Alternatively,
microprocessor complex. Furthermore, although each KSRP association with either the Drosha or Dicer
of the described mechanisms requires the RNA heli- complexes may be sufficient to alter the miRNA
cases p68 or p72, the miRNAs that are regulated are processing in response to cellular stimuli. For example,
not completely overlapping. We recently showed that lipopolysaccharide (LPS) treatment of macrophages
R-Smads recognize and directly associate with 20 dramatically increases mature miR-155 without signifi-
pri-miRNAs which contain a conserved sequence cantly altering the expression of the pri-miR-155 (68).
(50 -CAGAC-30 ) within the stem region (64). We Mobility shift assays indicate KSRP interacts with
found that direct association of R-Smads with pri-miR-155 and knockdown of KSRP prevents
pri-miRNA facilitates the recruitment of Drosha and LPS-mediated elevation of miR-155 (68). Altogether
DGCR8 and leads to efficient cropping of the these results suggest a role of KSRP in promoting
pri-miRNA and elevated expression of mature the post-transcriptional regulation of miR-155 in

385
B.N. Davis-Dusenbery and A. Hata

A hnRNP A1 B C
Lin28
KSRP

structural Drosha
Drosha
rearrangement
Drosha pri-miRNA
pri-miRNA
pri-miRNA
+
+
Lin28
KSRP
Lin28

pre-miRNA TUT4

Dicer
pre-miRNA
- Dicer pre-miRNA
+
NF90/NF40 UU
U
-
Drosha

miRNA:miRNA*
pri-miRNA miRNA:miRNA*

Fig. 3 RNA helicase-independent mechanisms of miRNA processing regulation. (A) hnRNP A1 recognizes the terminal loop of miRNAs and
promotes the structural remodelling of the stem region. Structural rearrangement generates a favourable Drosha-binding site and enhances
pri- to pre-miRNA processing. NF90 and NF40 are strongly associated with the stem region of pri-miRNAs, which precludes association with
the Drosha processing complex and thus inhibits the processing of miRNAs. (B) Similarly to hnRNP A1, KSRP binds to the loop region of
miRNAs and promotes both the Drosha and Dicer processing. (C) Binding of Lin28 to the terminal loop prevents the association of both
Drosha and Dicer with the pri- and pre-miRNAs, respectively. Additionally, Lin28 acts as a scaffold to promote the association of TUT4 with
the pre-miRNA. TUT4 promotes the 30 -uridinylation of pre-miRNA which is then rapidly degraded.

response to LPS. Interestingly, miR-155 is derived conserved nucleotide sequences in the hairpin loop.
from the non-coding BIC RNA which has long been This observation suggests that this region might be
associated with induction of lymphomas (69). BIC critical for the post-transcriptional regulation of let-7
(pri-miR-155) is strongly induced by B-cell receptor family of miRNAs (72, 73). While pri-let-7 is processed
signalling in a variety of cell types. Interestingly, in efficiently in vitro using cell extracts derived from dif-
the Burkitt-lymphoma-derived Ramos cell line, induc- ferentiated cells, processing is reduced in extracts
tion of BIC does not give rise to an increase in mature derived from undifferentiated cells. Affinity purifica-
miR-155 (70). This effect seems to be unique to the tion followed by mass spectrometry shows that
Ramos cell line as expression of pri-miR-155 in Lin-28 interacts with let-7 and inhibits processing
HEK293 or Hodgkin’s lymphoma cell lines gives rise (Fig. 3C) (72, 74). The physiological relevance of
to very high levels of miR-155 (70). The mechanism of let-7 regulation by Lin-28 is supported by the recipro-
this cell type-specific effect is unclear, however, in light cal expression of Lin-28 and mature let-7 during
of the results above, it may be interesting to examine if development.
KSRP levels or activity is altered in some lymphomas. The precise mechanism of Lin-28 inhibition of
In addition to the positive regulation of miRNA Drosha processing is still unclear. As Lin-28 has a
processing mediated by hnRNPs and KSRP discussed strong affinity for let-7, it is plausible that Lin-28 pre-
above, negative regulation of the Drosha processing of vents interaction with Drosha/DGCR8 by mutual ex-
some pri-miRNAs has also been reported. The let-7 clusion (Fig. 3C). Alternatively, similarly to hnRNP
family of miRNAs is encoded as multiple copies in A1 discussed above, the interaction of Lin-28 with
the genome; for example, the human genome contains the loop region could rearrange the secondary struc-
nine mature let-7 sequences derived from 12 precursors ture of the hairpin and inhibit Drosha processing.
(71). While most let-7 primary transcripts are highly Furthermore, as discussed further below (see:
expressed throughout development, mature let-7 is de- Control of Dicer Activity), recent reports suggest
tectable only in highly differentiated cells, suggesting a that Lin-28 inhibits the biogenesis of let-7 through
mechanism of post-transcriptional regulation during multiple mechanisms (Fig. 3C) (75, 76). It is interesting
differentiation (71). Analysis of the pri-miRNA se- to note that all three proteins (Lin-28, KSRP and
quences of the human let-7 family, as well as let-7 hnRNPs) interact with conserved nucleotides present
from different species indicates the presence of within the loop of pri-miRNAs (Fig. 3). The balanced

386
 Control of miRNA biogenesis

interaction of proteins with the pri-miRNA loop may Dicer may serve as an important control point in
allow for the fine tuning of miRNA levels during de- miRNA biogenesis. A careful analysis of the Dicer
velopment or in response to extra-cellular cues, such as 50 -UTR identified multiple alternatively spliced leader
growth factors. For example, although KSRP interacts exons that are expressed in a tissue specific manner
strongly with pri-let-7g in differentiated cells, high (83). Although differential splicing alters the transla-
levels of Lin28 block KSRP association in embryonic tion efficiency of Dicer in vitro, the specific roles of the
carcinoma p19 cells (67). various Dicer isoforms are unclear. Altered expression
Repression of Drosha processing of let-7 family of Dicer is observed in several types of human cancer,
members is also mediated by the nuclear factor 45 for example, Dicer is increased in prostate tumours, as
(NF45) and nuclear factor 90 (NF90) proteins (77). well as in a Burkitt’s lymphoma derived cell line
In vitro and in vivo pri-miRNA processing assays indi- (84—86). Conversely, Dicer expression is decreased in
cate that the NF90/NF45 complex reduces non-small cell—lung carcinoma, and reduced Dicer
pre-miRNA while increasing pri-miRNA levels. The levels correlate with poor prognosis (87). These con-
NF90/NF45 family of proteins shows strong associ- flicting trends may be indicative of cell type differences
ation with small ds RNAs, including the adenovirus or tumour stage. Analysis of precursor lesions of lung
VA RNA as well as some pri-miRNAs (78). adenocarcinoma showed increased Dicer expression,
Electrophoretic mobility shift assay analysis suggests while advanced invasive adenocarcinoma showed
that the high-affinity association of NF90/NF45 decreased Dicer levels (88).
complex with pri-miRNA precludes interaction with Depletion of the Dicer cofactors, TRBP or PACT,
the Drosha Complex, thus reducing production of decreases the steady state levels of Dicer protein
pre- and mature miRNA (Fig. 3A) (77). Unlike (89, 90). Truncation mutations of TRBP are found in
Lin-28, which specifically regulates let-7, the NF90/ carcinoma and are associated with decreased miRNA
NF45 complex seems to be more promiscuous as levels and dramatic destabilization of Dicer (91). Dicer
the processing of three additional miRNAs is also protein levels can be rescued by full-length TRBP,
modulated by the NF90/45 complex (78). suggesting that the interaction between Dicer and
TRBP is a critical determinant of Dicer stability.
Potential Control of Nuclear Export of Furthermore, the expression or activity of Dicer
can also be modulated by cellular-signalling pathways.
pre-miRNA
For example, MAPK/ERK signalling was found to
The export of pre-miRNA from the nucleus to the promote the phosphorylation of TRBP (92).
cytoplasm may be differentially regulated under cer- Phosphorylated TRBP enhances miRNA production
tain physiological conditions. For example, the hair- by increasing the stability of Dicer. Interestingly,
pins of pre-miR-105, -128 and -31 are detected in many increased abundance of Dicer is correlated with the
cells at relatively high abundance. However, under increase in growth-promoting miRNAs and decrease
some conditions the mature miRNA is undetectable of let-7 which has a tumour suppressor activity (92).
(79). For example, pre-miR-31 is expressed at compar-
able levels in the pancreatic cancer cell line HS766T Control of Dicer activity
and the MCF7 breast cancer cell line. However, Pre-miRNAs are often expressed at a low level relative
while HS766T cells show high levels of mature to pri-miRNA or mature miRNA, suggesting that
miR-31, no expression is detected in the MCF7 cells. Dicer is generally very efficient at processing
In situ hybridization of pre-miR-31 shows the expected pre-miRNA to mature miRNA (79, 93). Unlike
cytoplasmic localization in HS766T cells, while in Drosha which requires many accessory factors to pro-
MCF7 cells pre-miR-31 accumulated in the nucleolus mote processing, regulation of miRNAs at the Dicer
(79). Together, these results indicate that the step often involves inhibition of Dicer activity. Early
nuclear-cytoplasmic shuttling of pre-miR-31 is regu- evidence for post-transcriptional control of miRNA at
lated in a cell-type dependent manner; however, the the level of Dicer processing comes from studies of
mechanism of altered pre-miRNA export is unknown. miRNA expression in colorectal neoplasia. Mature
miR-143 and miR-145 are expressed at much lower
Regulation of the Second Processing Step levels in tumour samples than normal tissue.
However, the expression of pre-miR-143 or
from Pre- to Mature miRNA
pre-miR-145 is not significantly different in the
Control of Dicer expression tumour samples, suggesting that the Dicer processing
Following translocation into the cytoplasm, the step is inhibited in the colorectal tumour samples (94).
pre-miRNA is cleaved near the terminal loop by the miRNA processing by Dicer is also differentially regu-
RNase type III protein Dicer to generate a 22-nt ds lated during normal development and tissue specifica-
mature miRNA (Fig. 1) (80, 81). Dicer is highly con- tion. For example, while pre-miR-138 is ubiquitously
served throughout evolution and present in nearly all expressed in all tissues and HeLa cells, the mature
eukaryotic organisms. Similarly to Drosha, several miR-138 is only detected in adult mouse brain and
Dicer-associated proteins have been identified, includ- foetal liver (95). Potential alteration in nuclear export
ing TAR RNA-binding protein (TRBP) and protein is ruled out as the pre-miR-138 is effectively exported
kinase R-activating protein (PACT) (82). Association into the cytoplasm, therefore it is suggested that the
of TRBP and PACT with Dicer enhances Dicer stabil- regulation of Dicer processing of pre-miR-138 might
ity and processing activity (82). The total levels of be inhibited in many tissues. Furthermore, while

387
B.N. Davis-Dusenbery and A. Hata

pre-miR-138 is processed efficiently by Dicer in vitro, indicates an increase in Ago2 mRNA independent
addition of HeLa cell extracts inhibits the processing of the siRNA sequence (100). Ago2 mRNA and
of pre-miR-138 (95). HeLa extracts do not alter the protein expression is also found to be increased in a
processing of other pre-miRNAs which are normally subset of breast cancer cells. Epidermal growth factor
expressed in HeLa cells (95). These results suggest that treatment enhances Ago2 stability, suggesting that
HeLa cells, and other most tissues, express a yet to be Ago2 levels can be regulated at both the transcription-
identified factor which inhibits Dicer activity specific- al and post-transcriptional level (101). Furthermore,
ally on miR-138. increased Ago2 expression was associated with a
In addition to inhibition of the Drosha processing transformed phenotype in breast cancer cells (101).
step discussed above, Lin-28 also inhibits the Dicer Interestingly, Ago2 is found to be targeted for ubiqui-
processing of let-7 family members (Fig. 3C) (74, 96). tination and degradation by Lin-41 (102). Lin-41 is
Lin-28 shuttles between the nucleus and cytoplasm, expressed primarily in stem cells and undifferentiated
but is commonly enriched within the cytoplasm, sug- cell types. Co-immunoprecipitation studies indicate
gesting that the cytoplasm may be the primary location that Lin-41 directly interacts with Ago2 and overex-
of the Lin-28-let-7 interaction. Addition of purified pression of Lin-41-reduced miRNA-mediated mRNA
Lin-28 decreases the association of let-7 with Dicer, silencing (102). Reduction of Ago2 by Lin-41 may
and results in reduced processing by Dicer, suggesting serve to prevent expression of miRNAs and inhibit
that Lin-28 can compete with Dicer for access to let-7 cellular differentiation, thus maintaining a stem-cell-
(Fig. 3C) (74). Furthermore, Lin-28 also promotes the like phenotype (102). Interestingly, Lin-41 is a target
30 -polyuridylation of pre-let-7 by acting as a scaffold of let-7 which is highly regulated and important for
between let-7 and the terminal uridine transferase cellular differentiation (74). Similarly to lin-28, the
TUT4 (Fig. 3C) (75, 76). Polyuridylation of pre-let-7 levels of Lin-41 are inversely correlated with the level
inhibits Dicer processing and promotes the degrad- of let-7 (102). It is interesting to speculate that in con-
ation of pre-let-7 (76). Analysis of the Lin-28-binding ditions which are characterized by low levels of let-7,
site within the loop of let-7 identified a tetra-nucleotide such as lung cancer, Lin-41 may be increased which
sequence required for Lin-28/TUT4-mediated uridyla- could in turn result in reduced expression of Ago2.
tion. Interestingly, this motif is located within the ter- Indeed, immunohistochemical analysis of 68 gastric
minal loop of 15 additional pre-miRNAs, some of and colorectal tumours indicates a reduction in Ago2
which have also been shown to undergo uridylation protein in 40 and 35% of gastric and colorectal can-
(76). cers, respectively (103). It is interesting to speculate
that alterations in Ago2 might be a universal charac-
Control of the Argonaute teristic of poorly differentiated tumours.
Dicer is associated with several other proteins in a
complex termed the RISC-loading complex (RLC)
which allows the tight coupling of Dicer cleavage to Perspectives
the incorporation of miRNA into the RISC (90). miRNA biogenesis can be regulated at multiple steps
Cleavage by Dicer results in an unstable dsRNA com- by different regulatory mechanisms. Further investiga-
posed of the active guide strand (miRNA) and the pas- tion is expected to uncover many more mechanisms
senger (miRNA*) strand (Fig. 1). Selection of the and upstream signalling pathways that are critical for
miRNA strand which is incorporated into the RISC the maintenance or induction of miRNA levels. In the
can be regulated in a cell type-specific manner. The future it will be important to investigate how multiple
Argonaute (Ago) proteins are the primary component mechanisms of miRNA biogenesis control cooperate
of the RISC complex, and the effectors of to give rise to spatially and temporally controlled
miRNA-mediated repression of target mRNAs (97). miRNA expression. Furthermore, an understanding
The human genome contains eight Ago-family pro- of how miRNAs are regulated under physiologic con-
teins; Ago1-4 and Piwi1-4 (97). While all of the Ago ditions will allow better understanding of the patho-
proteins have the ability to interact with miRNAs and genesis of various human diseases and facilitate the
siRNAs, Ago2 is the only one with RNA cleavage development of a novel therapeutic approaches to
activity and is thought to play a critical role in
modify specific miRNA expression in vivo.
miRNA-mediated mRNA silencing (97). Total levels
of the Ago proteins within the cell also contribute to
global miRNA regulation and biogenesis. Ectopic ex- Acknowledgements
pression of Ago proteins results in a dramatic increase
We apologize to colleagues in the field for not being able to cite
in mature miRNAs (98). In contrast, MEFs and hem- many important papers due to the limitation of the article. We
atopoietic cells deleted in Ago2 gene exhibit reduced thank Drs Kohei Miyazono and Keiji Miyazawa for giving us the
levels of all mature miRNAs examined (98, 99). The opportunity to write this article. We also thank all past and current
dramatic increase in mature miRNA mediated by members of the Hata and Lagna Labs for helpful discussion, in
particular Dr Mun Chun Chan.
increased Ago expression could indicate that Ago pro-
teins are limiting in the cell and serve to stabilize
miRNA. If this is the case, one might expect that
Funding
Ago level may be linked to the level of miRNAs or National Institute of Health (RO1 HD042149 and RO1 HL082854
siRNAs present within the cell. Indeed, a retrospective to A.H. and T32 HL069770 to B.N.D.); American Heart Association
study of micro-array data from siRNA treated cells (0940095N to A.H.).

388
 Control of miRNA biogenesis

Conflict of interest 19. Bushati, N. and Cohen, S.M. (2007) MicroRNA func-
None declared. tions. Annu. Rev. Cell Dev. Biol. 23, 175—205
20. Cai, X., Hagedorn, C.H., and Cullen, B.R. (2004)
Human microRNAs are processed from capped, polya-
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