Structural and functional analysis of the interaction
between the nucleoporin Nup98 and
the mRNA export factor Rae1
Yi Ren, Hyuk-Soo Seo, Günter Blobel1, and André Hoelz1
Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065
Contributed by Günter Blobel, May 3, 2010 (sent for review April 2, 2010)
transport through nuclear pore complexes, and mRNP remodeling
events prior to translation. Ribonucleic acid export 1 (Rae1) and
Nup98 are evolutionarily conserved mRNA export factors that are
targeted by the vesicular stomatitis virus matrix protein to inhibit
host cell nuclear export. Here, we present the crystal structure of
human Rae1 in complex with the Gle2-binding sequence (GLEBS)
of Nup98 at 1.65 Å resolution. Rae1 forms a seven-bladed β-propel-
ler with several extensive surface loops. The Nup98 GLEBS motif
forms an ≈50-Å-long hairpin that binds with its C-terminal arm to
an essentially invariant hydrophobic surface that extends over
the entire top face of the Rae1 β-propeller. The C-terminal arm of
the GLEBS hairpin is necessary and sufficient for Rae1 binding,
and we identify a tandem glutamate element in this arm as critical
ditional conservedpatch with a positive electrostatic potential, and
we demonstrate that the complex possesses single-stranded RNA-
binding capability. Together, these data suggest that the Rae1•N-
up98 complex directly binds to the mRNP at several stages of the
mRNA export pathway.
mRNA export machinery ∣ fluorescence localization ∣ microscopy ∣
site-directed mutagenesis ∣ protein–protein interaction
neries. After transcription, the mRNA is spliced and packaged
into a messenger ribonucleoprotein particle (mRNP). The
mRNP includes mRNA export factors that provide access to
the nuclear pore complex (NPC) and allow for their translocation
to the cytoplasm. At the cytoplasmic face of the NPC, remodeling
events result in the partial disassembly of the mRNP prior to
Ribonucleic acid export 1 (Rae1) was initially discovered in
Schizosaccharomyces pombe in a genetic screen for proteins
involved in RNA export (4). The mammalian homolog of Rae1
was discovered independently by biochemical characterization
of a rat liver nuclear envelope subfraction (5). Because the iden-
tified protein was found to be UV-cross-linked in vivo to poly(A)
containing RNA it was termed mRNP41 (5). Finally, in a genetic
screen in Saccharomyces cerevisiae, the homolog of Rae1 was dis-
covered to be synthetically lethal in combination with a null mu-
nucleoporin Nup100 and was termed GLFG lethal 2 (Gle2) (6).
The next advance in the characterization of Rae1 was the
demonstration of the direct biochemical interaction between
Gle2 and one of the three GLFG nucleoporins in yeast,
Nup116 (7). The binding of Gle2 was mapped to a 57-residue
sequence stretch in the N-terminal region of Nup116 that was
termed Gle2-binding sequence (GLEBS) (7). The GLEBS motif
is absent in the other two GLFG nucleoporins of S. cerevisiae,
Nup100 and Nup145N (7–9). An evolutionary highly conserved
GLEBS motif was also found in the only GLFG nucleoporin
ukaryotic cells segregate their genetic material in the nucleus,
spatially separating the transcription and translation machi-
of vertebrates, Nup98 (10). Nup98 is a proto-oncogene that
has been identified in numerous leukemogenic fusions with a
variety of partner genes (11).
The Nup116 knockout yields a temperature-sensitive pheno-
type in which cells die at 37°C (12). Strikingly, electron micro-
scopic analyses revealed that at the nonpermissive temperature
the NPCs were clustered on one side of the nuclear envelope
and closed apparently by fusion of the nearby outer nuclear
envelope membrane (12). The fusion resulted in the formation
of a dome-shaped membrane seal over the cytoplasmic face of
the NPC, the accumulation of export substrates, and the appear-
ance of “herniated” nuclei (12). Subsequently, it was shown that
the removal of the GLEBS motif from Nup116 was sufficient
for the generation of herniated nuclei and for the loss of nuclear
envelope localization of Gle2 (7). These phenotypes could be res-
cued by the insertion of the Nup116 GLEBS motif into Nup100
(7). Together, these data indicate that the NPC recruitment of
Gle2 is essential for a normal morphology of the NPC with
respect to its surrounding outer nuclear envelope membrane.
A further advance in understanding the function of Rae1 in
RNA export was made with the demonstration that a viral
protein, the matrix (M) protein of the vesicular stomatitis virus
(VSV), inhibited nuclear export of host cell RNA by targeting
Nup98 and Rae1 (13–16). Interferon γ treatment of VSVinfected
cells was found to reverse the RNA export inhibition by up-reg-
ulation of Nup98 and Rae1 expression (15, 16), thereby under-
lining the importance of the interaction between Rae1 and
Nup98 for RNA export.
Rae1 contains seven WD40 repeats and is therefore predicted
to form a seven-bladed β-propeller (Fig. 1A). The β-propeller
domain is a classical protein–protein interaction platform that
is capable of mediating the association with several proteins
(17). The interaction of Rae1 with the Nup98 GLEBS motif
appears to be a critical association for RNA export. However,
Rae1 can interact with other proteins, not only in the nucleus
but also in the cytoplasm, and has also been reported to have
several functions in the formation of mitotic spindles (18–20).
Hence, Rae1 is a versatile protein that does not only function
as a component of the NPC.
The molecular mechanisms that underlie the manifold func-
tions of Rae1 remain poorly understood. In order to gain further
insight into Rae1’s function in mRNA export, we have deter-
mined the atomic structure of human Rae1 in complex with
the Nup98 GLEBS motif at 1.65 Å resolution. As anticipated,
Author contributions: Y.R., H.-S.S., and A.H. designed research; Y.R., H.-S.S., and A.H.
performed research; Y.R., H.-S.S., and A.H. analyzed data; Y.R., H.-S.S., G.B., and A.H. wrote
The authors declare no conflict of interest.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID code 3MMY).
1To whom correspondence may be addressed. E-mail: email@example.com or hoelza@
This article contains supporting information online at www.pnas.org/lookup/suppl/
10406–10411 ∣ PNAS ∣ June 8, 2010 ∣ vol. 107 ∣ no. 23www.pnas.org/cgi/doi/10.1073/pnas.1005389107
Rae1 forms a seven-bladed β-propeller domain that contains
several distinct surface loops. The GLEBS motif forms a hairpin
structure that interacts extensively with an evolutionarily
conserved surface on the top face of the Rae1 β-propeller. We
identify an invariant tandem glutamate element (Glu201 and
Glu202) in the Nup98 GLEBS motif to be essential for complex
formation with Rae1 in solution. Finally, we show that Nup116
variants that carry mutations in this element prevent Gle2 loca-
lization to the nuclear envelope and display a mild mRNA export
defect in vivo. Strikingly, the Rae1•Nup98GLEBScomplex contains
a highly conserved surface with a positive electrostatic potential
that could represent a binding site for RNA. We demonstrate bio-
chemically that the Rae1•Nup98GLEBScomplex binds single-
stranded RNA oligonucleotides.
Structure Determination. The Nup98 polypeptide chain encodes
one small structured domain at its C terminus, which possesses
autoproteolytic activity and thereby facilitates the evolutionarily
conserved, cotranslational cleavage of the Nup98 and Nup98-
Nup96 precursor proteins (21–23) (Fig. 1A and Fig. S1). More-
over, this domain anchors Nup98 at the cytoplasmic side of the
NPC by interacting with Nup88 (24). The remaining, unstruc-
tured ∼700 residue N-terminal part of Nup98 contains numerous
phenylalanine-glycine (FG) repeats and the GLEBS motif that
serve as docking sites for the mRNP export factors p15/TAP (for
Tip-associated protein) and for Rae1, respectively (7, 10, 25–28).
The complex between full-length human Rae1 and the 57-resi-
due GLEBS motif of Nup98 was formed by coexpression in Sf9
cells. The Rae1•Nup98GLEBScomplex crystallized in the triclinic
space group P1, with four complexes in the asymmetric unit.
The structure was solved by single-anomalous dispersion (SAD)
using anomalous X-ray diffraction data obtained from an
Os-derivative. The Rae1•Nup98GLEBSstructure was refined to
1.65 Å resolution with Rwork and Rfreevalues of 20.6% and
23.7%, respectively. For details of the crystallographic statistics,
see Table S1.
The Rae1•Nup98GLEBScomplex elutes as a single peak in size-
exclusion chromatography, corresponding to a heterodimer. Ana-
lytical ultracentrifugation determined the molecular weight to be
45.4 ? 7.5 kDa, confirming the single monomeric state with a 1∶1
stoichiometry in solution (theoretical molecular weight of
48.4 kDa, Fig. S2). Hence, in the following text, we focus on
the description of the Rae1•Nup98GLEBSheterodimer structure.
Structural Overview.The polypeptide chain of Rae1 can be divided
into a 30-residue N-terminal extended peptide segment (NTE)
followed by a canonical seven-bladed β-propeller domain (17)
(Fig. 1 B and C). The β-propeller domain contains a central tun-
nel along its pseudo-seven-fold axis and is decorated by several
long surface loops. In particular, the interblade connector 5D6A
and the 7BC loop project outward from the top face of the
The Nup98 GLEBS motif folds into an N-terminal 10-residue
coil region followed by two β-strands, β1 and β2, that are con-
nected via a 13-residue linker segment to the C-terminal α-helix
αA (Fig. 1B). Overall, the GLEBS motif forms an ≈50 Å long
hairpin structure, in which the antiparallel β-strands, β1 and
β2, the “β-tongue,” form the kink of the hairpin. No density is
observed for five residues of the β1-β2 connector that form
the tip of the β-tongue. Hence, these residues have been omitted
from the final model.
The Nup98 GLEBS hairpin binds to the top face of the Rae1
β-propeller domain and extends across the entire surface. The
GLEBS motif is anchored to the Rae1 β-propeller primarily
via two key interactions: (i) β2 of the GLEBS β-tongue interacts
with the 5D6A interblade connector, extending the β-tongue with
an additional parallel β-strand 5D′. (ii) the N-terminal tip of the
GLEBS helix αA inserts into the central tunnel of the Rae1
β-propeller. The Rae1-Nup98GLEBSinteraction is reinforced by
WD40 repeats (orange) are indicated. For Nup98, the GLEBS motif (magenta), the FG-repeat region (gray), the unstructured region (dark gray), the auto-
proteolytic domain (pink), and the C-terminal 6 kDa fragment (light gray) that is removed by cotranslational proteolysis are indicated. In an alternatively
spliced version, the Nup98-96 precursor, the 6 kDa fragment is replaced by Nup96, a protein that is embedded in the symmetric NPC core (Fig. S1). The arrows
indicate the sites of autoproteolytic cleavage. (B) Ribbon representation of the Rae1•Nup98GLEBScomplex. The Nup98 GLEBS motif is indicated in magenta.
For Rae1, the β-propeller domain (blue), the NTE (red), the 5D6A loop (green), and the 7BC loop (yellow) are indicated. A 90° rotated view is shown on the right.
(C) Schematic representation of the Rae1•Nup98GLEBSstructure, colored according to B. The blades of the Rae1 β-propeller are labeled from one to seven.
An asterisk indicates the Velcro-closure β-strand (17).
Structural overview of the Rae1•Nup98GLEBScomplex. (A)Domainorganizationof human Rae1 and human Nup98. For Rae1, the NTE (red) andthe seven
Ren et al.PNAS
June 8, 2010
the protruding Rae1 7BC loop that binds to a hydrophobic pock-
et, which is formed by the coil regions and helix αA of the GLEBS
motif. In total, 2;900 Å2of surface area are buried between Rae1
and the Nup98 GLEBS motif, involving 38 and 29 residues of
Rae1 and the GLEBS motif, respectively.
Rae1•Nup98GLEBScomplexes in the asymmetric unit involve
two FG-repeat-like sequence stretches of the Rae1 NTE that
bind to hydrophobic pockets in the surface of neighboring
Rae1 molecules (Fig. S3). These hydrophobic pockets may pro-
vide transient binding sites for the FG-repeats of Nup98 or other
Surface Properties. Many β-propeller domains allow for the simul-
taneous attachment of several binding partners (17). Rae1 was
identified as a component of the mRNA export machinery and
has been found to directly interact with poly(A)-containing
mRNA in cross-linking experiments conducted in HeLa cells
(5). Therefore, we analyzed the surface properties of Rae1
and the Rae1•Nup98GLEBScomplex to identify potential, addi-
tional interaction sites.
Rae1 features two large and essentially invariant surface
patches (Fig.2 and Fig. S4). The first patch is primarily hydropho-
The second patch is located on the side of the β-propeller, contig-
uous to the first patch, and is formed by surface residues in blades
five and six. This surface area has a highly positive electrostatic
potential. Strikingly, the binding of the Nup98 GLEBS places
eratinga cradle withapositive surfacepotential atitsdeepestsite.
The properties of this second surface patch are consistent with
those with a potential RNA-binding capability.
Structural Comparison. Several proteins that carry stretches of
high-sequence homology to the Nup98 GLEBS motif have been
identified (29). Among these proteins are the human spindle
checkpoint components Bub1 and BubR1 (in yeast Mad3) that
bind to the cell cycle arrest protein Bub3 (30, 31). Together with
other components, these proteins form an inhibitory complex at
the kinetochore that prevents the progression of the cell cycle
(32). Rae1 has been found to be capable of forming complexes
with the two GLEBS-like motifs of Bub1 and BubR1 (29). There-
fore, we compared our Rae1•Nup98GLEBSstructure to the
previously determined structures of Bub3 in complex with the
GLEBS-like sequences of Bub1 and Mad3 (33).
seven-bladed β-propellers(Fig.3).245equivalent Cαatomsofthe
two β-propeller cores superimpose with an rmsd of ≈1.4 Å. The
GLEBS-like Bub3 binding sequences of Bub1 and Mad3 are com-
than the Nup98 GLEBS motif. The Bub3•Bub1 and Bub3•Mad3
structures reveal that the binding mode of the GLEBS-like se-
quences of Bub1 and Mad3 are similar to that of the Nup98
GLEBS motif, involving the 5D6A and 7BC loops on the top sur-
face of the β-propeller. However, the shorter GLEBS-like se-
quences are composed of only the C-terminal arm of the
Nup98 GLEBS hairpin (GLEBS-C) and lack the N-terminal arm
linker segment between these two secondary structure elements
that varies in length among the three sequence motifs. Whereas
the 5D6A loop forms a similar interaction with the β2 strand in
all three cases, the interaction of the 7BC loop with the three
GLEBS motifs differs substantially. The lack of the GLEBS-N
7BC loop of Bub3 that partially covers and stabilizes the shorter
Rae1-Nup98GLEBSInterface. The edge of the Nup98 GLEBS hairpin
binds to the top face of the Rae1 β-propeller domain, primarily
involving the residues in the GLEBS-C arm. This result is in line
jority of the conserved residues are located in the GLEBS-C seg-
with Rae1: β2 forms a short parallel β-sheet with 5D′ of the 5D6A
face renditions of Rae1 and Rae1•Nup98GLEBS. The Rae1 surface that mediates
GLEBS motif surfaces are colored in blue and light magenta, respectively
(Right). As a reference, a ribbon representation of the Rae1•Nup98GLEBScom-
plex is shown and corresponds to the orientation of the left panel. The black
line in the left panel indicates the location of the side view surface. (B) The
surface representationsof Rae1and theRae1•Nup98GLEBScomplexare colored
according to multispecies sequence alignments (Fig. 4A and Fig. S3). The con-
servation at each position is mapped onto the surface and shaded in a color
gradient from yellow (60% similarity) to red (100% identity). (C) Rae1 and
Rae1•Nup98GLEBSsurface renditions, colored according to the electrostatic po-
tential, ranging from red (−10 kBT∕e) to blue (þ10 kBT∕e).
Surface properties of Rae1 and the Rae1•Nup98GLEBScomplex. (A) Sur-
complexes. The superposition of the Rae1 and Bub3 β-propeller domains is
shown in the right panel. Bub3 and Mad3 are colored in gray and pink,
respectively. The 5D6A interblade connectors and 7BC loops of the Rae1
and Bub3 β-propellers are indicated in different shades of green and yellow,
respectively. A 90° rotated view is shown in the lower panel.
Structural comparison between the Rae1•Nup98 and Bub3•Mad3
www.pnas.org/cgi/doi/10.1073/pnas.1005389107Ren et al.
interblade connector loop, and helix αA dips into the central tun-
nel of the Rae1 β-propeller (Fig. 1B). Whereas the residues in the
β2 strand are only moderately conserved, consistent with the for-
mation of a main-chain, hydrogen-bond network, the majority of
the residues in helix αA are involved in numerous side-chain con-
tactsandareinvariant from yeasttohuman(Fig.4B).Specifically,
the N terminus of helix αA is anchored at the central tunnel of
Rae1 by two key salt bridges that are formed between E201
and E202 of the Nup98 GLEBS, and R216 and R172 of Rae1, re-
spectively. Moreover, helix αA makes several additional highly
conserved interactions with Rae1, involving R204, D207, and
Y208. The interactions of the GLEBS-N arm involve the forma-
tion of the three-stranded β-sheet between Rae1 and the Nup98
by R239 and K258 of Rae1, forming an additional evolutionarily
conserved salt bridge between the two molecules. Finally, the in-
teraction between Rae1 and the Nup98 GLEBS is reinforced by
involving helix αA and the two coil regions.
The Nup98 GLEBS motif and the GLEBS-like sequences of
Mad3 and Bub1 are evolutionarily very divergent, preventing a
reliable alignment. In fact, the only conserved sequence feature
between the three structures refers to the two key salt bridges that
involve the tandem glutamate element and anchor helix αA at the
tunnel in the Rae1 β-propeller. All remaining interactions only
impose a loose restraint on the sequence.
Biochemical Analysis of the Interaction Between Rae1 and the Nup98
GLEBS Motif. The Nup98 GLEBS motif contains a unique N-term-
inal region (GLEBS-N) that is not found in the GLEBS-like
that the GLEBS-N segment only contributes to a minor extent to
the interaction with Rae1, we analyzed whether a “minimal”
Nup98 GLEBS region, as defined by the structural comparison
with the Bub3•Bub1 and Bub3•Mad3 structures, would be suffi-
cient for complex formation with Rae1. Indeed, in a pull-down
experiment, we found that the GLEBS-C arm is necessary and
sufficient for Rae1 binding, whereas the GLEBS-N region is
Our result is also in agreement with previous in vivo experiments
in which the overexpression of a similar GLEBS-C fragment
(residues 181–224) in HtTA cells is as sufficient to induce a
comparable mRNA export defect as is the entire GLEBS motif
(residues 150–224) (10).
We then tested if the tandem glutamate element which is a
shared structural feature between the GLEBS motifs of Nup98,
Mad3, and Bub1 is essential for complex formation between
Nup98 and Rae1. As predicted, the charge-reversal mutations
E201K and E202K abolish the interaction between the two pro-
teins. This result is in concurrence with previous mutagenesis ex-
periments that identified the salt bridge formed between E382 of
Mad3 and R197 of Bub3 as critical for complex formation in this
Rae1•Nup98GLEBSBinds to RNA in Vitro. Rae1 has been found to be
cross-linked to poly(A)-containing mRNA after the UV irradia-
tion of HeLa cells (5). The analysis of the Rae1•Nup98GLEBS
surface reveals an evolutionarily conserved basic surface patch
that supports the previously proposed RNA-binding capability.
Therefore, we analyzed whether the Rae1•Nup98GLEBScomplex
is able to bind to a degenerate, decameric, single-stranded
RNAoligonucleotide using anelectrophoreticmobility shiftassay
(Fig. 5). Indeed, the Rae1•Nup98GLEBSheterodimer shifts the
RNA band to lower mobility in a concentration-dependent
manner, indicating the formation of a Rae1•Nup98GLEBS•RNA
complex. This result demonstrates that Rae1•Nup98GLEBSdirectly
binds to single-stranded RNA with an approximate dissociation
constant in the low micromolar range.
GLEBS motif. The overall sequence conservation at each position is shaded in a color gradient from yellow (40% similarity) to red (100% identity) using the
Blosum62 weighting algorithm (40). The numbering of the residues and the secondary structure are according to human Nup98. The secondary structure is
indicated above the sequence as green arrows (β-strands), blue rectangles (α-helices), gray lines (coil regions), and gray dots (disordered residues). Dots below
the sequence indicate residues involved in Rae1 binding (magenta and green). Residues that are shown in B are highlighted with magenta dots. Asterisks
indicate the positions of the invariant Glu201 and Glu202 of the tandem glutamate element. (B) Details of the interaction between Rae1 and the Nup98 GLEBS
motif. The ribbon representation is colored according to Fig. 1B. The inset illustrates the position of the tandem glutamate element and is expanded on the
right. (C) Analysis of the interaction between Rae1 and GST-Nup98 GLEBS fragments and mutants. The C-terminal arm of the Nup98 GLEBS (GLEBS-C) is
necessary and sufficient for Rae1 binding. A double mutant in Nup98 GLEBS, E201K/E202K, abolishes the Rae1-Nup98GLEBSinteraction.
The tandem glutamate element of the Nup98 GLEBS motif is essential for the interaction with Rae1. (A) Multispecies sequence alignment of the Nup98
Ren et al. PNAS
June 8, 2010
of data implicate the Rae1•Nup98GLEBScomplex in mRNAexport
(4–10, 16, 28). By taking advantage of the structural infor-
mation, we examined the consequences of disrupting the
Rae1•Nup98GLEBScomplex in vivo using yeast as a model system
(Fig. 6). Deletion of the GLEBS motif in Nup116 (the yeast
homolog of Nup98) results in a temperature-sensitive growth de-
fect, as well as in the mislocalization of Gle2 (the yeast homolog
of Rae1). This observation is in agreement with previous findings
that the nuclear envelope localization of Gle2 is dependent on
the Nup116 GLEBS motif (7). We further tested the growth
and localization phenotypes of the Nup116 E154K/E155K double
mutant, which is equivalent to the Nup98 tandem glutamate ele-
ment mutant (E201K/E202K) that abolishes the interaction with
Rae1 in vitro (Fig. 4C). Similar to the Nup116 ΔGLEBS mutant,
this yeast Nup116 mutant results in a temperature-sensitive
growth phenotype and the loss of Gle2 nuclear envelope staining.
These data demonstrate that the mutation of two key glutamate
residues of the Nup116 GLEBS motif abolishes the Gle2-Nup116
interaction in vivo.
Based on our observation that the Rae1•Nup98GLEBSheterodi-
mer binds single-stranded RNA in solution, we hypothesized that
the disruption of the Gle2•Nup116GLEBScomplex may result in
mRNA export defects in yeast. To characterize the function of
the Gle2-Nup116 interaction in mRNA export, we analyzed
whether Nup116 mutants that fail to localize Gle2 to the nuclear
envelope also result in the nuclear retention of poly(A) mRNA by
fluorescence in situ hybridization (FISH), using an Alexa647-la-
beledoligo dT50probe (Fig.S6).Inatypicalwild-type population,
1.7 ? 0.9% of the cells display a marked nuclear FISH signal,
while 6.4 ? 2.2% of the cells are stained in the Nup116 knockout
strain. The introduction of the Nup116 ΔGLEBS or Nup116
E154K/E155K mutants to the Nup116 knockout strains shows a
nuclear mRNA accumulation in 6.1 ? 0.9% and 4.2 ? 1.0% of
the cells, respectively. These data demonstrate that the disruption
total poly(A) mRNA export defect.
The Rae1•Nup98 complex has distinct and versatile roles at
different stages of the cell cycle, including the nuclear export
of mRNAs and the regulation of the cell cycle progression (4–
10, 18–20, 28, 34). We have determined an atomic model that
resolves the interaction of Rae1 with the GLEBS motif of
Nup98. The analysis of our structure allowed for the identifica-
tion of a tandem glutamate element in the GLEBS motif that is
critical for the interaction with Rae1 in vitro, as well as for the
nuclear envelope localization of Gle2 (the yeast homolog of
Rae1) in vivo. Moreover, our structure reveals an evolutionarily
conserved surface patch with a positive surface potential. Strik-
ingly, the Rae1•Nup98GLEBSheterodimer is capable of directly
binding to single-stranded RNA oligonucleotides.
In the export of nuclear mRNA to the cytoplasm, the mRNA
is organized into an mRNP with the help of mRNA binding
proteins, including mRNA export factors, such as the p15/TAP
heterodimer (1–3). The targeting of the mature mRNP to the
NPC is facilitated by the interaction of the general mRNA export
factor p15/TAP with Nup98 (26). Photobleaching experiments in
living cells have yielded a residence time for Nup98 of about 3 h
(35). This finding, when correlated with the mRNP’s average
“dwell” time of several 100 ms at the NPC (36), suggests that
Nup98 stays associated with the NPC and conducts the sequential
export of many mRNPs. Nup98 is attached to the cytoplasmic
side of the NPC with its C-terminal domain, whereas the remain-
der of the protein is unstructured (24). This architecture allows
Nup98 to remain attached at the cytoplasmic side of the NPC,
while simultaneously reaching into the interior of the nucleus
with its flexible N-terminal unstructured region. Our finding that
the Rae1•Nup98GLEBSheterodimer is capable of RNA binding in
solution suggests that Nup98 may recruit mRNPs to the NPC not
growth assay performed using nup116Δ, Gle2-GFP cells transformed with
the indicated mCherry (mCh)-Nup116 constructs. Ten-fold serial dilutions
were spotted on SD-LEU plates and grown for 2–3 days at the indicated tem-
peratures. (B) In vivo localization of Gle2-GFP and mCh-Nup116 carried out at
30 °C. In the presence of full-length mCh-Nup116, Gle2-GFP is enriched at the
nuclear envelope. The ΔGLEBS and the tandem glutamate element (E154K/
E155K) Nup116 mutants result in a dispersed staining of Gle2-GFP through-
out the cell with no enrichment at the nuclear envelope. (Scale bar: 5 μm.)
In vivo analysis of the Gle2-Nup116GLEBSinteraction. (A) Yeast
assay was carried out, with a constant amount of degenerate 10-mer RNA
oligonucleotide (2 μM) and increasing concentrations of the Rae1•Nup98GLEBS
complex, as indicated. The RNA oligonucleotide was visualized with SYBR
Gold nucleic acid gel stain. The estimated dissociation constant of the inter-
action is in the low micromolar range.
Rae1•Nup98GLEBSbinds RNA in vitro. An electrophoretic mobility shift
www.pnas.org/cgi/doi/10.1073/pnas.1005389107Ren et al.
only by binding to p15/TAP (via its FG repeats) (26), but also by Download full-text
directly interacting with the RNA (via its association with Rae1).
The next step in mRNP export is the translocation through the
central channel of the NPC. This transit is followed by mRNP
remodeling, whereby proteins bound to nuclear mRNA are
dissociated and replaced by cytoplasmic mRNA binding proteins
(1–3). Key factors in this process are the DEAD-box helicase
hDbp5 (also known as DDX19), which is anchored to the cyto-
plasmic filament nucleoporin Nup214 and the hDbp5 ATPase
activating protein Gle1 (37–39). The weak RNA-binding affinity
of the Rae1•Nup98 heterodimer may also allow the complex to
be involved in this process by temporarily covering and thereby
protecting naked mRNA stretches prior to translation. However,
the Rae1•Nup98-RNA interaction may constitute only one of
many redundant mechanisms to protect the RNA, which would
explain our observation that disruption of the Gle2•Nup116
complex in yeast only yields a modest accumulation of poly(A)
mRNA in the nucleus. An alternative explanation would be that
the Gle2•Nup116-mediated export pathway is utilized only by a
subfraction of mRNAs. In this context, Gle2 may function as an
mRNP recruitment factor that allows for the preferential export
of a distinct mRNA subset by providing an additional binding site
to Nup116. In fact, Nup100 and Nup145N are two Nup116 homo-
logs that lack a GLEBS motif and may allow for the Gle2-
independent, but Mtr2/Mex67 (the yeast homolog of p15/TAP)-
mediated mRNP export.
The structure of the Rae1•Nup98GLEBScomplex reported here
provides an important piece in the mRNA export puzzle. Many
more structures of nucleoporins that constitute the mRNAexport
machinery at the cytoplasmic face of the NPC remain to be de-
termined. Together, these structures are expected to ultimately
provide a detailed, mechanistic understanding of the various
processes that accompany mRNA export and occur prior to
The details of protein expression, purification, crystallization, structure deter-
mination, protein interaction analysis, yeast experiments, and RNA-binding
assay are described in the SI Text published online. In short, Rae1 and the
Nup98 GLEBS motif were coexpressed in Sf9 insect cells using the pFastbac
Dual baculovirus system (Invitrogen) (Table S2). The Rae1•Nup98GLEBScomplex
were collected at the General Medicine and Cancer Institutes Collaborative
Access Team (GM/CA-CAT) beamline 23ID-B at the Advanced Photon Source,
Argonne National Laboratory. The structure was solved by SAD, using data
obtained from OsO4-derivatized crystals. Data collection and refinement
statistics are summarized in Table S1.
ACKNOWLEDGMENTS. We thank Erik Debler, Vivien Nagy, Johanna Napetsch-
nig, Alina Patke, Deniz Top, Pete Stavropoulos, and Kimihisa Yoshida for cri-
tical reading of the manuscript, and Stephanie Etherton for help with editing
the manuscript. Analytical ultracentrifugation was carried out by the Wads-
worth Center Biochemistry Core Facility. In addition, we thank Erik Debler for
help and Nagarajan Venugopalan (GM/CA-CAT) for support during data
collection. A.H. was supported by a grant from the Leukemia and Lymphoma
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