Original Paper |
|
|||
|
Acta Biochim Biophys |
||||
|
doi:10.1111/j.1745-7270.2008.00472.x |
Expression and characterization of rice putative PAUSED gene
Chengguo Yao1,3#, Liangfa Ge2,3#, Wei Li1,3, Botao Zhao1,3, Chaoqun Li4, Kangcheng Ruan1,3*, Hongxuan Lin2,3, and Youxin Jin1,3*
1 State Key Laboratory of Molecular Biology,
Institute of Biochemistry and Cell Biology, Shanghai 200031, China
2 State Key Laboratory of Plant Molecular
Genetics, Institute of Plant Physiology and Ecology, Shanghai 200032, China
3 Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai 200031, China
4 Department of Infectious Diseases, Ruijin
Hospital, Shanghai Second Medical University, Shanghai 200025, China
Received: May 08,
2008
Accepted: August
31, 2008
This work was
supported by grants from the National Natural Sciences Foundation of China (No.
30430210), the National Key Basic Research and Development Program (No.
2005CB724602) and the Chinese Academy of Sciences (KSCX1-YW-R-64,
KSCX2-YW-R-096)#These authors contributed
equally to this work
*Corresponding
author:
Youxin Jin: Tel,
86-21-54921222; Fax, 86-21-54921011; Email, [email protected]
Kangcheng Ruan:
Fax, 86-21-54921011; Email, [email protected]
In Arabidopsis, PAUSED (PSD)
encodes the ortholog of los1p/exportin-t, which mediates the nuclear export of
transfer RNA (tRNA) in yeast and mammals. However, in monocot plants such as
rice, knowledge of the corresponding ortholog is limited, and its effects on
growth development and productivity remain unknown. In this study, we verified
a rice transfer-DNA insertional mutant psd line and analyzed its
phenotypes; the mutant displayed severe morphological defects including
retarded development and low fertility compared with wild-type rice. Examining
intronless tRNA-Tyr and intron-containing pre-tRNA-Ala
expression levels in cytoplasmic and nuclear fraction with Northern blot
analysis between wild-type and mutant leaf tissue suggested that rice PSD might
be involved in tRNA export from the nucleus to the cytoplasm. Additionally,
reverse transcription-polymerase chain reaction analysis revealed that PSD transcript
was expressed throughout normal rice plant development, and subcellular
localization assays showed that rice PSD protein was present in both the
nucleus and cytoplasm. In summary, our data implied that the putative PSD
gene might be indispensable for normal rice development and its function might
be the same as that of Arabidopsis PSD.
Keywords PAUSED; tRNA export; development defects; rice
The transport of different RNA species from the nucleus to the
cytoplasm is fundamental to gene expression. A general paradigm has been
established in which RNA are exported through the nuclear pore complexes via
mobile export receptors after being produced in the nucleus. Small RNA, such as
transfer RNA (tRNA) and microRNA, follow a relatively simple pathway by
directly binding to export receptors, while large RNA, such as rRNA and mRNA,
are assembled into complicated ribonucleoprotein with exporters or other
specific adaptor proteins [1,2].
As a member of the karyopherin superfamily, exportin-t is the
principal tRNA exporter in vertebrate cells that binds to tRNA in a typical
Ran-GTPase-dependent manner. Properly processed 5¢ and 3¢ termini,
secondary and tertiary structural elements of tRNA, are decoded by exportin-t,
meaning that tRNA quality control is performed before export [3–5]. In plants, Arabidopsis
PSD is the ortholog of exportin-t and may function as tRNA exporter [6];
the psd mutant displays a series of growth and development defects,
including delayed leaf production and abnormal inflorescence morphology. Rice
is a model monocot plant that differs from Arabidopsis, which is representative
of dicot plants. The expression pattern of PSD homolog and its role in
normal rice plant development remain unknown. In this study, we employed a rice
transfer-DNA (T-DNA) insertional mutant to explore rice PSD to gain a
more comprehensive understanding of plant PSD gene annotation and to
verify the diversity and complexity of pathways for plant tRNA export [7].
Materials and Methods
Plant culture
Wild-type rice (Oryza sativa L. subsp. Japonica cv. Dongjin) and
T2 generation T-DNA insertional line (No. 1B-01410) seeds were obtained from
Plant Functional Genomics Laboratory (Kyoungbuk, Korea) [8–11]. The seeds
were germinated first and then transferred to farmland belonging to the
Shanghai Academy of Agricultural Sciences (Shanghai, China) in order to adapt
to natural growth conditions.
Sequence annotation and phylogenetic analysis
PSD gene sequences were downloaded from
the NCBI gene database. Exons were indicated with Vector NTI (Invitrogen,
Carlsbad, USA). Protein sequences showing obvious identity were downloaded from
the rice PSD BLAST result in the NCBI protein database (http://www.ncbi.nlm.nih.gov/blast).
Sequences were aligned using Clustalx1.83 (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/)
and further adjusted manually in BioEdit (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html)
[12,13]. Phylogenetic analysis was done using the maximum likelihood method by
tree-puzzle-5.2 (http://www.tree-puzzle.de/) [14].
Polymerase chain reaction (PCR) verification of the T-DNA
insertional mutant
Leaf genomic DNA was purified using traditional CTAB method [15].
Two common PCR were performed for the differentiation of wild-type,
heterozygote and homozygote plants. The primers were designed using the NCBI
database and web service (http://signal.salk.edu/cgi-bin/RiceGE).
Primers for rice genome (PCR product: 409 bp) are 5¢-TGCAGCTTATTTTCATTCA-3¢ (forward) and 5¢-GGCATTTGCTGAAGATTTA-3¢ (reverse);
primers for the T-DNA genome (PCR product: 543 bp) are 5¢-TCAGCCATTGGATCATAGT-3¢ (forward) and 5¢-CCTGTAAGATTTAGCACCC-3¢ (reverse). In
brief, the PCR was conducted for 30 cycles in a thermal controller (PTC-100;
Bio-Rad, Hercules, USA). Each amplification cycle consisted of 0.5 min at 94 ºC
for denaturizing, 0.5 min at 55 ºC for primer annealing and 1 min at 72 ºC for
extension.
Reverse transcription (RT)-PCR assay
Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad,
USA). Initially, 2 mg total RNA was reverse transcribed with gene-specific downstream
primer by Moloney murine leukemia virus (M-MLV) reverse transcriptase
(Promega, Madison, USA) in a total volume of 25 ml, and cDNA was generated
at 37 ºC for 30 min. Expression level was determined using Taq PCR MasterMix
system (Tiangen, Beijing, China) in a total volume of 25 ml reaction
mixture containing 12.5 ml 2´master mix, 0.5 ml (20 mM) sense primer,
0.5 ml (20 mM) antisense primer, 2 ml cDNA template and 9.5 ml distilled
water. The PCR was then performed for 28 amplification cycles in the thermal
controller (PTC-100; Bio-Rad). Each cycle consisted of 0.5 min at 94 ºC for
denaturizing, 0.5 min at 56 ºC for primer annealing and 1 min at 72 ºC for
extension. Primers were used as follows: Actin (PCR product: 336 bp): 5¢-TCCATCTTGGCATCTCTCAG-3¢ (forward) and 5¢-GTACCCTCATCAGGCATCTG-3¢ (reverse); PSD
(PCR product: 126 bp): 5¢–cgacagttgctcgttgat-3¢ (forward) and 5¢-TGCGATGGCAGGAAACAC-3¢ (reverse).
Subcellular localization assay
The full coding sequence used for transient expression of PSD
in onion epidermal cells was amplified from full length cDNA clone AK099895
using the forward primer 5¢-GGATCCGACGACCTCGAGCAGGCCAT-3¢ and reverse primer 5¢-GAGCTCCTATCTGAAGACAAGACTCC-3¢. The PCR
product was subcloned into the BamHI/SacI site of pA7-green
fluorescent protein (GFP) expression vector under control of the
enhanced cauliflower mosaic virus 35S promoter. The recombinant pA7-GFP–PSD
plasmid and pA7-GFP plasmid (used as control) were separately
transferred by bombardment into onion epidermal cells using a gene gun
(PDS-1000/He; Bio-Rad) according to the instruction manual. Transformed cells
were examined using a confocal microscope (Olympus FV500, Tokyo, Japan) after
incubation at 25 ºC for 24 h on Murashige and Skoog medium.
Cellular fractionation
Cellular fractionation was performed as described [6]. Briefly, leaf
tissue from 4-week-old seedings were frozen in liquid nitrogen and ground to a
powder with a mortar and pestle. Cell wall-disrupting buffer [10 mM potassium
phosphate (pH 7.0), 100 mM NaCl, 10 mM 2-mercaptoethanol, 1 M hexylene glycol]
was then added to make a thick slurry. This mixture was filtered through
Miracloth (Calbiochem, San Diego, USA) to remove large chunks of tissue and
centrifuged at 1500 g for 10 min at 4 ºC to
pellet nuclei and cell debris. After centrifugation, the
supernatant was collected and recentrifuged at 13,000 g for 15 min at 4
ºC. The supernatant of this second centrifugation was saved
for the cytoplasmic fraction. The first pellet was washed
with nuclei preparation buffer [10 mM potassium phosphate
(pH 7.0), 100 mM NaCl, 10 mM 2-mercaptoethanol, 1 M hexylene glycol, 10 mM MgCl2, 0.5% Triton X-100] and
centrifuged at 1500 g for 10 min at 4 ºC. After centrifugation, the
supernatant
was discarded and the pellet was washed with nuclei
preparation buffer. Washing and centrifugation were repeated
four to five times, and the final pellet was saved for the nuclear
fraction. RNA was extracted from the cytoplasmic and nuclear fractions with
TRIzol reagent [16].
Northern blot analysis for tRNA
A total of 10 mg cytoplasmic and 2 mg nuclear RNA were subjected to electrophoresis on an 8 M urea/12%
denaturing polyacrylamide gel, transferred to Hybond N+ membranes
(Amersham Pharmacia, Uppsala, Sweden) and then hybridized with [g–32P]-labeled probes. Oligonucleotide probes were labeled with T4
polynucleotide kinase (MBI Fermentas, St Leon-Rot, Germany). Hybridization and
washing were carried out at 40 ºC in the ExpressHyb Hybridization Solution
(Toyobo, Tokyo, Japan) according to the manufacturer’s instructions. The
membrane was air-dried and then measured by phosphorimager (Amersham
Pharmacia). 5S rRNA served as the loading control for the quantitative
calculation, and U6 small nuclear RNA was used as marker of nuclear RNA.
Probe sequences were as follows: 5S rRNA: 5¢-AGGACTTCCCAGGAGGTCACCC-3¢; U6 RNA:
5¢-TCGATTTGTGGGTGTCAT-3¢; tRNA-tyr:
5¢-ACCTGCCGGATTCGAACCAGCG-3¢; pre-tRNA-Ala:
5¢-CAAGAATGGGATTCGAACCCA-3¢.
Results
Genomic organization of rice putative PSD
Los1p/exportin-t proteins form a large family among many species,
such as yeast, animals and plants, containing xpo1 domain in the primary
structure [17,18]. By searching the rice genome database, we found that only
one homolog shares 51% sequence identity with Arabidopsis PSD according
to sequence alignment in NCBI. The homolog’s existence was previously validated
by rice full-length complementary DNA project (GenBank accession No. AK099895; http://cdna01.dna.affrc.go.jp/cDNA/).
Rice psd is located in
chr07 (25238070–25246227). The AUG translation start site and UAG stop codon are
respectively located at 151 and 3091 bp of the AK099895 clone. As shown in Fig.
1(A), the rice PSD protein-encoding region is comprised of 13 exons, which
span 7066 bp genomic sequences. Interestingly, the Arabidopsis PSD
encoding region is also comprised of 13 exons, and their distribution pattern
in the genome is almost the same, suggesting the mechanism of PSD gene
transcription and mRNA splicing among plants are much alike.
PSD homologs are conserved in evolution
Similar to other orthologs, the N-terminal of rice PSD contains xpo1
domain. The N-terminal is reportedly involved in Ran-GTPase binding and
interaction with leucine-rich nuclear export signal sequences, whereas the
C-terminal has been assumed to be a tRNA interaction region that contributes
to tRNA export [Fig. 1(B)] [19].
To better understand the position of rice PSD in the
los1p/exportin-t gene family, we searched for proteins with an identity
obviously similar to rice PSD in the NCBI protein database, and we then
sketched a phylogenetic tree. As expected, rice PSD is most identical to Arabidopsis
PSD and the degree of similarity depends on the distance between the
species [Fig. 1(C)]. Together with other homologs from human, mice and
so on, they form a clade with high bootstrap support. Additionally, proteins
from fungi form another independent clade. Reasonably, all these proteins come
from eukaryotes, indicating that the origin of these homologs possibly occurred
after the segregation of eukaryote and prokaryote.
Rice PSD was expressed throughout the development in normal
plant
In order to study the general spatio-temporal expression pattern of PSD
in normal rice plants, we subsequently examined the expression of PSD
in various tissues from different phases of wild-type rice by RT-PCR. As
expected, PSD was expressed throughout the development process [Fig.
2(A)], which is consistent with the fundamental nature of its possible
role in tRNA export.
Subcellular localization of the rice PSD protein in onion epidermal
cells
Transient expression of GFP gene fusion in onion epidermis
is a credible method for the investigating subcellular localization of GFP
protein in plant cells [20]. We used this method to study the subcellular
localization of rice PSD, and results showed that GFP and GFP-PSD were both
distributed throughout the cells [Fig. 2(B)], implying that PSD shuttle
between nucleus and cytoplasm to perform its function.
Knockout of rice putative PSD transcript by T-DNA insertion
caused severe development defects
To further investigate rice PSD function, we obtained T2
generation T-DNA insertional mutant line (1B-01410) seeds from Plant Functional
Genomics Laboratory to insert within the gene [Fig. 3(A)] [10,11].
Thirty-one homozygote plants were chosen with common PCR reaction. The
wild-type and heterozygote plants showed a sharp band (409 bp) with genomic
forward and reverse primers designed around the insertion site, while the
homozygote plant exhibited a negative result. Another pair of T-DNA primers
was used to differentiate heterozygote from wild-type plants; 19 heterozygote
plants showed an obvious PCR product of 543 bp while none of wild type did [Fig.
3(B)]. To confirm the knockout of PSD transcript in homozygote, we
examined the PSD expression in leaf tissue with RT-PCR, and results revealed
that T-DNA insertion completely disrupted the PSD expression [Fig.
3(C)].
Nearly all the homozygote plants displayed development retardation,
such as delayed panicle heading and reduced size in the first several development
stages, compared with the wild type [Fig. 3(D), upper]. These results
are not so obvious for the Arabidopsis psd mutant possibly
because of the different mutant pattern or the deviation of plant species. When
the plants grew mature, their height and appearance were almost indiscernible [Fig.
3(D), bottom]. However, for plants losing PSD function, the
seed-setting rate decreased significantly [Fig. 3(E,F)], as was nearly
the case for Arabidopsis psd mutant [21]. Taken together, loss of
function of PSD severely impaired the normal rice development process, and the
most notable sharing phenotype with Arabidopsis psd mutant
implies that the functions of the homologs are coupled.
Rice PSD might regulate the tRNA export in vivo
To examine the role of rice PSD in tRNA export, we examined the
distribution of intronless tRNA-Tyr and intron-containing pre-tRNA-Ala
(GGC) in the nuclear and cytoplasmic fractions of leaf tissue from
wild-type and psd double mutant with Northern blot analysis. As anticipated,
the mutant slightly decreased the level of both tRNA in the cytoplasm and
increased their levels in the nucleus. We were particularly interested in the
mutaton’s effect on pre-tRNA-Ala because accumulation of unspliced tRNA
can be used to diagnose defects in tRNA export in yeast. Moreover, as the Arabidopsis
psd mutant increased the level of unspliced tRNA-Tyr [22], the much
higher level of pre-tRNA-Ala in the nucleus of rice psd should
provide evidence of its regulatory role in tRNA export in vivo (Fig.
4).
Discussion
PSD is a member of the importin b family of nucleocytoplasmic
transport receptors. It is hard to predict the role for this superfamily. For
example, exportin 5, the mammalian ortholog of HASTY (HST) and another member
of importin b family, exports pre-microRNA, tRNA, a viral hairpin RNA and
proteins associated with these and other double-stranded RNA, while HST’s yeast
ortholog, Msn5p, exports several different types of phosphorylated proteins and
imports replication protein A [23]. Though rice PSD shares 51% sequence
identity with Arabidopsis PSD, the experimental validation is much more
persuasive and necessary to support the idea that they share the same
function. The ever-growing populations of T-DNA insertions in rice represent
such a powerful tool for study of gene function. In this study, we verified a
rice T-DNA insertional mutant psd and substantiated the function
assignment of rice PSD. The phenotypes of rice psd mutant were not
investigated in detail and other useful clues may have been missed. These
phenotypes are possibly caused by the loss of function of rice PSD. An
efficient way to confirm this point would be to construct the over-expressed
RNA interference vector to perform the complementation test, and then observe
the phenotypes of the transgenic plants. Considering that rice PSD is the
homolog of Arabidopsis PSD that provided our biochemical data about the
mutant, we assumed that these phenotypes likely originated from the impairment
of tRNA export and the resultant translation limitation, which might elucidate
rice PSD function to some extent.
In Arabidopsis, PSD transcript was observed in a
series of tissues such as roots, vegetative leaves, floral buds, shoot apex
and so on. Its omnipresence was also concluded in rice thus we did not
quantitatively measure the expression level. Similar omnipresence was also
concluded in Arabidopsis. RT-PCR application merely amplified part of
the PSD transcript, so we could not exclude the possibility that PSD
was differentially spliced in some tissue regions that we assayed. We also
could not rule out the transcriptional regulation of PSD at a tissue- or
cell-specific level. Further study of PSD regulation should increase our
understanding of its role in tRNA export pathways and its function in rice
development.
Inhibition of tRNA nuclear exporter in yeast and Arabidopsis
caused the accumulation of intron-containing pre-tRNA, leading to the
prediction that pre-tRNA splicing could occur in the cytoplasm [22], and the
case is the same for rice. Based on the expression and characterization of
rice PSD, we proposed a simple hypothetical model for the involvement of PSD in
the process of tRNA nuclear export. Intronless and intron-containing pre-tRNA
were transcribed, processed, edited in the nucleus and then exported to the
cytoplasm in the presence of PSD and other associated factors through the
nuclear pore complex. In the cytoplasm, the tRNA cargo was released for later
splicing (for pre-tRNA), translation or aminoacylation.
If rice PSD indeed functions as tRNA exporter in vivo, we
speculated that it might not be the only tRNA export receptor in rice; though
there were no other homologs according to BLAST up-to-date rice genome
database. Also, notably, null allele of this gene was viable. Actually, Arabidopsis
psd mutant slightly increased the level of intronless tRNA-Met in
the nucleus, but it did not affect the cytoplasmic level of this tRNA [6],
which demonstrated that it is not absolutely required for this process. Based
on sequence alignment and conservation analysis, we tentatively annotated the
function domain of rice PSD. However, it was rather difficult to predict the in
vivo interaction mechanism due to a lack of a consistent in vitro
assay to recreate events that occur during tRNA generation, processing and
RNA-protein assembly, especially in plants [24]. Moreover, we do not have a
profound structural knowledge of tRNA export. Deciphering the complicated
interaction between PSD with tRNA is therefore still a major challenge [25–27].
References
1 Kohler A, Hurt E. Exporting RNA from the
nucleus to the cytoplasm. Nat Rev Mol Cell Biol 2007, 8: 761–773
2 Rodriguez MS, Dargemont C, Stutz F. Nuclear
export of RNA. Biol Cell 2004, 96: 639–655
3 Arts GJ, Kuersten S, Romby P, Ehresmann B,
Mattaj IW. The role of exportin-t in selective nuclear export of mature tRNAs.
EMBO J 1998, 17: 7430–7441
4 Li J, Chen X. PAUSED, a putative
exportin-t, acts pleiotropically in Arabidopsis development but is dispensable
for viability. Plant Physiol 2003, 132: 1913–1924
5 Lipowsky G, Bischoff FR, Izaurralde E, Kutay
U, Schafer S, Gross HJ, Beier H et al. Coordination of tRNA nuclear
export with processing of tRNA. RNA 1999, 5: 539–549
6 Park MY, Wu G, Gonzalez-Sulser A, Vaucheret
H, Poethig RS. Nuclear processing and export of microRNAs in Arabidopsis. Proc
Natl Acad Sci U S A 2005, 102: 3691–3696
7 Simos G, Hurt E. Transfer RNA biogenesis: a
visa to leave the nucleus. Curr Biol 1999, 9: 238–241
8 An S, Park S, Jeong DH, Lee DY, Kang HG, Yu
JH, Hur J et al. Generation and analysis of end sequence database for
T-DNA tagging lines in rice. Plant Physiol 2003, 133: 2040–2047
9 Jeon JS, Lee S, Jung KH, Jun SH, Jeong DH,
Lee J, Kim C et al. T-DNA insertional mutagenesis for functional
genomics in rice. Plant J 2000, 22: 561–570
10 Jeong DH, An S, Kang HG, Moon S, Han JJ, Park
S, Lee HS et al. T-DNA insertional mutagenesis for activation tagging in
rice. Plant Physiol 2002, 130: 1636–1644
11 Jeong DH, An S, Park S, Kang HG, Park GG, Kim
SR, Sim J et al. Generation of a flanking sequence-tag database for
activation-tagging lines in japonica rice. Plant J 2006, 45: 123–132
12 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin
F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for
multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res
1997, 15: 4876–4882
13 Hall TA. BioEdit: a user-friendly biological
sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic
Acids Symp Ser 1999, 41: 95–98
14 Schmidt HA, Strimmer K, Vingron M, von
Haeseler A. TREE-PUZZLE: maximum likelihood phylogenetic analysis using
quartets and parallel computing. Bioinformatics 2002, 18: 502–504
15 Richards E, Reichardt M, Rogers S. Preparation
of genomic DNA from plant tissue. Curr Protoc Mol Biol 2001, 5: 2–3
16 Pawlowski K. Kunze R. Vries S, Bisseling T.
Isolation of total, poly(A) and polysomal RNA from plant tissues. Plant Mol
Biol Manual 1994, D5: 11–13
17 Kutay U, Lipowsky G, Izaurralde E, Bischoff
FR, Schwarzmaier P, Hartmann E, Görlich D. Identification of a tRNA-specific
nuclear export receptor. Mol Cell 1998, 1: 359–369
18 Arts GJ, Fornerod M, Mattaj IW. Identification
of a nuclear export receptor for tRNA. Curr Biol 1998, 8: 305–314
19 Kuersten S, Arts GJ, Walther TC, Englmeier L,
Mattaj IW. Steady-state nuclear localization of exportin-t involves RanGTP
binding and two distinct nuclear pore complex interaction domains. Mol Cell
Biol 2002, 22: 5708–5720
20 Hanson MR, Köhler RH. GFP imaging: methodology
and application to investigate cellular compartmentation in plants. J Exp Bot
2001, 52: 529-539
21 Hunter CA, Aukerman MJ, Sun H, Fokina M,
Poethig RS. PAUSED encodes the Arabidopsis exportin-t ortholog.
Plant Physiol 2003, 132: 2135–2143
22 Hopper AK, Shaheen HH. A decade of surprises
for tRNA nuclear-cytoplasmic dynamics. Trends Cell Biol 2008, 18: 98–104
23 Shibata S, Sasaki M, Miki T, Shimamoto A,
Furuichi Y, Katahira J, Yoneda Y. Exportin-5 orthologues are functionally
divergent among species. Nucleic Acids Res 2006, 34: 4711–4721
24 Yao C, Zhao B, Li W, Li Y, Qin W, Huang B, Jin
Y. Cloning of novel repeat-associated small RNAs derived from hairpin
precursors in Oryza sativa. Acta Biochim Biophys Sin 2007, 39: 829–834
25 McGuire AT, Mangroo D. Cex1p is a novel
cytoplasmic component of the Saccharomyces cerevisiae nuclear tRNA
export machinery. EMBO J 2007, 26: 288–300
26 Ghavidel A, Kislinger T, Pogoutse O, Sopko R,
Jurisica I, Emili A. Impaired tRNA nuclear export links DNA damage and
cell-cycle checkpoint. Cell 2007, 131: 915–926
27 Hutten S, Kehlenbach RH. CRM1-mediated nuclear
export: to the pore and beyond. Trends Cell Biol 2007, 17: 193–201