From the Department of Biochemistry, Graduate Program
in Genetics and Molecular Biology and ¶ Graduate Program in
Biochemistry, Cell, and Developmental Biology, Emory University School
of Medicine, Atlanta, Georgia 30322
Received for publication, July 26, 2002, and in revised form, November 26, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mature poly(A) RNA transcripts are exported from
the nucleus in complex with heterogeneous nuclear ribonucleoproteins
(hnRNPs). Nab2p is an essential Saccharomyces cerevisiae
hnRNP protein that interacts with poly(A) RNA and shuttles between the
nucleus and cytoplasm. Functional Nab2p is required for export of
poly(A) RNA from the nucleus. The Nab2 protein consists of the
following four domains: a unique N-terminal domain, a glutamine-rich
domain, an arginine-glycine (RGG) domain, and a zinc finger domain. We generated Nab2p deletion mutants to analyze the contribution of each
domain to the in vivo function of Nab2p. We first tested whether the deletion mutants could replace the essential
NAB2 gene. We then examined the impact of these mutations
on Nab2p localization, poly(A) RNA localization, and association of
Nab2p with poly(A) RNA. Our analyses revealed that the N-terminal
domain is required for nuclear export of both poly(A) RNA and Nab2p. We
confirm that the RGG domain is important for Nab2p import in vivo. Finally, the zinc finger domain is critical for the
interaction between Nab2p and poly(A) RNA in vivo. Our data
support a model where Nab2p associates with poly(A) RNA in the nucleus
through the zinc finger domain and facilitates the export of the
poly(A) RNA through protein interactions mediated by the N-terminal domain.
The eukaryotic cell is divided into functional compartments that
separate important cellular processes. This compartmentalization prevents cross-talk between these cellular processes in an
inappropriate or unregulated manner. For example, the membrane-bound
nucleus serves at least two critical purposes: to sequester and protect the genetic material and to separate the nuclear processes of transcription and pre-mRNA processing from protein translation in
the cytoplasm. As a result of this sequestration, the eukaryotic cell
has evolved highly regulated mechanisms to actively transport macromolecules into and out of the nucleus (1, 2). Proteins that
function within the nucleus are imported through large proteinaceous nuclear pore complexes that are embedded within the nuclear envelope (3). Similarly, macromolecules, including
mRNP1 complexes, consisting
of mRNA and hnRNP proteins, are exported from the nucleus through
nuclear pores (4).
Mature, fully processed mRNAs are selectively exported from the
nucleus (4-6). Thus, nuclear export of poly(A) RNA is extremely complex because it depends on numerous pre-mRNA processing events that must be completed within the nucleus prior to export (6). Nascent
transcripts are co-transcriptionally modified by proteins that mediate
5'-capping (7), 3'-cleavage, polyadenylation (7, 8), and splicing (9).
During pre-mRNA processing, the maturing mRNAs associate with
many heterogeneous nuclear ribonucleoproteins (hnRNPs) (4), which may
serve as markers for the completion of pre-mRNA processing events
(5). Successful completion of these processing steps results in a
mature mRNA species that is ready to be exported from the nucleus
to the cytoplasm where it can be translated into protein. Over the
course of pre-mRNA processing, some of the hnRNP proteins
dissociate from the maturing transcript in the nucleus, whereas others
that shuttle between the nucleus and the cytoplasm (4) remain
associated with the transcript to form an mRNP complex that is exported
to the cytoplasm (4-6).
Many transport events, such as classical nuclear protein import, are
mediated by a family of conserved transport receptors, known as
karyopherins (10), that recognize targeting signals within the
transport cargo (11). However, mRNA export does not appear to use
members from this family of transport receptors (4). Instead, proteins
such as Saccharomyces cerevisiae Mex67p/Mtr2p (TAP/p15 in
mammalian cells) specifically associate with mature mRNA and act as
poly(A) RNA export factors (5, 6). Therefore, the association of
mRNA with a combination of hnRNPs and mRNA export factors
culminates in the export of mature mRNA transcripts (5, 6).
The volume, complexity, and timing of proteins that associate with and
dissociate from the maturing transcript during the pre-mRNA
processing stages make it difficult to simply map the route that
mRNA must travel in order to be exported from the nucleus. Unraveling this complexity requires detailed analyses of the proteins within the poly(A) RNA-hnRNP complexes. To this end, we have analyzed the S. cerevisiae hnRNP protein, Nab2p. Nab2p,
nuclear abundant poly(A) RNA
binding protein 2, is an essential protein that
was identified as a poly(A) RNA-binding protein (12). As with other shuttling hnRNP proteins (13-16), Nab2p localizes to the nucleus at
steady state (12) but shuttles between the nucleus and the cytoplasm
(17-19). Nab2p is imported into the nucleus by a transport receptor
from the karyopherin family, Kap104p (20). Although the mechanism of
Nab2p export is unknown, it relies on ongoing synthesis of poly(A) RNA
and the arginine methyltransferase, Hmt1p (17). Importantly, in the
absence of a functional Nab2 protein, poly(A) RNA accumulates within
the nucleus (17, 21). Furthermore, Nab2p binds directly to poly(A) RNA
in vitro (12, 21), and cells lacking Nab2p accumulate
poly(A) RNA with hyperadenylated poly(A) tails suggesting a role for
Nab2p in polyadenylation termination (21). Taken together, these
findings support a model where Nab2p plays a role in ensuring proper
export of mature mRNA from the nucleus.
In support of this model, the Nab2 protein contains domains similar to
those found in other proteins that function in RNA metabolism. Nab2p
can be divided into four distinct domains: a unique N-terminal domain,
a glutamine-rich domain, an RGG (arginine-glycine-glycine) domain, and
a seven repeat zinc finger domain encompassing the entire C-terminal
half of the protein (see Fig. 1) (12). The RGG domain is found in many
proteins involved in RNA metabolism, including the yeast hnRNPs, Npl3p
and Hrp1p, and the mammalian hnRNP A1 protein (12, 16, 22, 23). In
Nab2p, the RGG domain is the site of methylation by the yeast arginine
methyltransferase, Hmt1p (17). This domain also overlaps with the
nuclear localization signal/Kap104p-binding site in Nab2p (19, 24). In
addition, this domain is a putative RNA binding domain (12). The zinc finger repeats,
CX5CX4-6CX3H,
resemble those found within the largest subunit of RNA polymerases
I-III (12). This is a putative site for both protein-RNA interactions
(12, 25) and protein-protein interactions (26-29).
For this study, we have generated a series of NAB2 mutants,
and analyzed these variant proteins to provide insight into the function of each domain. We find that the N-terminal domain is important for efficient export of both Nab2p and poly(A) RNA from the
nucleus. The RGG domain of Nab2p is important, but not absolutely required, for import of Nab2p into the nucleus. Finally, the zinc finger domain is essential for Nab2p function, and our results suggest
that the last three zinc finger repeats are critical for the
interaction with poly(A) RNA in vivo.
Strains, Plasmids, and Chemicals--
DNA manipulations were
performed according to standard methods (30), and all yeast media were
prepared by standard procedures (31). All yeast strains and plasmids
used are described in Table I. Chemicals
were obtained from Sigma, US Biological, or Fisher unless otherwise
noted.
Construction and Functional Analysis of nab2 Mutant
Alleles--
Overlap PCR procedures were performed to generate the
NAB2 deletion mutants (32). Proper construction of all
mutants was confirmed by sequencing of the resulting clones. The
function of each mutant was assessed through a plasmid shuffle
technique using 5-fluoroorotic acid (5-FOA) (33). NAB2
deletion cells (ACY429) maintained by a wild-type
URA3-NAB2 plasmid (pAC636) and also containing a
LEU2 plasmid carrying the mutant allele of NAB2
to be tested were grown to saturation, counted, and serially diluted in
distilled H2O to obtain 10,000, 1000, 100, 10, or 1 cell
per 3 µl. These dilutions were spotted onto URA Immunoblot Analysis--
Cultures were grown to saturation (or
as indicated) and then harvested by centrifugation at 3000 rpm for 3 min. Cell pellets were washed twice with distilled H2O and
once in PBSMT (PBS, 2.5 mM MgCl2, 5% Triton
X-100). Cells were then resuspended in 500 µl of PBSMT supplemented
with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride and 3 µg/ml each of aprotinin, leupeptin, chymostatin, and
pepstatin). One volume of glass beads was added to each sample, and
cells were lysed with 60-s pulses in a bead beater (Biospec Products).
The lysate was clarified by centrifugation at 13,000 rpm and assayed
for total protein concentration using a Bio-Rad protein assay kit.
Immunoblot analysis was performed as described previously (34). Nab2
protein was detected with a polyclonal anti-Nab2p antibody (1:50,000)
(17); GFP-tagged proteins were detected using a polyclonal anti-GFP
antibody (1:5000) (35), and Arf1p was detected using a polyclonal
anti-Arf1p antibody (1:1000) (36).
Microscopy--
All microscopy was carried out using filters
from Chroma Technology and an Olympus BX60 epifluorescence microscope
equipped with a photometrics Quantix digital camera. For direct
fluorescence, cells expressing GFP-tagged proteins were grown to 1 × 107 cells per ml, and live cells were then examined for
the GFP signal through a GFP optimized filter. All images were captured
using IP Lab Spectrum software.
Nuclear Protein Export Assay--
Nuclear export of Nab2p was
examined as described previously (37). The wild-type and mutant
Nab2-GFP fusion proteins were expressed in nup49-313
(ACY480) yeast (38). Cells were grown to mid-log phase at 25 °C in
minimal media lacking uracil and supplemented with 2% glucose. To
inhibit protein synthesis, cycloheximide (100 µg/ml) was added to
each sample for 1 h at 25 °C. Cultures were then either
maintained at 25 °C or shifted to 37 °C for 5 h. The GFP
signal was examined in live cells by direct fluorescence microscopy as
described above.
Fluorescence in Situ Hybridization (FISH)--
The FISH protocol
was adapted from Wong and co-workers (39, 40). Cells expressing
wild-type Nab2p (pAC717), Indirect Immunofluorescence--
Indirect immunofluorescence was
performed according to Wong et al. (39). Cultures were grown
to log phase and cells were prepared for immunofluorescence and adhered
to slides as described for FISH. Cells were then fixed onto slides by
methanol/acetone treatment, blocked with PBS/BSA (1× PBS, 0.5% BSA),
and then incubated with an anti-GFP antibody (1:1000) (35) overnight.
Samples were subsequently washed with PBS/BSA and incubated with
FITC-conjugated anti-rabbit secondary antibody (1:1000) (Roche
Molecular Biochemicals) for 2 h at room temperature. Samples were
washed and stained with DAPI (1 µg/ml). Finally, sample wells were
washed several times with PBS, dried, treated with antifade, and sealed.
Poly(A) RNA/Protein Cross-linking--
The protocol
for UV cross-linking was adapted from Krebber et al. (41).
Cultures were grown to mid-log phase and shifted to 18 °C until
cultures reached early saturation. Cell pellets were washed once and
resuspended in PBS. Proteins and RNA were cross-linked by UV (
The amount of protein bound to poly(A) RNA was quantified by
fluoroimaging using Labworks software from UVP BioImaging Systems. The
amount of protein in the bound and lysate lanes of each immunoblot was
quantified using non-linear equations. The background density was
subtracted from each lane resulting in a value corresponding to density
of total Nab2 protein in each lane. The amount of bound protein was
calculated by setting each lysate value to 1. The fold difference in
the amount of bound protein for the mutant proteins as compared with
wild-type Nab2p was calculated by setting the amount of bound wild-type
Nab2p to 1 for each matched experiment. Experiments were performed and
analyzed independently three to four times. Standard deviations were
calculated for each set of experiments.
Characterization of Nab2p Deletion Mutants--
To probe the
function of each domain of Nab2p, we generated Nab2p mutants that
precisely deleted each of the major predicted domains (Fig.
1). Because NAB2 is essential
(12), a plasmid shuffle technique was used to assess the function of
each of the deletion mutants (Fig.
2A). The positive control for
growth is the wild-type NAB2, and the negative control is
the vector alone. Cells expressing a deletion of the N-terminal domain
(
To confirm that each of the mutant proteins was expressed, quantitative
immunoblot analysis of the GFP-tagged wild-type and mutant Nab2
proteins was performed. Because cells expressing the zinc finger domain
deletion mutant (
We next examined poly(A) RNA localization in NAB2 mutant
cells to determine which domains of Nab2p are critical for poly(A) RNA
export. In wild-type cells, poly(A) RNA localizes throughout the cell
at both 30 and 18 °C (Fig. 2C). In contrast,
To examine the steady state localization of each of the mutant Nab2
proteins, GFP-tagged mutant and wild-type proteins were expressed from
the NAB2 promoter on a low copy plasmid in wild-type cells
(Fig. 3A). Similar to
wild-type Nab2p-GFP, the
The
Since wild-type Nab2p shuttles between the nucleus and the cytoplasm
(17-19), we tested whether the NAB2 deletion mutant
proteins could also shuttle. To assess Nab2p shuttling, a
NUP49-based nuclear export assay was used (37). This assay
exploits a temperature-sensitive mutant of a nuclear pore component,
nup49-313 (38). At the non-permissive temperature,
nup49-313 cells are defective in global protein import, whereas macromolecular export is unaffected (38, 44). The export of
nuclear proteins can be monitored in these cells because they have
ongoing nuclear export under conditions where re-import is blocked.
Thus, appearance of protein in the cytoplasm of the nup49-313 mutant cells indicates that the normally nuclear
localized protein can be exported from the nucleus. To ensure that any
cytoplasmic accumulation of protein observed is because of protein
export rather than new protein synthesis, cycloheximide is added to the cultures prior to the shift to the non-permissive temperature.
In this assay, wild-type Nab2p-GFP can be observed in the cytoplasm
(Fig. 3D) as has been demonstrated previously (17, 18). Because the NUP49 nuclear export assay relies on monitoring
the appearance of the protein of interest in the cytoplasm, a
prerequisite for this assay is that the protein of interest must be
localized strictly to the nucleus under steady state conditions. For
this reason, only the Analysis of the N-terminal Domain of Nab2p--
Our experiments
show that the Nab2p N-terminal deletion mutant (
Because the NLS-N(Nab2p)-GFP protein is detected in the cytoplasm of
wild-type cells, the N-terminal domain of Nab2p could either act as a
nuclear export signal or an inhibitor of import of the NLS-GFP reporter
protein. To distinguish between these possibilities, we took advantage
of the rat7-1 temperature-sensitive mutant (42). If the
N-terminal domain can act as an export signal, we would predict that
NLS-N(Nab2p)-GFP protein export would be blocked, and the protein would
accumulate within the nucleus of rat7-1 cells at the
non-permissive temperature. However, if the N-terminal domain inhibits
import of the NLS-GFP protein, we would expect no change in
localization for the NLS-N(Nab2p)-GFP protein in rat7-1
cells as compared with wild-type cells at 37 °C. In rat7-1 cells at the permissive temperature (25 °C), the
NLS-N(Nab2p)-GFP protein is localized to the nucleus with some
cytoplasmic signal (Fig. 4K). In contrast, the
NLS-N(Nab2p)-GFP protein is detected only within the nucleus at the
non-permissive temperature (37 °C) (Fig. 4M). As a
control, the NLS-GFP protein is strictly nuclear in rat7-1
cells at both 25 and 37 °C, and the NLS-NES-GFP reporter protein
accumulates in the nucleus in rat7-1 cells at 37 °C (Fig. 4, I and Q). Together, these results suggest that
the N-terminal domain contains within it a nuclear export signal that
could facilitate Nab2p export from the nucleus. Our analysis of the
The finding that the
Analysis of the Analysis of the Nab2p Zinc Finger Domain--
Deletion of the
entire C-terminal zinc finger domain yields a non-functional Nab2
protein (see Fig. 2A). Closer analysis of the zinc finger
domain reveals that the seven CCCH repeats cluster into two groups, a
set of four repeats and a set of three repeats. To dissect further the
function of the zinc finger domain, we generated mutants that deleted
the first four repeats (
By using the GFP-tagged zinc finger deletion mutants, we examined the
steady state localization of each protein in wild-type cells. As
demonstrated earlier, wild-type Nab2p-GFP is localized within the
nucleus at steady state, and the zinc finger deletion mutant protein
(
To characterize further the zinc finger deletion mutants, we used
in vivo cross-linking experiments to examine whether these mutant proteins interact with poly(A) RNA. Because deletion of the zinc
finger repeats results in a non-functional protein (see Fig.
7A), we performed these experiments in wild-type cells
expressing GFP-tagged alleles of Nab2p, This study characterizes the domains of the essential S. cerevisiae hnRNP, Nab2p. We generated a series of deletion mutants of NAB2 and tested whether each mutant could replace the
essential Nab2 protein in vivo. To characterize the impact
of the mutations on Nab2p function, we examined the effect of these
deletions on Nab2p localization, poly(A) RNA export, and interaction
between Nab2p and poly(A) RNA. Our conclusions are summarized in Fig. 9A. From our analysis, we have
characterized the in vivo roles for three functionally
important domains of Nab2p: the N-terminal, RGG, and zinc finger
domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains and plasmids
glucose
and 5-FOA plates and incubated at 37, 30, and 18 °C. Cells
expressing wild-type NAB2 or vector alone served as controls.
N (pAC1152),
QQQP (pAC1115), or
RGG
(pAC957) as their only copy of NAB2 were grown to log phase
at 18, 25, 30, and 37 °C. Cells were fixed in 5.2% formaldehyde
(J. T. Baker Inc.) for 90 min, washed twice with 0.1 M
potassium phosphate, and washed once and resuspended in P-solution (0.1 M potassium phosphate, pH 6.5, and 1.2 M
sorbitol). Samples were then incubated with 25 µM
dithiothreitol and digested with 1 mg/ml zymolyase. Cells were applied
to Teflon-coated slides (Cell Point) treated with 0.1% polylysine.
Cells were subsequently permeabilized for 5 min with 0.5% igepal,
equilibrated for 2 min with 0.1 M triethanolamine, pH 8.0, and incubated for 10 min with 0.1 M triethanolamine, pH
8.0, with 0.25% acetic anhydride to block polar groups. The slide was
then incubated for 1 h at 37 °C in prehybridization buffer
(50% deionized formamide, 4× SSC, 1× Denhardt's solution, 125 µg/µl tRNA, 10% dextran sulfate, and 0.5 mg/ml boiled
single-stranded DNA). The digoxigenin-labeled oligo(dT)50
probe was applied to the wells for overnight incubation at 37 °C.
Wells were washed several times with dilutions of SSC (2, 1, and 0.5×)
before incubation in antibody blocking buffer (0.1 M Tris,
pH 9.0, 0.15 M NaCl, 5% heat-inactivated fetal calf serum,
and 0.3% Triton X-100) for 1 h at room temperature. Fluorescein isothiocyanate (FITC)-conjugated anti-digoxigenin antibody (Roche Molecular Biochemicals) was diluted 1:200 in antibody blocking buffer
and applied to the wells for overnight incubation at room temperature.
The wells were washed several times with salt washes (antibody wash 1:
0.1 M Tris, pH 9.0, and 0.15 M NaCl; antibody wash 2: 0.1 M Tris, pH 9.5, 0.1 M NaCl, 50 mM MgCl2). DAPI was added at a final
concentration of 1 µg/ml for 1 min. The slides were washed, dried,
treated with antifade, and sealed with clear nail polish.
= 365 nm) irradiating the cells four times for 2.5 min each time using
a Stratalinker (Stratagene). Cells were collected, washed with PBS, and
resuspended in lysis buffer (20 mM Tris, pH 7.5, 50 mM LiCl, 1% SDS, 1 mM EDTA, and protease and
RNase inhibitors) with glass beads. Samples were vortexed for 20 min at
4 °C to lyse the cells and clarified by centrifugation at 13,000 rpm
for 15 min. Lysates were incubated with LiCl, oligo(dT) binding buffer
(10 mM Tris, pH 7.4, 1 mM EDTA, 0.5% SDS, 0.5 mM LiCl), 0.2 g of oligo(dT)25-cellulose
(New England Biolabs), and RNase inhibitor (Promega) for 30 min at room
temperature. The complexes bound to the
oligo(dT)25-cellulose were washed, resuspended in oligo(dT)
binding buffer, and poured into a polyprep column (Bio-Rad). Complexes
were eluted from the oligo(dT)25-cellulose with elution
buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 0.05%
SDS). The eluate was heated at 65 °C for 10 min, cooled on ice for
10 min, supplemented with 0.5 M LiCl, and loaded back onto
the cellulose column. The column was washed with oligo(dT) binding
buffer, and complexes were eluted with elution buffer. The eluate was
subsequently concentrated by extraction with butanol. The RNA-protein
complexes were precipitated with 0.2 M LiCl and 100%
ethanol overnight at
80 °C and resuspended in RSB (10 mM Tris, pH 7.4, 100 mM NaCl, 2.5 mM MgCl2, 1 mM CaCl2,
and protease inhibitors). Units of RNA in each sample were measured at
A260. Samples were treated with RNase and
micrococcal nuclease at 30 °C for 30 min. For each sample, 35 units
of RNA in gel loading dye was resolved on a SDS-PAGE gel and
immunoblotted with anti-Nab2p antibody (1:50,000) (17), anti-GFP
antibody (1:5000) (35), or anti-Arf1p antibody (1:1000) (36).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N) of Nab2p as their only form of Nab2p grow slowly at 30 and
37 °C and are not viable at 18 °C. In contrast, deletion of the
glutamine-rich domain (
QQQP) causes no apparent growth defect at any
temperature examined. Cells expressing a deletion of the RGG domain
(
RGG) of Nab2p grow slowly at 18 and 37 °C but show no growth
defect at 30 °C. Deletion of the entire zinc finger domain (
CCCH)
yields a non-functional protein that cannot replace the essential Nab2 protein (Fig. 2A). Identical results were obtained when each
of the Nab2 deletion mutant proteins was C-terminally tagged with GFP
and analyzed in this assay (data not shown).
View larger version (24K):
[in a new window]
Fig. 1.
Schematic of Nab2 mutant
proteins. Full-length Nab2p is shown at the top, and
the four domains within the wild-type protein are indicated by the
shading. All deletion mutants are diagrammed accordingly
with the internal deletions indicated. The amino acids deleted in each
mutant are indicated in parentheses next to their notation.
Deletion notation: N deletes the N-terminal domain;
QQQP deletes the glutamine-rich domain;
RGG
deletes the RGG domain;
CCCH deletes the entire zinc
finger domain;
C4 deletes the first four zinc finger
repeats;
C3 deletes the last three zinc finger repeats;
and
CT deletes the last 47 amino acids of Nab2p.
View larger version (92K):
[in a new window]
Fig. 2.
Functional analysis of the Nab2p deletion
mutants. A, a plasmid shuffle technique was used to
assess the function of each deletion mutant. NAB2 deletion
cells (ACY429) maintained by a URA3-NAB2 plasmid
(pAC636) were transformed with plasmids carrying vector alone (pAC3),
wild-type NAB2 (pAC717), or one of the indicated deletion
mutants. Cells were spotted onto a control plate (URA
glucose) or selective media (5-FOA) at 37, 30, and 18 °C. Vector
alone and wild-type NAB2 served as the negative and positive
growth controls. B, wild-type cells (ACY192) expressing
wild-type (pAC753) or mutant GFP-tagged Nab2 proteins were prepared for
immunoblot analysis, and lysates were probed with an anti-GFP antibody
(35). C, NAB2 deletion cells (ACY429) expressing
wild-type NAB2 (pAC717) or each of the NAB2
deletion mutants were grown to log phase at 30 °C, incubated at 30 or 18 °C for 3 h, and prepared for FISH. Poly(A) RNA was
detected using an oligo(dT) probe. The nucleus is indicated by DAPI
staining of the chromatin. Corresponding DIC images are shown.
CCCH) as their only copy of NAB2 are
inviable, we analyzed the expression of each of the mutant proteins in
a wild-type background where the endogenous NAB2 gene is
intact. As shown in Fig. 2B, all the mutant proteins were
expressed at their predicted sizes at levels comparable with that of
wild-type Nab2p-GFP. Similar results were obtained for the untagged
mutant proteins when they were detected with an anti-Nab2p antibody
(data not shown). Furthermore, we analyzed the expression of the
N-GFP mutant protein, which confers strong cold sensitivity, at both
30 and 18 °C. The GFP-tagged N-terminal deletion mutant was
expressed at the same level as wild-type Nab2p-GFP at both temperatures
tested (data not shown).
N mutant cells show nuclear accumulation of bulk poly(A) RNA at both 30 °C,
where they grow very slowly, and 18 °C, where they do not grow at
all (Fig. 2C). Cells that express the
QQQP mutant have no
apparent defect in poly(A) RNA export (Fig. 2C). Cells
expressing
RGG accumulate poly(A) RNA in the nucleus at 18 °C in
~50% of the population but export poly(A) RNA efficiently at
30 °C. Localization of poly(A) RNA could not be analyzed in cells
expressing the
CCCH mutant as the only copy of NAB2
because these cells are inviable (see Fig. 2A). Taken
together, these results demonstrate that the N-terminal domain and RGG
domain of Nab2p are both required for efficient export of poly(A) RNA
from the nucleus.
N-GFP and
QQQP-GFP mutant proteins were
localized in the nucleus at steady state. The zinc finger deletion
mutant (
CCCH-GFP) protein was localized in the nucleus with a low
level also present in the cytoplasm. We determined that the nuclear
localization of
CCCH-GFP is dependent on the Nab2p import receptor,
Kap104p (20) (data not shown), suggesting that, despite the large
deletion, this protein is sufficiently folded to be targeted to the
nucleus by the same mechanism as wild-type Nab2p. In contrast to the
other mutant proteins, the
RGG-GFP mutant protein is localized
throughout the cell (Fig. 3A). We also observed this
mislocalization with an untagged
RGG protein detected with an
anti-Nab2p antibody (data not shown). These results are consistent with
previous work (19, 20, 24) demonstrating that the RGG domain mediates the interaction between Nab2p and its import receptor, Kap104p. Identical localization patterns were observed when cells were fixed,
and GFP-tagged Nab2 proteins were visualized using an anti-GFP antibody
(Fig. 3B).
View larger version (98K):
[in a new window]
Fig. 3.
Localization of the Nab2p deletion
mutants. A, wild-type (pAC753) or each mutant Nab2-GFP
protein was expressed in wild-type cells (ACY192), and their
localization was examined by direct fluorescence microscopy.
Corresponding DIC images are shown. B, wild-type (pAC753) or
each mutant Nab2-GFP protein was expressed in wild-type cells (ACY192).
The cells were fixed and prepared for indirect immunofluorescence, and
Nab2p-GFP proteins were detected with an anti-GFP antibody (35). The
nuclei are indicated by the DAPI-stained chromatin. Corresponding DIC
images are shown. C, wild-type (ACY192) or rat7-1
(ACY194) cells expressing RGG-GFP (pAC980) were grown to log phase
at 25 °C and shifted to 37 °C for 15 min, and GFP-tagged proteins
were viewed by direct fluorescence microscopy. Corresponding DIC images
are shown. D, the nuclear export assay was performed using
nup49-313 cells (ACY480) as described under "Experimental
Procedures." Cells expressing Nab2p-GFP (pAC719),
N-GFP (pAC1050),
or
QQQP-GFP (pAC1051) were treated with cycloheximide and shifted to
37 °C. The localization of GFP-tagged proteins was analyzed by
direct fluorescence microscopy. Corresponding DIC images are
shown.
RGG-GFP mutant protein, which lacks the Kap104p-binding site
(19, 24), is localized throughout the cell at steady state (Fig. 3,
A and B). Based solely on this observation, it is
difficult to determine whether this mutant protein never enters the
nucleus or, alternatively, whether the mutant protein still shuttles
but enters the nucleus at a slower rate than wild-type Nab2p so that
the steady state localization is now cytoplasmic. To distinguish
between these two possibilities, we localized the
RGG-GFP protein in
cells containing a temperature-sensitive allele (rat7-1) of
the nucleoporin, Rat7p/Nup159p (42). rat7-1 cells are
defective in macromolecular export at the non-permissive temperature (37 °C) (41-43). If the
RGG-GFP protein is imported into the
nucleus, then it should accumulate in the nucleus of rat7-1
cells at 37 °C, where export is blocked. However, if
RGG-GFP
never enters the nucleus, there should be no difference in localization
of
RGG-GFP in the rat7-1 cells as compared with wild-type
cells, and the protein should remain in the cytoplasm. As shown in Fig. 3C, the
RGG-GFP protein accumulates within the nucleus of
the rat7-1 cells following a 15-min shift to the
non-permissive temperature. These results show that
RGG-GFP is, in
fact, imported into the nucleus in an RGG-independent manner.
Furthermore, this experiment demonstrates that the export of Nab2p from
the nucleus is dependent on the nucleoporin, Rat7p/Nup159p.
N and
QQQP mutant proteins, which are both strictly nuclear at steady state, could be analyzed using this assay.
As shown in Fig. 3D, the
N-GFP mutant protein remains within the nucleus indicating that export of this mutant protein is
blocked. In contrast, the
QQQP-GFP mutant protein is detected in the cytoplasm demonstrating that deletion of this domain does not
impact Nab2p export from the nucleus (Fig. 3D). Thus,
analysis of the Nab2p deletion mutants reveals that the N-terminal
domain of Nab2p is required for export of both poly(A) RNA and Nab2p from the nucleus.
N) is not
efficiently exported from the nucleus (see Fig. 3D). One
possible explanation for this defect is that this N-terminal domain may
contain a signal for Nab2p export. To test this possibility, we
generated a nuclear localized reporter protein containing an SV40
bipartite nuclear localization signal (NLS) fused to two GFP molecules
(NLS-GFP)2 (45) and a
modified NLS-GFP protein that also incorporates the N-terminal domain
(amino acids 1-97) of Nab2p (NLS-N(Nab2p)-GFP). Unlike the strictly
nuclear NLS-GFP protein, the NLS-N(Nab2p)-GFP shows some cytoplasmic
signal (Fig. 4, A and
C). The
NLS-NES-GFP3 reporter
protein, which contains the classical leucine-rich NES (41), is
exported from the nucleus more efficiently than the NLS-N(Nab2p)-GFP
reporter protein (Fig. 4E).
View larger version (60K):
[in a new window]
Fig. 4.
The N-terminal domain of Nab2p can direct
nuclear export. Wild-type (ACY192) cells expressing either NLS-GFP
(pAC1057), NLS-N(Nab2p)-GFP (pAC1193), or an NLS-NES-GFP (pAC1345)
control protein were grown to log phase, and protein localization was
examined by direct fluorescence of GFP (A, C, and
E). NLS-GFP (pAC1057), NLS-N(Nab2p)-GFP (pAC1193), and
NLS-NES-GFP (pAC1345) were independently expressed in rat7-1
cells (ACY194). The cells were maintained at 25 °C
(G, K, and O) or shifted to 37 °C
(I, M, and Q), and the localization of
the GFP-tagged proteins was examined by direct fluorescence microscopy.
Corresponding DIC images are shown (B, D,
F, H, J, L, N,
P, and R).
N mutant suggests a model where Nab2p associates with poly(A) RNA in
the nucleus (12), and the N-terminal domain of Nab2p facilitates export of both poly(A) RNA and Nab2p.
N Nab2 mutant protein cannot exit the nucleus
may explain why poly(A) RNA export is blocked in these mutant cells
(see Fig. 2C). Perhaps, the
N mutant protein still binds
poly(A) RNA within the nucleus but then cannot exit the nucleus and
consequently accumulates in the nucleus bound to poly(A) RNA. This
explanation is only valid if the
N Nab2p mutant binds to poly(A)
RNA. To determine whether the
N Nab2 protein binds to poly(A) RNA,
we performed an in vivo RNA/protein cross-linking experiment. Cells expressing either wild-type or
N Nab2p were UV
cross-linked, and poly(A) RNA complexes were isolated. Nab2p present in
the lysate (L), bound (B), or unbound
(U) fraction was detected by immunoblot analysis. For each
experiment, an equal amount of cross-linked RNA from each sample was
analyzed (Fig. 5A), and the
amount of Nab2 protein bound was quantified (Fig. 5B) by
fluoroimaging of the immunoblots as described under "Experimental Procedures." Wild-type Nab2p is detected in complex with poly(A) RNA
in this experiment as indicated by the band in the bound fraction (Fig.
5A, middle row, lanes 2 and
5, and 5B). The
N mutant protein also binds to
poly(A) RNA at both 18 °C and 30 °C (Fig. 5A,
top row, lanes 2 and 5, and
B). In fact, more
N Nab2p is detected in complex with
poly(A) RNA than wild-type Nab2p, which could be a result of increased
levels of
N Nab2p in the nucleus because
N does not appear to
exit the nucleus (see Fig. 3D). The cytoplasmic GTPase,
Arf1p, was used as a control for nonspecific binding. The anti-Arf1p
immunoblot from the wild-type cross-linking experiment is shown in Fig.
5A (bottom row). In addition, we did not observe any nonspecific Nab2p binding to the oligo(dT) column when UV cross-linking was not performed prior to isolation of poly(A) RNA (data
not shown).
View larger version (47K):
[in a new window]
Fig. 5.
In vivo cross-linking of
N to poly(A) RNA. NAB2 deletion
cells (ACY429) expressing only wild-type Nab2p (pAC717) or
N Nab2p
(pAC1152) were grown to saturation, diluted, and incubated at 30 or
18 °C. Cells were collected by centrifugation and cross-linked by UV
irradiation. Cell lysates were incubated with oligo(dT)-cellulose and
washed. Complexes bound to the cellulose were eluted and examined by
SDS-PAGE and immunoblotting. L indicates lysate,
B indicates bound/eluted fraction, and U
indicates unbound fraction. A typical experiment is shown in
A, and quantification of the data from two independent
experiments is shown in B. A, poly(A) RNA-protein
complexes were examined from cross-linked cells expressing either the
N (top row) or wild-type Nab2p (middle and
bottom rows). Immunoblots were probed with an anti-Nab2p
antibody (17) (top and middle rows) or a control
anti-Arf1p antibody (36) (bottom row). B, the
amount of bound mutant Nab2 protein was quantified as described under
"Experimental Procedures" and compared with the corresponding band
for bound wild-type Nab2p, which was set to 1.0. The error
bars indicate the S.E. in the data.
N mutant supports a model where Nab2p binds poly(A)
RNA in the nucleus and the N-terminal domain mediates a critical
interaction that facilitates export of poly(A) RNA-mRNP complex
to the cytoplasm. This model predicts that if the wild-type and
N
Nab2 proteins are co-expressed, the
N Nab2 protein should compete
with wild-type Nab2p for binding to poly(A) RNA. Consequently, cells
that overexpress
N Nab2p, even in the presence of wild-type Nab2p,
should accumulate poly(A) RNA in the nucleus. To test this prediction,
we examined poly(A) RNA localization and cell growth in wild-type cells
that overexpress
N Nab2p. For these experiments,
N
NAB2 was placed under the control of a galactose-inducible promoter. As shown in Fig. 6A,
wild-type cells that overexpress
N Nab2p accumulate poly(A) RNA in
the nucleus (Fig. 6A, panel J) whereas cells that
overexpress wild-type Nab2p export poly(A) RNA to the cytoplasm (Fig.
6A, panel D). Because overexpression of
N in wild-type cells blocks the essential process of poly(A) RNA
export, we predict that overexpression of
N should also inhibit cell
growth. Thus, growth of wild-type cells either expressing (Gal plate)
or not expressing (Glu plate)
N Nab2p was examined. Cells that
overexpress
N Nab2p grow more slowly than cells that express either
wild-type Nab2p or an empty vector (Fig. 6B, Gal plate). A dominant negative mutant of yeast Ran
(gsp1R79E (46)) was included as a control (Fig.
6B, Gal plate). Immunoblot analysis demonstrates
that the galactose-inducible Nab2 and
N proteins are expressed at
similar levels upon induction with galactose (data not shown). These
results are consistent with the predictions of our model.
View larger version (76K):
[in a new window]
Fig. 6.
Overexpression of N
blocks poly(A) RNA export. Wild-type cells (ACY192) were
transformed with plasmids containing galactose-inducible
NAB2 (pAC888),
N (pAC1248), vector (pAC17), or
a control gsp1R79E protein (pAC422) (46). A,
cells were grown in glucose or galactose and prepared for FISH
analysis. The nuclei are indicated by the DAPI-stained chromatin.
Corresponding DIC images are shown. B, cells were serially
diluted and spotted onto media containing glucose (no expression) or
galactose (expression).
C4), the last three repeats (
C3), or the
last 47 amino acids of Nab2p (
CT), which lie outside of the zinc
finger domain (see Fig. 1). The C-terminal mutants were expressed in
nab2 cells that were maintained by a plasmid-borne copy
of NAB2, and a plasmid shuffle was used to assess the
function of each mutant as compared with wild-type Nab2p (Fig.
7A). Cells expressing any of
the zinc finger deletion mutants (
CCCH,
C4, or
C3) as their
only copy of NAB2 were inviable indicating that these mutant
proteins are non-functional. In contrast, deletion of the last 47 amino
acids (
CT) has no effect on Nab2p function because cells expressing
this mutant grew well at all temperatures tested (18, 25, 30, and
37 °C) (Fig. 7A and data not shown). As shown in Fig.
7B, all the C-terminal mutant proteins were expressed.
Furthermore, the size of each mutant protein was consistent with the
predicted molecular weight.
View larger version (91K):
[in a new window]
Fig. 7.
Analysis of deletion mutants within the zinc
finger domain of Nab2p. A, plasmid shuffle technique
was used to examine the function of each deletion within the zinc
finger domain. NAB2 deletion cells (ACY429) maintained by a
URA3-NAB2 plasmid (pAC636) were transformed with
plasmids carrying either vector alone (pAC3), wild-type NAB2
(pAC1110), or each of the C-terminal deletion mutants. Cells were
grown, counted, and serially spotted onto URA glucose or
selective media (5-FOA) at 30 °C. Vector alone and wild-type
NAB2 served as negative and positive controls for growth.
B, wild-type cells (ACY192) expressing wild-type (pAC719) or
mutant GFP-tagged Nab2 proteins were prepared for immunoblot analysis,
and lysates were probed with an anti-GFP antibody (35). C,
wild-type (pAC719) or each mutant Nab2-GFP protein was expressed in
wild-type cells (ACY192), and localization was examined by direct
fluorescence microscopy. Corresponding DIC images are shown.
D, the nuclear export assay was performed using
nup49-313 cells (ACY480) as described under "Experimental
Procedures." Cells expressing Nab2p-GFP (pAC719),
C4-GFP
(pAC1155),
C3-GFP (pAC1154), or
CT-GFP (pAC1153) were treated
with cycloheximide and shifted to 37 °C. Localization of the
GFP-tagged proteins was analyzed by direct fluorescence microscopy.
Corresponding DIC images are shown.
CCCH), which is missing the entire zinc finger domain, is localized
primarily within the nucleus with some cytoplasmic signal (Figs.
3A and 7C). The steady state localization of the three C-terminal deletion proteins (
C4,
C3, and
CT) is nuclear (Fig. 7C). Therefore, deletion of each part of the zinc
finger domain results in a non-functional protein that is properly
localized to the nucleus. Furthermore, we determined that the nuclear
import of each of these deletion mutant proteins is dependent on the Nab2p import receptor, Kap104p (20) (data not shown), suggesting that
these proteins are sufficiently folded to be imported into the nucleus
by the same mechanism as full-length Nab2p. Because the smaller zinc
finger deletion variants (
C3,
C4, and
CT) were strictly
nuclear, we could determine the impact of these deletions on Nab2p
shuttling using the NUP49-based nuclear export assay. As
shown in Fig. 7D, the
C4 and
CT proteins are exported to the cytoplasm of the nup49-313 mutant cells at 37 °C
in a manner similar to wild-type Nab2p. However, deletion of the last
three zinc finger repeats (
C3) blocks nuclear export of Nab2p. These results indicate that the last three zinc finger repeats are more important for Nab2p export than the first four repeats.
CCCH,
C3, and
C4.
Wild-type Nab2p-GFP interacts with poly(A) RNA in our experiments (Fig.
8A, lane 2). Analysis of the zinc finger domain shows that deletion of the entire
zinc finger domain (
CCCH) abolishes all Nab2p-poly(A) RNA binding
(Fig. 8A, lane 5, and 8B), whereas
deletion of the last three repeats (
C3) greatly diminishes, but does
not completely eliminate, the interaction between Nab2p and poly(A) RNA
(Fig. 8A, lane 8, and 8B). Deletion of
the first four repeats (
C4) modestly diminishes the Nab2p/poly(A)
RNA interaction (Fig. 8A, lane 11, and Fig.
8B).
View larger version (43K):
[in a new window]
Fig. 8.
In vivo cross-linking of the zinc
finger deletion mutants to poly(A) RNA. Wild-type cells (ACY192)
expressing Nab2p-GFP (pAC719), CCCH-GFP (pAC1048),
C3-GFP
(pAC1153), or
C4-GFP (pAC1154) were grown to saturation, diluted,
and incubated at 30 °C. Cells were collected by centrifugation and
cross-linked by UV irradiation. Cell lysates were incubated with
oligo(dT)-cellulose and washed, and complexes bound to the cellulose
were eluted and examined by SDS-PAGE and immunoblotting. L
indicates lysate, B indicates bound/eluted fraction, and
U indicates unbound fraction. A typical experiment is shown
in A, and quantification of the data from three independent
experiments is shown in B. A, poly(A) RNA-protein complexes
were examined from cross-linked cells expressing the wild-type
Nab2p-GFP (top row, lanes 1-3) or
CCCH-GFP
(top row, lanes 4-6),
C3-GFP (bottom
row, lanes 7-9), or
C4-GFP (bottom
row, lanes 10-12) mutants. Immunoblots were probed
with an anti-GFP antibody (35). B, the amount of bound
mutant Nab2 proteins was quantified and compared with the corresponding
band for bound wild-type Nab2p, which was set to 1.0. The error
bars indicate the standard deviations in the data.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (30K):
[in a new window]
Fig. 9.
Model for Nab2p function. A,
the functions ascribed to each domain are indicated. The N-terminal
domain of Nab2p mediates an interaction with a protein to facilitate
efficient Nab2p and poly(A) RNA export. The glutamine-rich domain is
dispensable for the Nab2p functions analyzed here. The RGG domain is
methylated (17) and is required for binding to Kap104p (19, 24). The
zinc finger domain interacts with poly(A) RNA. B, model of
the relationship between the RGG domain of Nab2p and arginine
methylation-dependent nuclear export. In the nucleus, Nab2p
binds a nuclear binding partner in an arginine
methylation-dependent manner. Methylation of Nab2p disrupts
this interaction allowing Nab2p export (top). In cells
lacking HMT1, unmethylated Nab2p remains bound to the
nuclear binding partner tethering Nab2p in the nucleus and preventing
Nab2p export (middle). RGG Nab2p does not interact with
the nuclear binding partner regardless of Hmt1p function, and thus
export of
RGG Nab2p occurs (bottom).
Cells expressing the N-terminal deletion mutant of Nab2p (N) as the
only copy of NAB2 accumulate both poly(A) RNA and Nab2p within the nucleus. Because our experiments also demonstrate that deletion of the N-terminal domain does not decrease the interaction between poly(A) RNA and Nab2p, we propose that the
N protein binds
poly(A) RNA and sequesters it within the nucleus. Consistent with this
idea, we find that overexpression of the non-shuttling
N protein in
wild-type cells causes accumulation of poly(A) RNA in the nucleus. In
addition, the N-terminal domain can act as a weak nuclear export signal
in the context of a heterologous protein, SV40 NLS-GFP-GFP. Taken
together, these data could suggest that the N-terminal domain of Nab2p
mediates a critical interaction required for export of both Nab2p and
poly(A) RNA. However, because the
C3 mutant protein, which contains
an intact N-terminal domain but has decreased interaction with poly(A)
RNA, is also not exported from the nucleus, we conclude that the export
signal within the N-terminal domain is necessary but not sufficient for
export of the full-length Nab2 protein.
We show here that the RGG domain is important, but not absolutely
required, for the essential function of Nab2p. Cells expressing the RGG
deletion mutant grow well at 30 °C, but slowly at 18 °C, where a
defect in poly(A) RNA export is observed. We have no evidence that the
RGG domain plays a direct role in mediating poly(A) RNA export.
Instead, we believe that the primary role of the RGG domain is to
target Nab2p to the nucleus as suggested previously (19, 20, 24).
Although we predict that the poly(A) RNA export defect observed in
RGG cells at 18 °C results from a decreased nuclear pool of
Nab2p, we cannot rule out the possibility that the RGG domain plays
direct roles both in nuclear targeting of Nab2p and in
Nab2p-facilitated poly(A) RNA export.
It is necessary to reconcile the results of this study with previous
work that demonstrates that, as for other essential arginine-methylated yeast hnRNP proteins (16), the arginine methyltransferase, Hmt1p, is
required for export of full-length Nab2p from the nucleus (17). The
inference from this finding is that arginine methylation of Nab2p is
essential for its export from the nucleus. Although several studies
(16, 17) have demonstrated that the HMT1 gene is essential for export of arginine-methylated hnRNP proteins, none of these studies
have demonstrated that it is methylation of the actual hnRNP being
studied that is required for its export. Thus, it could be methylation
of any number of described methylation targets or as yet undiscovered
targets that are actually required for hnRNP export. In the case of
Nab2p, a number of observations must be reconciled with our model for
export. The RGG domain of Nab2p is methylated in an
Hmt1p-dependent manner, and Hmt1p is required for Nab2p
export (17), but the RGG domain is not essential for Nab2p export as
the RGG protein is apparently exported from the nucleus. One model
(Fig. 9B) consistent with these observations is that Nab2p
interacts with a protein in the nucleus via the RGG domain, and this
interaction is regulated by arginine methylation. The simplest idea is
that this interaction is disrupted by methylation. Once this
interaction is disrupted, Nab2p can exit the nucleus. For the Nab2
protein that lacks the RGG domain, the interaction with the nuclear
protein is absent, and now there is no dependence on arginine
methylation for export. Obviously, further work will be required to
understand how methylation influences hnRNP function.
The RGG protein can still enter the nucleus arguing for the
existence of a Kap104p-independent mechanism for import of Nab2p. This
idea of an alternate import pathway is supported by the following observations. Nab2p is essential for cell viability (12) which demonstrates that it is absolutely essential for some cellular process.
As suggested previously for other hnRNPs, such as Hrp1p (13), it is
likely that the essential function of Nab2p is within the nucleus (17,
21), and therefore import of Nab2p into the nucleus should be essential
for cell viability. However, the known Nab2p import receptor, Kap104p,
is not essential (47, 48) suggesting that in the absence of Kap104p
there must be another mechanism to target Nab2p into the nucleus. This
is consistent with our finding that the RGG domain is not absolutely
required for Nab2p import. Thus, it is likely that an alternate import receptor or adaptor protein can recognize another domain within Nab2p
and mediate its import. There is precedent for the existence of
multiple import mechanisms for essential nuclear proteins because a
number of different karyopherins have been identified that can mediate
the import of other essential nuclear proteins such as the TATA-binding
protein (49, 50) and histones (51, 52). Further studies will be
required to characterize the alternate import pathway for Nab2p.
Our study highlights the importance of the zinc finger domain for Nab2p
function because deletions within this domain result in a
non-functional protein. Although the deletions could impact the overall
folding of the Nab2 protein, it is important to note that all of the
C-terminal deletion mutants are imported into the nucleus in a
Kap104p-dependent manner suggesting that they are
sufficiently folded to be correctly recognized by the major Nab2p
import receptor. We propose that the C-terminal domain, consisting of
seven zinc finger repeats clustered into two groups, is essential for
the interaction between Nab2p and poly(A) RNA in vivo.
Support for this idea comes from previous in vitro data showing that deletion of the zinc finger domain specifically eliminates Nab2p binding to poly(A) homopolymers (12) and our in vivo
cross-linking data where deletion of the entire zinc finger domain
completely abolishes the interaction between Nab2p and poly(A) RNA.
Significantly, deletion of the last three zinc finger repeats (C3)
decreases the interaction between Nab2p and poly(A) RNA to ~10% of
wild-type levels. This finding suggests that the last three zinc finger repeats comprise the primary binding site for poly(A) RNA in
vivo. Interestingly, Hector et al. (21) have recently
isolated a cold-sensitive allele of NAB2,
nab2-21, which contains a deletion corresponding to most of
the C3 domain. They found that cells expressing this mutant accumulate
poly(A) RNA within the nucleus (21). This mutant phenotype could be
consistent with our finding that the last three zinc finger repeats of
Nab2p are required for binding poly(A) RNA in vivo.
The data from these analyses suggest a model where efficient export of poly(A) RNA from the nucleus requires both the N-terminal domain of Nab2p and Nab2p association with poly(A) RNA via the zinc finger domain. In this model, Nab2p is imported into the nucleus primarily by Kap104p (20), which binds to the RGG domain of Nab2p (19, 24). Once in the nucleus, Nab2p dissociates from Kap104p and binds to poly(A) RNA; a previous study has shown that Nab2p release from Kap104p requires both binding to poly(A) RNA and the GTP-bound form of the small GTPase, Ran (19). We predict that the zinc finger domain of Nab2p mediates binding to poly(A) RNA, which could cause a conformational change in Nab2p to facilitate dissociation from Kap104p. Nab2p then associates with the maturing mRNA transcript within the nucleus, and as recent evidence suggests, Nab2p may play a role in polyadenylation termination of the pre-mRNA (21). It is likely that Nab2p bound to poly(A) RNA associates with other hnRNP proteins to form an mRNP complex that is exported from the nucleus. We propose that the N-terminal domain of Nab2p mediates an interaction that facilitates the efficient export of the mRNP complex. Finally, Nab2p is released from the mRNP complex in the cytoplasm and is recycled back into the nucleus by Kap104p.
From this study, we conclude that Nab2p requires both binding to
poly(A) RNA via the zinc finger domain and interactions with the
N-terminal domain to facilitate proper export of poly(A) RNA from the
nucleus. Ultimately, in order to understand the detailed steps required
for poly(A) RNA processing and export, it will be necessary to define
the protein complexes that mediate each step in the process.
Furthermore, it will be important to understand how the individual
proteins within these complexes interact with one another and with the
poly(A) RNA substrate. The nab2 mutants that we have
generated will be useful in characterizing the relationships between
Nab2p and other poly(A) RNA-binding proteins and in identifying the
critical interactions required for export of the mRNP complex from the nucleus.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank R. Kahn and P. A. Silver for the generous gifts of anti-Arf1p antibody and anti-Npl3p antibody, respectively. We also thank H. Krebber for advice regarding the in vivo cross-linking experiment, A. E. Hodel for advice concerning analysis of the zinc finger domain of Nab2p, and A. C. Berger and A. Lange for comments on the manuscript. We also thank the members of the Corbett lab for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the National Institutes of Health (to A. H. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Member of the Summer Undergraduate Research Experience program, which is supported by the Howard Hughes Medical Institute and the National Science Foundation, at Emory University.
Supported by the Emory Minority Graduate Fellowship.
** To whom correspondence should be addressed: 4117 Rollins Research Center, Emory University, 1510 Clifton Rd., N.E., Atlanta, GA 30322. Tel.: 404-727-4546; Fax: 404-727-3549; E-mail: acorbe2@emory.edu.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M207571200
2 M. T. Harreman, G. Truscott, and A. H. Corbett, unpublished data.
3 M. Hodel and A. Hodel, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: mRNP, mRNA and heterogeneous nuclear ribonucleoprotein complex; hnRNP, heterogeneous nuclear ribonuclear particle protein; FISH, fluorescence in situ hybridization; GFP, green fluorescent protein; 5-FOA, 5-fluoroorotic acid; FITC, fluorescein isothiocyanate; DAPI, 4',6-diamidino-2-phenylindole; PBS, phosphate buffered saline; NLS, nuclear localization sequence; BSA, bovine serum albumin; DIC, differential interference contrast.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Quimby, B. B., and Corbett, A. H. (2001) Cell. Mol. Life Sci. 58, 1766-1773[Medline] [Order article via Infotrieve] |
2. |
Macara, I. G.
(2001)
Microbiol. Mol. Biol. Rev.
65,
570-594 |
3. |
Rout, M. P.,
and Aitchison, J. D.
(2001)
J. Biol. Chem.
276,
16593-16596 |
4. | Lei, E. P., and Silver, P. A. (2002) Dev. Cell 2, 261-272[Medline] [Order article via Infotrieve] |
5. | Reed, R., and Hurt, E. (2002) Cell 108, 523-531[Medline] [Order article via Infotrieve] |
6. |
Zenklusen, D.,
Vinciguerra, P.,
Strahm, Y.,
and Stutz, F.
(2001)
Mol. Cell. Biol.
21,
4219-4232 |
7. | Cramer, P., Srebrow, A., Kadener, S., Werbajh, S., de la Mata, M., Melen, G., Nogues, G., and Kornblihtt, A. R. (2001) FEBS Lett. 498, 179-182[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Akker, S. A.,
Smith, P. J.,
and Chew, S. L.
(2001)
J. Mol. Endocrinol.
27,
123-131 |
9. | Hastings, M. L., and Krainer, A. R. (2001) Curr. Opin. Cell Biol. 13, 302-309[CrossRef][Medline] [Order article via Infotrieve] |
10. | Wozniak, R. W., Rout, M. P., and Aitchison, J. D. (1998) Trends Cell Biol. 8, 184-188[CrossRef][Medline] [Order article via Infotrieve] |
11. | Marelli, M., Dilworth, D. J., Wozniak, R. W., and Aitchison, J. D. (2001) Biochem. Cell Biol. 79, 603-612[CrossRef][Medline] [Order article via Infotrieve] |
12. | Anderson, J. T., Wilson, S. M., Datar, K. V., and Swanson, M. S. (1993) Mol. Cell. Biol. 13, 2730-2741[Abstract] |
13. |
Kessler, M. M.,
Henry, M. F.,
Shen, E.,
Zhao, J.,
Gross, S.,
Silver, P. A.,
and Moore, C. L.
(1997)
Genes Dev.
11,
2545-2556 |
14. | Flach, J., Bossie, M., Vogel, J., Corbett, A. H., Jinks, T., Willins, D. A., and Silver, P. A. (1994) Mol. Cell. Biol. 14, 8399-8407[Abstract] |
15. | Henry, M. F., and Silver, P. A. (1996) Mol. Cell. Biol. 16, 3668-3678[Abstract] |
16. |
Shen, E. C.,
Henry, M. F.,
Weiss, V. H.,
Valentini, S. R.,
Silver, P. A.,
and Lee, M. S.
(1998)
Genes Dev.
12,
679-691 |
17. |
Green, D. M.,
Marfatia, K. A.,
Crafton, E. B.,
Zhang, X.,
Cheng, X.,
and Corbett, A. H.
(2002)
J. Biol. Chem.
277,
7752-7760 |
18. | Duncan, K., Umen, J. G., and Guthrie, C. (2000) Curr. Biol. 10, 687-696[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Lee, D. C.,
and Aitchison, J. D.
(1999)
J. Biol. Chem.
274,
29031-29037 |
20. |
Aitchison, J. D.,
Blobel, G.,
and Rout, M. P.
(1996)
Science
274,
624-627 |
21. |
Hector, R. E.,
Nykamp, K. R.,
Dheur, S.,
Anderson, J. T.,
Non, P. J.,
Urbinati, C. R.,
Wilson, S. M.,
Minvielle-Sebastia, L.,
and Swanson, M. S.
(2002)
EMBO J.
21,
1800-1810 |
22. | Siomi, H., and Dreyfuss, G. (1995) J. Cell Biol. 129, 551-560[Abstract] |
23. | Liu, Q., and Dreyfuss, G. (1995) Mol. Cell. Biol. 15, 2800-2808[Abstract] |
24. | Truant, R., Fridell, R. A., Benson, E. R., Herold, A., and Cullen, B. R. (1998) Eur. J. Cell Biol. 77, 269-275[Medline] [Order article via Infotrieve] |
25. |
Lai, W. S.,
Kennington, E. A.,
and Blackshear, P. J.
(2002)
J. Biol. Chem.
277,
9606-9613 |
26. | Leon, O., and Roth, M. (2000) Biol. Res. 33, 21-30[Medline] [Order article via Infotrieve] |
27. | Yano, R., and Nomura, M. (1991) Mol. Cell. Biol. 11, 754-764[Medline] [Order article via Infotrieve] |
28. | Yano, R., Oakes, M., Tabb, M. M., and Nomura, M. (1992) Mol. Cell. Biol. 12, 5640-5651[Abstract] |
29. |
Berg, J. M.
(1990)
J. Biol. Chem.
265,
6513-6516 |
30. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual , 3rd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
31. | Adams, A., Gottschling, D. E., Kaiser, C. A., and Stearns, T. (1997) Methods in Yeast Genetics , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
32. | Elion, E. A. (1993) in Current Protocols in Molecular Biology (Ausubel, F. E. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struhl, K., eds), Vol. 1 , pp. 3.17.1-3.17.10, John Wiley & Sons, Inc., New York |
33. | Boeke, J. D., Truehart, J., Natsoulis, G., and Fink, G. (1987) Methods Enzymol. 154, 164-175[Medline] [Order article via Infotrieve] |
34. | Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract] |
35. |
Seedorf, M.,
Damelin, M.,
Kahana, J.,
Taura, T.,
and Silver, P. A.
(1999)
Mol. Cell. Biol.
19,
1547-1557 |
36. |
Kahn, R. A.,
Clark, J.,
Rulka, C.,
Stearns, T.,
Zhang, C. J.,
Randazzo, P. A.,
Terui, T.,
and Cavenagh, M.
(1995)
J. Biol. Chem.
270,
143-150 |
37. | Lee, M. S., Henry, M., and Silver, P. A. (1996) Genes Dev. 10, 1233-1246[Abstract] |
38. | Doye, V., Wepf, R., and Hurt, E. C. (1994) EMBO J. 13, 6062-6075[Abstract] |
39. | Wong, D. H., Corbett, A. H., Kent, H. M., Stewart, M., and Silver, P. A. (1997) Mol. Cell. Biol. 17, 3755-3767[Abstract] |
40. | Amberg, D. C., Goldstein, A. L., and Cole, C. N. (1992) Genes Dev. 6, 1173-1189[Abstract] |
41. |
Krebber, H.,
Taura, T.,
Lee, M. S.,
and Silver, P. A.
(1999)
Genes Dev.
13,
1994-2004 |
42. | Gorsch, L. C., Dockendorff, T. C., and Cole, C. N. (1995) J. Cell Biol. 129, 939-955[Abstract] |
43. | DelPriore, V., Snay, C. A., Bahr, A., and Cole, C. N. (1996) Mol. Biol. Cell 7, 1601-1621[Abstract] |
44. | Schlenstedt, G., Hurt, E., Doye, V., and Silver, P. A. (1993) J. Cell Biol. 123, 785-798[Abstract] |
45. |
Hodel, M. R.,
Corbett, A. H.,
and Hodel, A. E.
(2001)
J. Biol. Chem.
276,
1317-1325 |
46. | Kent, H. M., Moore, M. S., Quimby, B. B., Baker, A. M., McCoy, A. J., Murphy, G. A., Corbett, A. H., and Stewart, M. (1999) J. Mol. Biol. 289, 565-577[CrossRef][Medline] [Order article via Infotrieve] |
47. | Schaaff-Gerstenschlager, I., Schindwolf, T., Lehnert, W., Rose, M., and Zimmermann, F. K. (1995) Yeast 11, 79-83[Medline] [Order article via Infotrieve] |
48. | Entian, K. D., Schuster, T., Hegemann, J. H., Becher, D., Feldmann, H., Guldener, U., Gotz, R., Hansen, M., Hollenberg, C. P., Jansen, G., Kramer, W., Klein, S., Kotter, P., Kricke, J., Launhardt, H., Mannhaupt, G., Maierl, A., Meyer, P., Mewes, W., Munder, T., Niedenthal, R. K., Ramezani Rad, M., Rohmer, A., Romer, A., Hinnen, A., et al.. (1999) Mol. Gen. Genet. 262, 683-702[Medline] [Order article via Infotrieve] |
49. |
Morehouse, H.,
Buratowski, R. M.,
Silver, P. A.,
and Buratowski, S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12542-12547 |
50. |
Pemberton, L. F.,
Rosenblum, J. S.,
and Blobel, G.
(1999)
J. Cell Biol.
145,
1407-1417 |
51. |
Mosammaparast, N.,
Jackson, K. R.,
Guo, Y.,
Brame, C. J.,
Shabanowitz, J.,
Hunt, D. F.,
and Pemberton, L. F.
(2001)
J. Cell Biol.
153,
251-262 |
52. |
Mosammaparast, N.,
Guo, Y.,
Shabanowitz, J.,
Hunt, D. F.,
and Pemberton, L. F.
(2002)
J. Biol. Chem.
277,
862-868 |
53. | Winston, F., Dollard, C., and Ricupero-Hovasse, S. L. (1995) Yeast 11, 53-55[Medline] [Order article via Infotrieve] |
54. |
Belgareh, N.,
Snay-Hodge, C.,
Pasteau, F.,
Dagher, S.,
Cole, C. N.,
and Doye, V.
(1998)
Mol. Biol. Cell
9,
3475-3492 |
55. |
Sikorski, R. S.,
and Hieter, P.
(1989)
Genetics
122,
19-27 |