Download exportin-5 mediates their nuclear export

Update
156
TRENDS in Cell Biology
Vol.14 No.4 April 2004
MicroRNA precursors in motion: exportin-5
mediates their nuclear export
V. Narry Kim
School of Biological Sciences and Institute of Molecular Biology and Genetics, Seoul National University, Seoul, 151-742, Korea
The discovery of microRNAs (miRNAs) has changed the
paradigm of gene regulation, leaving us with numerous
exciting questions regarding what these molecules do
and how they originate. A model for miRNA biogenesis
has emerged recently, yet several key factors – including the identity of the miRNA nuclear export receptor –
remained unknown. However, recent studies have
shown that exportin-5 (Exp5), a Ran-dependent importin-b-related transport receptor, mediates nuclear
export of miRNA precursors (pre-miRNAs).
MicroRNAs (miRNAs) constitute a large family of noncoding small RNAs , 22 nucleotides (nt) in length [1]. Over
three hundred miRNA genes have been reported in diverse
eukaryotic organisms, and the number is predicted to
increase by up to several hundred per species. Our
understanding of miRNA originates from studies of the
developmentally regulated miRNAs lin-4 and let-7 in
Caenorhabditis elegans [1]. By binding and inhibiting
the translation of the target mRNAs, the lin-4 and let-7
RNAs control the timing of larval development [1]. Recent
advances have revealed that miRNAs function in a
variety of regulatory pathways, including cell proliferation, apoptosis, fat metabolism, hematopoiesis and
organogenesis [1].
MiRNAs are transcribed as long primary transcripts
(termed pri-miRNAs) [2], which are first cropped into
, 70-nt stem – loop precursors (pre-miRNAs) in the
nucleus by the RNase-III-type protein Drosha [3]
(Figure 1). Following this initial processing, pre-miRNAs
get exported to the cytoplasm and are subject to a second
processing event to generate the final product of ,22-nt by
another RNase III termed Dicer. Owing to compartmentalization of the two processing events, nuclear export of premiRNAs is a crucial step in miRNA biogenesis [2].
Nuclear transport occurs through nuclear pore complexes (NPCs) that are large proteinaceous channels
embedded in the nuclear membrane [4]. Soluble transport
receptors recognize specific sequences in substrates
(cargos) and escort them through NPCs by interacting
with proteins in NPCs termed nucleoporins. The majority
of receptors belong to a single family of nuclear transport
receptors (NTRs), with the prototypes being importin b
and transportin. Members of the NTR family share a key
cofactor – the small GTPase Ran. The export receptor
forms a complex cooperatively with the cargo and the
GTP-bound form of Ran (RanGTP) in the nucleus and then
Corresponding author: V. Narry Kim ([email protected]).
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undergoes translocation to the cytoplasm. Upon export,
hydrolysis of GTP to GDP on Ran results in release of the
cargo from the export complex. Noncoding RNAs such as
transfer RNAs (tRNAs), small nuclear RNAs (snRNAs)
and ribosomal RNAs (rRNAs) are exported by NTR family
receptors [4]. For example, exportin-t recognizes common
features of mature tRNAs and constitutes the major
pathway for tRNA export [5,6].
Intriguingly, no consensus sequences can be found in
pre-miRNAs. Therefore, the common export factor, if there
is any, is likely to recognize a structural motif common in
all pre-miRNAs. Analysis of the precursor microRNA premiR-30a showed that pre-miR-30a is a stem – loop of 63-nt
containing a 2-nt 30 overhang [3] (Figure 2). Other premiRNAs were also found to contain a short 30 overhang [7],
which is likely to represent a key structural feature in
nuclear export. Typically, pre-miRNAs comprise a stem of
, 24-bp, a loop of variable size, and a 30 overhang of , 2-nt.
It is important to note that the ‘pre-miRNA’ sequences
presented in most literature and databases are only
predicted sequences and that the exact ends of these
stem – loops should be experimentally determined.
Exportin-5 (Exp5) was originally identified based on its
homology to CRM1/exportin 1 [8] and later reported to be a
minor export receptor for tRNAs [9,10]. Another substrate
for Exp5 is the adenoviral RNA VA1, a 160-nt non-coding
RNA that inhibits the double-stranded-dependent kinase
PKR [11]. Analysis of cis-acting elements for nuclear
export in VA1 revealed a structural motif termed ‘minihelix motif ’ comprising a .14-bp stem and a 3 – 8-nt 30
overhang [12] (Figure 2), whose nucleotide sequences are,
however, not important for nuclear export of this RNA. The
main cellular cargo(s) for Exp5 has been a subject of debate
because, in normal conditions, tRNA is mostly transported
by Exp-t. Here, we discuss the recent identification of Exp5
as the export receptor for pre-miRNAs.
Does Exp5 export pre-miRNAs?
Because pre-miRNAs form minihelix-like structures and
serve as export cargos, Exp5 became a promising candidate as the pre-miRNA export receptor. This was proven by
Lund and colleagues in a series of elegant microinjection
experiments in Xenopus oocytes [13]. First, they showed
that pre-miRNA export is dependent on RanGTP and that
different pre-miRNAs compete with each other but not
with tRNA or U1 snRNA. These data suggest that premiRNAs are exported by a common NTR family receptor
that is different from Exp-t (the receptor for tRNA) or
Update
TRENDS in Cell Biology
miRNA gene
(polycistronic)
Transcription
157
Vol.14 No.4 April 2004
miRNA gene
(monocistronic)
shRNA
expression cassette
pol III
?
pri-miRNA
Drosha
Cropping
pre-miRNA
(60–100-nt stem–loop
with ~2-nt 3′ overhang)
UU
shRNA
(50–70-nt stem–loop
with ~2-nt 3′ overhang)
Exp5
Export
Nucleus
Cytoplasm
NPC
shRNA
pre-miRNA
UU
Dicing
Dicer
siRNA duplex (~22-nt)
miRNA duplex (~22-nt)
UU
Strand selection
?
Mature miRNA
(miRNP)
siRNA (RISC)
Cap
pA
Inhibit translation
Cap
pA
Degrade target mRNA
TRENDS in Cell Biology
Figure 1. Model for miRNA biogenesis. MiRNA genes are transcribed by an unidentified polymerase to generate the primary transcripts, referred to as pri-miRNAs. The
initiation step (cropping) by Drosha results in pre-miRNAs of , 70-nt, which are exported by Exp5. Upon export, Dicer participates in the second step (dicing) to produce
miRNA duplexes. The duplex is separated and usually one strand is selected as a mature miRNA, whereas the other strand is degraded. Final products act as guide molecules in translational control or cleavage of certain mRNAs. The question marks indicate unidentified biogenesis factors.
CRM1 (the receptor for U1 snRNA). Purified receptor
proteins were then injected into the nuclei of oocytes that
had been saturated with unlabeled pre-miRNA and tRNA.
Injection of Exp5 resulted in cytoplasmic accumulation of
pre-miRNA but not tRNA or U1 snRNA. Exp-t overcame
the blockade of tRNA export but not that of pre-miRNAs.
Gel retardation assays showed that Exp5 binds to premiRNA directly in a RanGTP-dependent manner and that
the affinity for pre-miRNA is much higher than that to
tRNA, strongly indicating that the main cellular cargos for
Exp5 are not tRNAs but pre-miRNAs. Considering that
miRNAs have an abundance approaching 50 000 copies
per cell [14], pre-miRNAs are likely to be the main cargos
for Exp5. Moreover, RNAi against Exp5 in HeLa cells
resulted in reduction in the concentration of let-7a,
offering additional evidence that Exp5 is important for
miRNA biogenesis. The new method developed to measure
miRNA levels in this study appears to be more sensitive
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and convenient compared with other conventional
methods such as northern blot analysis.
Through different experimental approaches, Cullen
and colleagues described the crucial role of Exp5 in the
export of pre-miRNAs [15]. RNAi of Exp5 was followed by a
reporter assay using plasmids containing target sites for
miR-30a or miR-21 in the 30 UTR that demonstrated that
Exp5 is required for not only the biogenesis but also the
function of miRNAs. More direct evidence was given
determining the nuclear/cytoplasmic level of pre-miRNA
after RNAi. When the cells were depleted of Exp5, the premiR-30a level was reduced drastically in the cytoplasm.
Rather unexpectedly, RNAi of Exp5 did not result in
nuclear accumulation of pre-miRNA. This result suggests
that pre-miRNA is relatively unstable and that premiRNA is stabilized by Exp5.
Notably, Cullen and colleagues also showed that Exp5 is
required for efficient production of siRNAs from short
158
(a) Pre-miR-30a
(b) shRNA
against p53
Update
TRENDS in Cell Biology
UC
GUGAAG
UGUAAACAUCC GACUGGAAGCU
C
C
ACGUUUGUAGG--CUGACUUUCGG
A
CG
GUAGAC
GACUCCAGUGGUAAUCGAC U U C A
A
CUGAGGUCACCAUUAGAUG A
G AG
UU
G AU A
G
A
U
A
A
G
A
G CGA
A
A
G
U
GGGC CUCUUCCGU GUCUG
UCGCA GGGU UCAUGGCGGACG CCGGG UU
A
CUCG GAGGGGGCA CAGACUGCAGCGU CCCA AGUGCCGCCUGC GGCCU GG
C
G
A
UUUUC
C
A
C
A CCC
A U
U G GC
C
G
U
G C
U A
Minihelix motif
G U
C G
G C
C G
C G
C U
G
U
CAA
(c) VA1 RNA
TRENDS in Cell Biology
Figure 2. Export substrates for Exp5. Shown are sequences and predicted structures of (a) microRNA miR-30a, (b) short hairpin (sh) RNA against p53 [22], and (c)
VA1 RNA.
hairpin RNAs (shRNAs) (Figure 1). ShRNAs are transcribed from a polymerase III (pol III) promoter and
contain a 19 – 29-bp stem and a , 2-nt 30 overhang (UU)
(Figure 2). Because of the similarity in the structure, shRNA
appears to be recognized as pre-miRNA by cellular machinery including Exp5 and Dicer. This result further
strengthens the idea that siRNAs and miRNAs are indistinguishable in human cells. The practical implication of this
finding is that, for efficient RNAi, it might be important to
optimize shRNA design for enhanced binding to Exp5.
Depletion of Exp5 by RNAi resulted in a significant
reduction but not full loss of mature miRNA, leaving the
question is Exp5-dependent export the only pathway or is
there an alternative pathway(s). This is a problem that one
almost always gets with knockdown experiments due to
the nature of RNAi, especially when the target protein or
the product of the protein is stable. Indeed, mature miRNA
appears to have long half-life because RNAi for three days
against Drosha or Dicer resulted in a similar reduction and
it took longer to achieve further decreases [3]. Yi et al.
circumvented this problem by intensive RNAi and ectopic
expression of miR-30a [15]. Because pri-miR-30a was
expressed transiently after Exp5 was knocked-down, more
dramatic reduction of mature miRNA levels could be seen,
strongly indicating that Exp5 is the major, if not the only,
receptor for the export of pre-miRNAs.
This conclusion was confirmed by a more recent report by
Gorlich and colleagues [16]. Injecting anti-Exp5 antibody
into the nuclei of Xenopus oocytes completely blocked premiRNA export. This study also showed that Exp5 binds to
dsRNA in a RanGTP-dependent manner in vitro, indicating
that the stem of pre-miRNA might be the primary binding
site for Exp5. Note, however, that correct end structure is
also important for efficient export of pre-miRNAs in vivo [13].
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Vol.14 No.4 April 2004
Concerted action of Drosha, Exp5 and Dicer
The steps in miRNA biogenesis appear to be well
coordinated by two RNase III proteins and one exportin.
Drosha initiates miRNA processing by specific cropping of
the stem– loop precursor in the nucleus (Figure 1). Like
other RNase-III-type endonucleases, Drosha cuts dsRNA
to create a short (,2-nt) 30 overhang. The resulting
structure that is similar to a minihelix is then recognized
by Exp5. Upon export, pre-miRNA is released from the
export complex and handed over to another RNase III,
Dicer. Dicer is known to have a preference towards a
terminus of dsRNA [17]. Indeed, Dicer cleaves , 22-nt
from the terminus of the pre-miRNA stem [18] and from
the base of shRNA [19]. Interestingly, the ends of premiRNAs often correspond to those of mature miRNAs
[3,7], indicating that Drosha creates one end of miRNA and
then Dicer generates the other end by measuring , 22-nt
from the first end. Therefore, Drosha produces optimal
substrates for Exp5 and Dicer, thereby facilitating
biogenesis and predetermining the sequences of the
mature miRNA.
Concluding remarks
The identification of Exp5 as the export factor for premiRNA is an important step towards understanding
the regulatory mechanism of miRNA biogenesis. Evidence suggests that miRNA expression might be
regulated at the level of RNA maturation. Some
miRNAs accumulate as long forms (70 – 100-nt) at
early stages of embryonic development in the fruit fly
and sea urchin [20,21]. It remains to be determined
which step is controlled and how this control is
achieved. One interesting possibility is that some premiRNAs might be retained in the nucleus until export
is triggered by a certain developmental signal.
Another important question is how miRNAs and their
precursors are recognized as such. Because there is no
conserved sequence element either in the mature miRNA
or in its precursors (pri- and pre-), Exp5 and other
processing factors should recognize the common structural
elements. The papers described above identified the
structural element comprising a . 14-nt stem and a
short 30 overhang [12,13,15,16]. It would be interesting
to understand the structural basis of the interaction
between Exp5 and pre-miRNA, which is likely to reveal
novel RNA-binding motif(s). Also important is finding the
processing element(s) present in pri-miRNA, which is
required for binding to Drosha because Drosha-mediated
cropping is the first, and most important, step in determination of mature miRNA sequences.
Other steps in miRNA biogenesis still remain
uncharacterized. For instance, little is known about
transcription of miRNAs, even though the regulation of
miRNA biogenesis is likely to occur mainly at the
transcriptional level. Characterization of the transcription machinery will help understand miRNA biogenesis
and provide clues to miRNA function. Information on
miRNA gene structure is also important because this
will greatly facilitate prediction of miRNA genes and
understanding of eukaryotic genomes.
Update
TRENDS in Cell Biology
Acknowledgements
This work was supported by the Korea Research Foundation (KRF-2002 –
041-C00204) and the BK21 Research Fellowship from the Ministry of
Education and Human Resources Development of Korea.
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doi:10.1016/j.tcb.2004.02.006
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