Howard Hughes Medical Institute and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
(e-mail: culle002{at}mc.duke.edu)
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Summary |
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Key words: Gene expression, mRNA, Nuclear pore complex, Nuclear RNA export
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Introduction |
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Although the nucleocytoplasmic transport of proteins and RNAs share many
features in common, and indeed all nuclear RNA export is protein mediated,
here I nevertheless focus exclusively on the subset of nuclear export pathways
used by different classes of RNA molecule. Although I deal primarily with
nuclear RNA export in metazoan cells, it is clear that these export pathways
are highly conserved among eukaryotes and I will therefore also rely
extensively on data obtained in the yeast Saccharomyces cerevisiae,
which has proven to be a powerful genetic system to identify critical
components of several nuclear RNA export pathways. Readers interested in the
mechanisms underlying protein nuclear export and, particularly, import are
directed to reviews in this area
(Görlich and Kutay, 1999;
Nakielny and Dreyfuss,
1999
).
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Fundamental aspects of nucleocytoplasmic transport |
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Careful proteomic analysis has shown that the yeast NPC consists of 30
distinct proteins termed nucleoporins
(Rout et al., 2000
); the
somewhat larger metazoan NPCs may consist of
50 or more components.
Although the majority of these nucleoporins are stationary, some NPC
components are mobile and might, therefore, interact with nucleocytoplasmic
transport factors prior to docking. A subset of NPC components contain
characteristic domains featuring multiple phenylalanineglycine (FG) repeats.
These function as transient hydrophobic docking sites for nucleocytoplasmic
transport factors. The NPCs contain an aqueous channel that is
9 nm in
diameter when at rest but can expand up to
25 nm during active transport.
The fact that the NPC remains partially open when not engaged in active
transport means that small (
40 kDa) proteins and RNAs can move between the
nucleus and cytoplasm by passive diffusion. However, this process is
inefficient, and even small proteins and RNAs that need to cross the nuclear
membrane are generally actively transported
(Zasloff, 1983
;
Breeuwer and Goldfarb,
1990
).
The bulk of cellular nucleocytoplasmic transport is mediated by factors
that belong to a single family of nuclear transport receptors termed
karyopherins or importins/exportins. Different members of this protein family
bind to distinct cargo molecules, or to adapter proteins that in turn bind
cargo, and also share the ability to interact with specific nucleoporins.
However, the most important characteristic of karyopherins is their shared
dependence on the biological activity of a key cofactor, the small GTPase Ran
(reviewed by Görlich and Kutay,
1999; Nakielny and Dreyfuss,
1999
; Allen et al.,
2000
).
The Ran-specific GTPase-activating protein (RanGAP) is localized to the
cytoplasm whereas the Ran-specific guanine-nucleotide-exchange factor (RanGEF)
is localized in the nucleus. As a result, cytoplasmic Ran exists predominantly
in an inactive, GDP-bound form, whereas nuclear Ran is largely bound to GTP.
Nuclear import factors belonging to the karyopherin family bind their protein
cargo in the cytoplasm, in the absence of Ran-GTP, translocate through the NPC
and then release their cargo upon binding to nuclear Ran-GTP
(Fig. 1). Conversely, nuclear
export factors of the karyopherin class, such as Crm1 and Exportin t (Exp-t),
require Ran-GTP for cargo binding. Therefore, these factors bind their cargo
in the nucleus and then release it upon translocation to the cytoplasm, where
hydrolysis of the Ran bound GTP is induced by Ran-GAP
(Fig. 1). Because the large
majority of nuclear import and export is mediated by karyopherin family
members, interference with the Ran-GTP gradient across the nuclear membrane
inhibits many transport processes, including the nuclear export of most RNAs.
The major exception to this generalization is bulk mRNA export, which is not
dependent on Ran and is, in fact, mediated by nuclear export factors that are
not members of the karyopherin family of proteins
(Clouse et al., 2001;
Herold et al., 2001
).
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Several different types of RNA are exported from the nucleus, including
transcripts synthesized by RNA polymerase I (Pol I: large rRNAs), RNA
polymerase II [Pol II; mRNAs and some uridine-rich small nuclear RNAs (U
snRNAs)] and RNA polymerase III (Pol III; tRNAs and 5S rRNA). Competition
experiments in microinjected Xenopus oocytes have shown that these
RNAs largely use distinct pathways to exit the nucleus [e.g. an excess of
microinjected tRNA inhibits tRNA export but not U snRNA export
(Jarmolowski et al., 1994)].
Efforts to identify the critical protein components that define each of these
distinct nuclear RNA export pathways have used a number of distinct yet
complementary approaches, including microinjection assays in Xenopus
oocytes, genetic analysis in yeast cells, biochemical analyses of vertebrate
cell extracts and, more recently, RNA interference to inactivate specific
genes in metazoan cells. One system that has proven unexpectedly fruitful in
identifying human nuclear RNA export factors has been the analysis of the
nuclear mRNA export pathways accessed by different primate retroviruses
(Cullen, 1998
).
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Crm1-dependent nuclear RNA export |
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Crm1-dependent nuclear export of mRNAs
It is perhaps ironic that Crm1 was first identified as a factor required
for the nuclear export of late HIV-1 mRNAs because Crm1, although required for
the export of a wide variety of cellular protein and RNA substrates, actually
plays at most a very limited role in cellular mRNA export
(Fischer et al., 1995;
Fornerod et al., 1997
;
Bogerd et al., 1998
;
Neville and Rosbash, 1999
). In
addition to HIV-1, several other retroviruses encode adapter proteins that
recruit Crm1 to late viral mRNAs. These include the Rev proteins encoded by
all members of the lentivirus family, the distinct human T-cell leukemia virus
Rex protein (Bogerd et al.,
1998
) and the K-Rev protein encoded by a family of unrelated human
endogenous retroviruses termed the HERV-Ks
(Yang et al., 1999
). The
reason these retroviruses encode adapter proteins that recruit Crm1 to
incompletely spliced retroviral mRNAs is that eukaryotic cells have a
stringent proofreading mechanism to ensure that mRNAs that retain complete
introns (i.e. pre-mRNAs) do not leave the nucleus. This primarily reflects the
recognition of splice sites by a subset of splicing factors, termed commitment
factors, that can retain pre-mRNAs in the nucleus
(Chang and Sharp, 1989
;
Legrain and Rosbash, 1989
).
Although advantageous to the cell, this proofreading mechanism presents a
problem for retroviruses that, as an integral part of their life cycle, must
express both fully spliced and incompletely spliced variants of the same
initial transcript in the infected cell cytoplasm
(Cullen, 1998
). Retroviruses
therefore have developed mechanisms that overcome nuclear retention by
promoting the sequence-specific recruitment of cellular nuclear export factors
to these mRNAs.
Because Crm1 function can be specifically inhibited by the drug leptomycin
B (LMB), it is simple to demonstrate that nuclear export of the late mRNAs
encoded by HIV-1 depends on Crm1 function whereas nuclear export of bulk
cellular poly(A)+ RNA does not
(Fornerod et al., 1997).
Nevertheless, export of a small subset of cellular mRNAs might also rely on
Crm1. One line of evidence supporting this hypothesis comes from studies of
the AU-rich elements (AREs) found in the 3' untranslated regions of many
human genes involved in cell signaling
(Brennan et al., 2000
). AREs
bind to the protein HuR, which in turn interacts with two nucleocytoplasmic
shuttle proteins, pp32 and APRIL. Both pp32 and APRIL contain leucine-rich
NESs and interact with Crm1. Importantly, Brennan et al. reported that
inhibition of Crm1 function using LMB results in the selective nuclear
accumulation of mRNAs that contain AREs (e.g. fos mRNA) whereas the
subcellular distribution of bulk poly(A)+ RNA is unaltered
(Brennan et al., 2000
). These
data imply that a specific subset of cellular mRNAs are substrates for
Crm1-mediated nuclear mRNA export, at least under certain conditions.
A second line of evidence suggesting that Crm1 functionally interacts with
some cellular mRNAs comes from an analysis of members of the NXF family of
human nuclear export factors (Yang et al.,
2001). Bulk nuclear mRNA export in metazoan cells is mediated by a
member of the NXF protein family termed NXF1 or, more commonly, Tap
(Table 1, see below). Tap is
not a karyopherin, and Tap-mediated nuclear export does not require its
interaction with any karyopherin. Instead, Tap contains two domains that can
directly interact with components of the NPC, including one located at the
C-terminus. Both of these NPC-binding domains play a critical role in
mediating Tap-dependent nuclear mRNA export
(Kang and Cullen, 1999
;
Braun et al., 2002
).
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While Tap is expressed ubiquitously in all cells and tissues, two closely
related proteins, NXF2 and NXF3, display highly tissue-specific expression
patterns (Yang et al., 2001).
Analysis of its biological activity showed that NXF3, like Tap, is a
nucleocytoplasmic shuttle protein that can export nuclear mRNA efficiently
when tethered via a heterologous RNA-binding motif. However, NXF3 entirely
lacks the C-terminal NPC-binding domain that is critical for Tap function.
This conundrum was resolved by the demonstration that NXF3, unlike Tap,
contains a leucine-rich NES that directly interacts with Crm1
(Yang et al., 2001
).
NXF3-dependent, but not Tap-dependent, nuclear mRNA export is therefore
effectively blocked by LMB. NXF3 associates with poly(A)+ mRNA in
vivo and therefore probably functions as a tissue specific, Crm1-dependent
nuclear mRNA export factor. However, no mRNAs dependent on NXF3 for their
nuclear export have been identified thus far.
Crm1-dependent nuclear export of U snRNAs
The small nuclear ribonucleoprotein particles (snRNPs) are RNA-protein
complexes that play a critical role in the splicing of nuclear pre-mRNAs. In
higher eukaryotes, the U snRNA components of the snRNPs are synthesized in the
nucleus but, with the exception of U6, then assembled into mature snRNPs in
the cytoplasm and reimported into the nucleus. The snRNAs that undergo this
cytoplasmic assembly step (snRNAs U1, U2, U4 and U5) are synthesized by Pol II
and, like mRNA, acquire a 7-methylguanosine (m7G) cap
cotranscriptionally. This cap serves as the critical export signal for these U
snRNAs (Izaurralde et al.,
1995).
The m7G cap is bound by the nuclear cap-binding complex (CBC),
consisting of the proteins CBP20 and CBP80, and CBC binding is required for U
snRNA export (Izaurralde et al.,
1995). Because U snRNA export depends on Crm1 function, one or
both components of the CBC heterodimer might in theory serve as targets for
Crm1 binding. However, neither CBP20 or CBP80 contains a leucine-rich NES and
neither protein directly interacts with Crm1 in vitro. It was therefore clear
that the CBC:Crm1 interaction must involve at least one additional component.
The identity of this factor, termed PHAX for phosphorylated adapter for RNA
export, was recently resolved in a series of elegant experiments performed by
the Mattaj laboratory (Fig.
2).
Ohno et al. showed that the Ran-GTP-bound form of Crm1 and PHAX bind, in a
highly cooperative manner, to the CBC, which is in turn bound to the U snRNA
m7G cap (Ohno et al.,
2000). Importantly, PHAX binding requires phosphorylation at
as-yet-undefined residues. Once this U snRNA ribonucleoprotein complex
migrates through the NPC to the cytoplasm, it is disassembled following
hydrolysis of the Ran-GTP component and de-phosphorylation of PHAX. PHAX is
then reimported into the nucleus, where it is re-phosphorylated and can again
participate in U snRNA binding and export
(Ohno et al., 2000
). The
directionality of U snRNA export is therefore assured not only by the Ran
cycle (Fig. 1) but also by the
compartmentalization of the as-yet-unidentified nuclear kinase and cytoplasmic
phosphatase that act on PHAX.
Crm1-dependent nuclear export of rRNAs
Three rRNAs (28S, 18S and 5.8S) are transcribed in the nucleus by RNA
polymerase I as a single large pre-rRNA that is then extensively processed to
yield mature rRNAs. The fourth rRNA, 5S rRNA, is transcribed by RNA Pol III
and requires only modest post-transcriptional processing. In a complex and
highly ordered process, the 28S, 5.8S and 5S rRNA species are then assembled,
together with 50 ribosomal proteins, to form the 60S preribosomal subunit; the
18S rRNA is assembled with 33 ribosomal proteins to give the 40S preribosomal
subunit (reviewed by Venema and Tollervey,
1999). These subunits are then separately exported to the
cytoplasm.
Initial clues to the mechanism underlying nuclear export of the
preribosomal subunits came from genetic screens in S. cerevisiae,
which showed that export of both subunits depends on Ran and is therefore
likely to be mediated by one or more karyopherins
(Hurt et al., 1999;
Moy and Silver, 1999
). Genetic
screens also identified a protein called Nmd3p as critical for a late step in
the biosynthesis of 60S, but not 40S, preribosomal subunits
(Ho and Johnson, 1999
).
Although Nmd3p is not a ribosomal protein per se, it associates with 60S
preribosomal subunits in the nucleus through a direct interaction with the
large subunit ribosomal protein Rpl10p
(Gadal et al., 2001
). Nmd3p
shuttles between nucleus and cytoplasm in a Crm1-dependent manner and contains
two C-terminal NESs similar to that in HIV-1 Rev
(Ho et al., 2000
;
Gadal et al., 2001
).
Importantly, nuclear export of 60S preribosomal subunits can be inhibited not
only by the Crm1-specific inhibitor LMB but also by overexpression of Nmd3p
mutants lacking a functional NES. Nuclear export of the 60S preribosomal
subunit is therefore mediated by the Nmd3p-dependent recruitment of Crm1
(Fig. 2). Because assembly of
Nmd3p onto the 60S preribosome is a very late step in 60S preribosome
assembly, Crm1 is only recruited to 60S preribosomes that are ready for export
and assembled appropriately.
Nuclear export of the 40S preribosomal subunit is also Crm1 dependent
(Moy and Silver, 2002).
However, Nmd3p does not bind to 40S subunits and does not play a direct role
in mediating their export. Efforts to demonstrate a direct interaction between
40S preribosomal subunits and Crm1 have so far been unsuccessful. Recruitment
of Crm1 to nuclear 40S preribosomal subunits therefore probably depends on an
asyet-unidentified adapter that has an analogous role to Nmd3p
(Fig. 2).
Although 5S rRNA is normally assembled directly into large ribosomal
subunits in the cell nucleus and therefore does not reach the cytoplasm on its
own, an interesting exception exists in amphibian oocytes
(Nakielny et al., 1997). These
highly specialized cells express an oocyte-specific 5S rRNA that migrates to
the cytoplasm, where it is stored pending the onset of vitellogenesis, when it
is reimported into the nucleus and incorporated into 60S subunits. A major
cytoplasmic 5S rRNA storage particle, the 7S particle, consists of 5S rRNA and
the transcription factor TFIIIA, which also plays a critical role in the
transcription of 5S rRNA. Interestingly, amphibian TFIIIA contains a
functional leucine-rich NES that is apparently absent in mammalian TFIIIA
(Fridell et al., 1996
).
Nuclear export of 5S rRNA is specifically inhibited in Xenopus
oocytes upon saturation of the Crm1 export pathway by nuclear injection of
high levels of a leucine-rich NES peptide
(Fischer et al., 1995
). In
this differentiated cell type, Crm1 thus probably also mediates nuclear export
of 7S storage particles containing TFIIIA and 5S rRNA
(Fig. 2).
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Other karyopherin-dependent nuclear RNA export pathways |
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The first clue to the identity of the nuclear export factor for tRNAs was
the identification, in a yeast genetic screen, of Los1p, a protein that is
important for production of suppressor tRNAs
(Hopper et al., 1980). Los1p
is a member of the karyopherin family, and its human homolog, Exp-t, binds
directly and specifically to mature tRNA molecules, but only in the presence
of Ran-GTP (Arts et al., 1998a
;
Kutay et al., 1998
)
(Fig. 2). Although the ability
of Exp-t to bind to an RNA, rather than to a protein target, so far appears to
be unique among karyopherins, in common with other karyopherins, Exp-t can
bind to specific nucleoporins and shuttle between nucleus and cytoplasm.
A key prediction for the tRNA export factor is that it should be specific
only for mature tRNAs. In fact, Exp-t binds very poorly to tRNAs that bear
incorrect 5' or 3' ends or are inappropriately modified
(Arts et al., 1998b;
Lipowsky et al., 1999
).
Unexpectedly, Exp-t does bind to tRNAs that retain an intron and can export
these from injected oocyte nuclei. However, because intron removal normally
occurs prior to 5'- and 3'-end processing at physiological levels
of tRNA expression (Lund and Dahlberg,
1998
), this may not normally present a problem. Two groups have
also suggested that Exp-t selectively binds to and exports tRNAs that have
been aminoacylated in the nucleus (Lund
and Dahlberg, 1998
; Sarkar et
al., 1999
), although aminoacylation is clearly not an absolute
requirement for export (Arts et al.,
1998b
). Because aminoacylation would occur efficiently only with
mature tRNAs, this would, however, represent an effective proofreading step to
ensure that only functional tRNAs are exported from the nucleus.
Nuclear export of micro RNAs and other small non-coding RNAs
Eukaryotic cells, and particularly metazoan cells, encode a wide range of
other small non-coding RNAs, whose functions are frequently unknown. Several
of these are predominantly localized to the cytoplasm: the 100 nt long
human Y RNAs, which form part of the Ro ribonucleoprotein particle; the
160 nt long VA RNAs encoded by adenoviruses, which act as inhibitors of
host antiviral responses; and micro RNAs (miRNAs), a diverse class of small
non-coding RNAs that appear to play important roles in post-transcriptional
gene regulation (Peek et al.,
1993
; Liao et al.,
1998
; Ambros,
2001
). Y and VA RNAs are transcribed by Pol III, but the
polymerase(s) responsible for miRNA transcription is not yet known.
Although both Y and VA RNAs are small, highly structured molecules, they do
not share any significant sequence similarity. However, both feature a
terminal RNA helix that is necessary and sufficient for nuclear export
(Gwizdek et al., 2001;
Rutjes et al., 2001
). The
sequence of this `terminal minihelix' appears to be irrelevant, although a
double-stranded region of >10 bp, and its location at the end of the RNAs,
are essential. Export of the Y and VA RNAs depends on the integrity of the Ran
system, which implicates a karyopherin, but is not dependent on Crm1 or Exp-t
(Gwizdek et al., 2001
;
Rutjes et al., 2001
). The
mechanism underlying the nuclear export of these small, non-coding RNAs and
other small RNAs bearing a terminal helical region therefore remains largely
unclear.
All known miRNAs are single stranded and 22 nt in length when fully
mature but are initially transcribed as longer precursor RNAs (pri-miRNAs),
that can be several hundred nucleotides in length. Embedded within the
pri-miRNA is the structured miRNA precursor (pre-miRNA), which is an
70
nt RNA stem-loop structure containing the mature miRNA sequence as part of the
stem (Lee et al., 2002
). miRNA
synthesis involves the initial excision of the
70 nt pre-miRNA from the
longer pri-miRNA, which gives rise to a short RNA stem-loop bearing a terminal
RNA helix (Lee et al., 2002
).
This pre-miRNA is then exported to the cytoplasm, where it is further
processed, by the unusual dicer ribonuclease, to yield the mature
22 nt
miRNA. At present, nothing is known about the mechanism underlying the nuclear
export of the pre-miRNA precursor. However, the close structural similarity of
this small, structured RNA to other small RNAs that are actively exported
through terminal minihelix recognition strongly suggests that the pre-miRNA
utilizes the same nuclear export pathway as the Y and VA RNAs
(Fig. 2). Indeed, the integrity
of the pre-miRNA basal stem, but not the sequence, is essential for miRNA
expression in transfected cells (Zeng and
Cullen, 2003
).
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Nuclear export of mRNAs |
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Evidence implicating Mex67p as a potential nuclear mRNA export factor
initially came from the observation that yeast cells lacking functional Mex67p
rapidly accumulate poly(A)+ RNA in the nucleus
(Segref et al., 1997). Mex67p
was subsequently found to bind to a second essential export factor, Mtr2p.
This in turn induces binding of Mex67p-Mtr2p to nucleoporins and is required
for nucleocytoplasmic shuttling of the Mex67p-Mtr2p heterodimer and export of
poly(A)+ RNA (Santos-Rosa et
al., 1998
; Strässer et
al., 2000
).
Meanwhile, investigation of the nuclear export of the incompletely spliced
mRNA encoded by the retrovirus Mason Pfizer Monkey Virus (MPMV) had identified
Tap as a key nuclear mRNA export factor in metazoan cells. Like other
retroviruses, MPMV requires nuclear export of incompletely spliced mRNAs (see
above). Rather than encoding adapter proteins such as Rev that recruit Crm1,
however, several simple retroviruses, such as MPMV, instead encode a
cis-acting RNA sequence, the constitutive transport element (CTE), that is
sufficient to induce nuclear export in the absence of any viral gene product
(Bray et al., 1994). Evidence
suggesting that the cellular cofactor that binds to the CTE also functions in
cellular mRNA export came from microinjection experiments in Xenopus
oocytes, which showed that an excess of CTE RNA blocks cellular mRNA export
but does not affect Crm1-dependent RNA export or tRNA export
(Pasquinelli et al., 1997
;
Saavedra et al., 1997
).
Grüter et al. subsequently identified the cellular cofactor for the MPMV
CTE as Tap, the human ortholog of Mex67p, using a biochemical approach
(Grüter et al.,
1998
).
The binding partner for Tap is Nxt
(Katahira et al., 1999).
Although Nxt does not show any obvious sequence similarity to Mtr2p, it is
nevertheless functionally closely analogous. Thus, nucleoporin binding by Tap
is greatly enhanced by formation of a Tap-Nxt heterodimer, as is
nucleocytoplasmic shuttling (Fribourg et
al., 2001
; Lévesque et
al., 2001
; Wiegand et al.,
2002
). Indeed, Tap mutants that cannot form Tap-Nxt heterodimers
do not support nuclear mRNA export. Finally, although human Tap and Nxt can at
least partially rescue nuclear mRNA export in Mex67p- and/or Mtr2p-deficient
yeast cells, both human proteins are required
(Katahira et al., 1999
). This
strongly suggests that Tap and Nxt are the true orthologs of Mex67p and Mtr2p,
respectively (Table 1).
Tap and Mex67p proteins share a similar domain organization: an N-terminal
leucine-rich region (LRR), a central domain that shows sequence similarity to
the nuclear import receptor NTF2 and finally a C-terminal domain that displays
a UBA (ubiquitin associated) fold. The central domain mediates the interaction
with Nxt and Mtr2p. The UBA domain is critical for interaction with
nucleoporins and hence Tap-mediated nucleocytoplasmic transport
(Kang and Cullen, 1999;
Katahira et al., 1999
;
Bachi et al., 2000
).
Interestingly, recent data suggest that the nucleoporin-binding functions of
the central Nxt-binding domain and the UBA domain are functionally equivalent,
that is, Tap variants containing two central domains but no UBA domain are
partially active in mediating nuclear mRNA export, and vice versa
(Braun et al., 2002
). In
contrast, mutants of Tap containing only one copy of either domain are
inactive. The N-terminal LRR domain is required for CTE RNA binding by Tap and
is likely also to be critical for the sequence-non-specific recruitment of Tap
to cellular mRNAs (Kang and Cullen,
1999
; Liker et al.,
2000
). Indeed, the LRR domain becomes dispensable for Tap-mediated
nuclear RNA export when Tap is tethered to target RNAs via a heterologous
RNA-binding domain. In contrast, both the central NTF-2-like domain and the
C-terminal UBA-like domain remain essential in this fusion protein context
(Wiegand et al., 2002
).
RNA interference has now revealed that, like Mex67p and Mtr2p, Tap and Nxt
are essential for poly(A)+ RNA nuclear export
(Tan et al., 2000;
Herold et al., 2001
;
Wiegand et al., 2002
). The
Tap-Nxt heterodimer thus plays a critical, evolutionarily conserved role in
mediating the nuclear export of cellular mRNAs in a wide range of eukaryotic
organisms (Fig. 3).
How Tap-Nxt is recruited to mature nuclear mRNAs remains uncertain. Unlike
non-coding RNAs, mRNAs do not share any obvious RNA sequence or structure
except for the 5' m7G cap and the 3' poly(A) tail.
However, neither of these two post-transcriptional modifications is critical
for mRNA export in injected oocytes (although the process of 3' end
formation is important, see below)
(Jarmolowski et al., 1994).
Recruitment of the Tap-Nxt mRNA exporter is therefore likely to occur during
the synthesis and/or the post-transcriptional processing of mRNAs
(Fig. 3).
Coupling of mRNA splicing with nuclear export
The large majority of genes encoded by humans and other metazoans contain
introns. In general, mRNAs containing introns give rise to a higher level of
the encoded protein than do the equivalent intronless, cDNA-derived mRNAs. The
mechanisms underlying this difference are complex because splicing can
strongly influence not only the efficiency of mRNA 3' end formation but
also mRNA stability and even translation
(Ryu and Mertz, 1989;
Niwa et al., 1990
;
Matsumoto et al., 1998
).
Nevertheless, for most human genes, splicing is not essential for detectable
mRNA and protein synthesis. In fact, a recent survey of 15 genes showed that
the presence of an excisable intron enhanced gene expression in human cells by
an average of 6.3±4.7 fold with a range of from 1.5- to over 20-fold
(S. Lu and B. R. Cullen, unpublished).
The first evidence suggesting that splicing also enhances the efficiency of
mRNA export came from the demonstration that several intron-containing mRNAs
are exported more efficiently from microinjected Xenopus oocyte
nuclei than the same RNAs injected in an intronless, cDNA form
(Luo and Reed, 1999). Luo and
Reed also compared the export of an RNA that had been subjected to splicing in
a nuclear extract with those of a cDNA version of the same RNA that had simply
been incubated in nuclear extract. Remarkably, ribonucleoprotein (RNP)
complexes formed on these in principle identical RNA molecules displayed
distinct mobilities on non-denaturing gels, the spliced RNP complex migrating
more slowly. In addition, the RNA contained within the spliced RNP complex was
exported more efficiently from injected oocyte nuclei than the RNA contained
within the RNP that lacked splicing factors. Intron removal thus appeared to
lead to the deposition of specific proteins that targeted the spliced mRNA for
efficient nuclear export.
That splicing somehow tags an mRNA was confirmed by the discovery of the
exon junction complex (EJC), a protein complex deposited 20-24 nucleotides
5' of the site of intron removal
(Kataoka et al., 2000;
Le Hir et al., 2000
). Although
the composition of the EJC remains to be fully determined, it minimally
consists of the proteins SRm160, RNPS1, Y14, Magoh and a protein termed Aly or
Ref. SRm160 appears to facilitate appropriate mRNA 3' end formation
(McCracken et al., 2002
)
whereas RNPS1 and Y14 appear to be involved in the regulation of
nonsense-mediated decay (reviewed by Wagner and Lykke-Anderson, 2002). Aly has
been proposed to facilitate nuclear mRNA export by directly interacting with
Tap (Fig. 3).
The first evidence that Tap and Aly interact came from studies in yeast.
Strässer and Hurt, and Stutz et al. demonstrated a direct interaction
between Yralp, the yeast ortholog of metazoan Aly
(Table 1), and Mex67p in vitro
and also showed that yeast Yralp mutants are impaired in nuclear
poly(A)+ RNA export
(Strässer and Hurt, 2000;
Stutz et al., 2000
). Yralp was
also shown to bind to mRNA molecules both in vitro and in vivo
(Strässer and Hurt, 2000
;
Lei and Silver, 2002
). Zhou et
al. subsequently demonstrated that human Aly is a nucleocytoplasmic shuttle
protein that is selectively recruited during in vitro mRNA splicing
(Zhou et al., 2000
). Most
importantly, recombinant Aly was found to selectively enhance nuclear mRNA
export in microinjected oocytes and to specifically interact with recombinant
human Tap in vitro (Zhou et al.,
2000
; Rodrigues et al.,
2001
). Finally, Le Hir et al. showed that the EJC provides a
binding site for the Tap-Nxt heterodimer, thus providing a potential
explanation for how splicing could enhance nuclear mRNA export
(Le Hir et al., 2001
).
An interesting question that had remained unresolved was how splicing leads
to the deposition of the EJC and, more specifically, how Aly is recruited to
mRNAs during splicing. Again, yeast genetics provided the initial
breakthrough. Strässer and Hurt demonstrated a genetic interaction
between Yralp, the yeast Aly homolog, and Sub2p
(Strässer and Hurt,
2001), a member of the DEAD-box family of RNA helicases that had
previously been shown to play an important role in spliceosome assembly
(Fleckner et al., 1997
;
Libri et al., 2001
). Sub2p and
Yralp directly interact both in vitro and in vivo, and loss of Sub2p function
blocks poly(A)+ RNA nuclear export in yeast cells. In metazoan
cells, Aly specifically interacts with UAP56, the human homolog of Sub2p, and
recruitment of Aly to spliced mRNAs depends on this interaction
(Luo et al., 2001
)
(Fig. 3). Gatfield et al.
subsequently showed, using RNA interference, that UAP56 is also essential for
nuclear mRNA export in Drosophila
(Gatfield et al., 2001
). UAP56
associates with mRNAs in the nucleus and accompanies the resultant mRNP
complexes to the NPC (Kiesler et al.,
2002
).
On the basis of these data, one can envision a simple hypothesis for how Tap-Nxt is recruited to mRNAs in a splicing-dependent manner in metazoan cells (Fig. 3). During splicing, UAP56 facilitates spliceosome assembly and then recruits Aly to the resultant EJC. Aly in turn recruits the Tap-Nxt heterodimer, which then targets the spliced mRNP complex to the NPC and, hence, to the cytoplasm. Unfortunately, considerable recent evidence suggests that this attractive hypothesis is at best an oversimplification.
One problem with the above proposal is that it is not clear that splicing
is essential for nuclear mRNA export. Although Luo and Reed reported that
splicing can markedly enhance mRNA export in microinjected oocytes
(Luo and Reed, 1999), a
difficulty with this experimental approach is that only the nuclear export of
preformed RNAs was analyzed. Therefore, if the effect of splicing on mRNA
export factor recruitment is largely or entirely redundant with the
recruitment of the same export factors during transcription and/or mRNA
3' end formation (Fig.
3), this assay will give the misleading result that splicing is
uniquely critical for nuclear mRNA export. More importantly, there is no
agreement that splicing is indeed essential for nuclear export of mRNA
molecules, even in Xenopus oocytes. Rodrigues et al. reported that
splicing does not enhance the nuclear export of mRNAs derived from the
Ftz or ß-globin genes, although splicing does enhance
the nuclear export of a very short RNA derived from the adenovirus major late
region (Rodrigues et al.,
2001
). Ohno et al. have also presented evidence against a critical
role for splicing in mRNA nuclear export in Xenopus oocytes
(Ohno et al., 2002
).
In transfected human cells, splicing can result in a significant
enhancement in the expression of the majority of genes (see above). However,
the absence of introns reduces mRNA levels equivalently in both the nucleus
and cytoplasm; a selective defect in nuclear mRNA export should affect
cytoplasmic levels more significantly. Also, the poor expression of intronless
mRNAs is not significantly enhanced by insertion in cis of the MPMV CTE, a
high affinity-binding site for Tap. Splicing thus does not appear to have a
major effect on the nuclear export efficiency of mRNAs transcribed in metazoan
cells (S. Lu and B. R. Cullen, unpublished; A. Nott, S. H. Meislin and M. J.
Moore, personal communication). A final argument against a critical role for
the EJC in mediating nuclear mRNA export comes from recent RNA interference
experiments (Gatfield and Izaurralde,
2002) showing that known components of the EJC, including SRm160,
Y14, RNPS1 and even Aly, are not required for bulk export of nuclear
poly(A)+ RNA in Drosophila. In contrast, Tap, Nxt and
UAP56 are all clearly essential. These data therefore again suggest that
splicing, and the EJC, are not critical for nuclear mRNA export and further
imply that the role of metazoan Aly in mediating the recruitment of Tap-Nxt to
mature mRNAs must, at minimum, be redundant
(Fig. 3).
A second problem with the hypothesis that mRNA nuclear export depends on
splicing is that recruitment of UAP56, and probably also of Aly and Tap, to
mRNA transcripts appears to occur independently of splicing in vivo. Sub2p and
Yralp are essential for nuclear export of the intronless mRNAs encoded by most
yeast genes (Strässer and Hurt,
2000; Strässer and Hurt,
2001
; Jensen et al.,
2001a
), and Yralp binding to mRNAs can clearly occur in the
absence of splicing (Lei and Silver,
2002
). In metazoan cells, recruitment of UAP56 to mRNAs has also
been shown to occur independently of splicing
(Kiesler et al., 2002
), and
elimination of either UAP56 or Tap expression by RNA interference again blocks
the nuclear export of not only spliced but also intronless mRNAs
(Gatfield et al., 2001
;
Herold et al., 2001
). Finally,
in Xenopus oocytes, microinjection of antibodies against Aly inhibits
the nuclear export of both spliced and intronless mRNAs
(Rodrigues et al., 2001
). In
total, the above experiments therefore strongly argue that splicing is not a
prerequisite for the recruitment of nuclear mRNA export factors.
If splicing does not direct mRNAs into the export pathway, then what does?
Surprisingly, a recent paper suggests that an unstructured region of
sufficient length can act as a dominant determinant of mRNA identity. Ohno et
al. inserted an intron into U1 snRNA and found that the U1 snRNA was spliced
and then exported exclusively through the Tap-dependent mRNA export pathway,
rather than the Crm1 pathway (Ohno et al.,
2002). This result is obviously consistent with the proposal that
splicing facilitates Tap recruitment. Surprisingly, however, insertion of
random exonic sequences also targeted U1 snRNA into the mRNA export pathway,
regardless of whether these sequences were inserted in a sense or antisense
orientation. Unstructured RNA, inserted into the otherwise highly structured
U1 snRNA, therefore appears to be sufficient to induce the recruitment of
Tap-Nxt (Fig. 3). These data
suggest that the mRNA nuclear export pathway could represent the default
nuclear export pathway followed by unstructured RNA molecules that are either
not recognized by other RNA export factors and/or not actively retained in the
nucleus by proofreading proteins, such as splicing commitment factors.
Coupling of transcription with mRNA nuclear export
Pre-mRNA processing is coupled to transcription, the C-terminal domain
(CTD) of Pol II providing a platform for recruitment of RNA-processing factors
during transcription (reviewed by
Neugebauer, 2002).
Transcription of mRNAs facilitates their capping, splicing and polyadenylation
and might also directly promote the recruitment of nuclear export factors. The
first evidence for this hypothesis came from chromatin immunoprecipitation
assays, which showed that yeast proteins involved in mRNA export are recruited
to mRNAs co-transcriptionally (Lei et al.,
2001
). More recently, Strässer et al. have shown that Yra1p
and Sub2p are stoichiometrically associated with a yeast protein complex, THO,
that plays a key role in transcription elongation
(Strässer et al., 2002
).
Similarly, human Aly and UAP56 associate with the human THO complex.
Strässer et al. proposed that this complex of mRNA export and
transcription factors, which they termed the transcription/export (TREX)
complex, plays an important role in mediating the co-transcriptional
recruitment of nuclear export factors such as UAP56 and Aly, and hence
presumably Tap-Nxt, to mRNAs
(Strässer et al., 2002
)
(Fig. 3).
Although the hypothesis that export factor recruitment occurs
co-transcriptionally is attractive, it leaves unresolved the issue of how
pre-mRNAs avoid being exported to the cytoplasm. For example, the incompletely
spliced mRNAs encoded by the retrovirus MPMV depend on the cis-acting CTE RNA
for nuclear export, and the CTE acts as a high-affinity binding site for
Tap-Nxt (Grüter et al.,
1998) (Fig. 3). If
the Tap-Nxt heterodimer is indeed recruited to mRNAs, including MPMV RNAs,
co-transcriptionally, then it is unclear why the nuclear export of this mRNA
would be dependent on the subsequent recruitment of the same Tap-Nxt
heterodimer by the CTE.
Coupling of 3' end formation with mRNA nuclear export
Microinjection assays in Xenopus oocytes indicate that insertion
of a stretch of adenine residues at the 3' end of an mRNA does not
significantly enhance its export
(Jarmolowski et al., 1994).
Conversely, experiments in transfected human cells have demonstrated that mRNA
molecules bearing 3' ends formed by ribozyme cleavage are not
efficiently exported to the cytoplasm
(Eckner et al., 1991
;
Huang and Carmichael, 1996
).
Together, these data suggest that it is the process of mRNA 3'
end-formation, rather than the poly(A) stretch itself, that facilitates
nuclear mRNA export (Fig. 3).
Moreover, in yeast, mutational inactivation of nuclear export factors such as
Mex67p or Mtr2p results in the accumulation of transcripts at the site of
transcription and in their hyperadenylation
(Hilleren and Parker, 2001
;
Jensen et al., 2001b
).
Conversely, several yeast mutants that express defective forms of mRNA
3' processing factors, including for example the poly(A) polymerase,
exhibit defective mRNA export (Hammell et
al., 2002
). Recently, Lei and Silver have demonstrated that
recruitment of Yra1p to yeast mRNAs is dependent on appropriate 3' end
formation, even if the mRNA contains an intron
(Lei and Silver, 2002
).
Although these data clearly indicate some form of tight mechanistic coupling
between mRNA 3' end formation and nuclear export, its molecular basis
remains unresolved.
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Acknowledgments |
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References |
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