1 Department of Molecular Biology, Massachusetts General Hospital, 50 Blossum
Street, Boston, MA 02114, USA
2 The Dana-Farber Cancer Institute, 1 Jimmy Fund Way, Boston, MA 02115,
USA
* Author for correspondence (e-mail: pamela_silver{at}dfci.harvard.edu )
Accepted 7 May 2002
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Summary |
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Key words: Ribosome, Nuclear export, In situ hybridization
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Introduction |
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All macromolecular transport into or out of the nucleus is believed to
occur through the nuclear pore complex (NPC). In yeast, the nuclear pore is a
60 MDa complex composed of proteins called nucleoporins (reviewed by
Davis, 1995;
Fabre and Hurt, 1997
). The
dimensions of the nuclear pore channel are such that only one ribosomal
subunit could pass through at any given time. Several models have been put
forth for how individual proteins pass through the NPC on their way into the
nucleus (Ben-Efraim and Gerace,
2001
; Ribbeck et al.,
1999
; Rout et al.,
2000
). However, the precise mechanism for nuclear exit of large
ribonucleoprotein complexes such as the ribosomal subunits remains
unknown.
One key determinant of the directionality of nuclear transport is the
nucleotide-bound state of the small GTPase Ran (reviewed by
Koepp and Silver, 1996).
Directionality is determined by a GTPase-activating protein, in yeast termed
Rnaq, and by a guanine exchange factor, in yeast termed Prp20 (Rcc1 in
mammals). Rna1 is localized in the cytoplasm while Prp20 is inside the
nucleus. Consequently, the concentration of Ran-GDP is elevated in the
cytoplasm while the concentration of Ran-GTP is elevated in the nucleoplasm.
The nucleotide-bound state of Ran affects its interactions with nuclear
transport factors containing a Ran-binding domain. One family of yeast
Ran-binding proteins includes Yrb1 (RanBP1 in mammals), a protein that resides
in the cytoplasm, and Yrb2 (RanBP3 in mammals), a protein that resides in the
nucleus (Mueller et al., 1998
;
Schlenstedt et al., 1995
;
Taura et al., 1997
). The
second family of Ran-binding proteins consists of transport receptors termed
importins, exportins or karyopherins (reviewed by
Görlich and Kutay,
1999
).
Importins/karyopherins bind cargo in the cytoplasm and enter the nucleus
through the NPC. Once inside the nucleus, the protein cargo dissociates from
its importer. This dissociation is driven by Ran-GTP. Conversely, proteins are
exported out of the nucleus by binding to exportins in association with
Ran-GTP. Once in the cytoplasm the export complex dissociates when the GTP of
Ran is hydrolyzed (Görlich and Kutay,
1999).
Crm1/Xpo1 (or exportin) is the major karyopherin that exports
various cargoes out of the nucleus. Crm1 is perhaps best known for exporting
proteins that have the consensus leucine-rich nuclear export sequence (NES)
(Fornerod et al., 1997;
Fukuda et al., 1997
;
Neville et al., 1997
;
Stade et al., 1997
). Crm1 also
exports some proteins that do not bear the consensus leucine-rich NES. A case
in point is snurportin, a protein involved in U snRNP import
(Huber et al., 1998
;
Paraskeva et al., 1999
).
Additionally, Crm1 exports some of its cargo through adapter proteins. Crm1
exports U snRNAs and the HIV intron-containing RNA through the PHAX
(phosphorylated adapter for RNA export) and
HIV Rev adapters, respectively (Askjaer et
al., 1998
; Ohno et al.,
2000
). Recently, Crm1 has been shown to be important for the
nuclear export of the large ribosomal subunit using the NES-bearing adaptor
Nmd3 (Gadal et al., 2001
;
Ho et al., 2000b
). No such
adaptor has yet been determined for the small subunit, but analysis of a
crm1/xpo1-1 mutant suggested that export of the small subunit relies
on the NES/Crm1 pathway (Moy and Silver,
1999
).
Yrb2 is required for the efficient nuclear export of proteins containing a
leucine-rich NES (Taura et al.,
1998). In vitro studies show that Yrb2 can bind to Ran-GTP but not
Ran-GDP (Noguchi et al.,
1997
). Yrb2 contains the phenylalanineglycine repeat motifs that
are also found on many nucleoporins, but Yrb2 is not a stable component of the
NPC (Taura et al., 1998
).
YRB2 is not an essential gene in yeast, but cells lacking
YRB2 are slower growing at 15°C and accumulate the NES reporter
protein in the nucleus (Taura et al.,
1998
). The mammalian Yrb2 homologue, RanBP3, has been shown to
promote the interaction of Crm1 with its substrates and thus affect export
(Englmeier et al., 2001
;
Lindsay et al., 2001
).
In order to study the export of the small ribosomal subunit in yeast, we
previously reported the implementation of a novel assay to monitor the
distribution of nascent small ribosomal subunits by in situ analysis. The
small ribosomal subunit is exported as a 43S particle containing 20S pre-rRNA.
In the cytoplasm, the 20S pre-rRNA is cleaved to produce the mature 18S rRNA
and a 209 base fragment, the 5' ITS1 RNA
(Stevens et al., 1991;
Trapman et al., 1975
;
Udem and Warner, 1973
).
Defects in the nuclear export of the small ribosomal subunit can be detected
by localizing the 5' ITS1 RNA by fluorescent in situ hybridization;
5' ITS1 RNA accumulates in the nucleoplasm in these cells. Furthermore,
in cells defective in small ribosomal subunit export, the 20S pre-rRNA does
not mature to the 18S rRNA (Moy and
Silver, 1999
). With this assay, we previously reported that export
of the small ribosomal subunit depended on the nucleotide-bound state of Ran
and certain nucleoporins. We have now further refined the assay and extended
its use to a number of export factor and nucleoporin mutants. In addition, we
have used the assay to screen a large collection of temperature-sensitive
mutants for small subunit export defects.
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Materials and Methods |
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rRNA pulse-chase experiments
In each of the following experiments, cells were grown a density of
1-2x107 cells/ml in met- dropout media. Cultures
of FY23 and PSY2070 were grown at 30°C, diluted by 1:10 with fresh media,
and shifted to 15°C for 24 hours. 5x108 cells were
concentrated to a volume of 3 ml and pulse labeled with 250 µCi of
[methyl-3H]methionine for 5 minutes to methylate rRNA (70-85
Ci/mmol, 1 mCi/ml; NEN, Boston, MA). 500 µl of culture was transferred to a
new tube, 1 ml of ice-cold media was added, the tube was centrifuged,
supernatants were removed, and the cell pellets were frozen on dry ice. Chase
was initiated by adding 150 µl unlabeled methionine at concentration of 20
mg/ml to the 2.75 ml of culture. At time points of 5, 10, 20, 40 and 60
minutes of chase, 500 µl of culture were removed and processed as described
above. RNA was isolated by the hot acid phenol method
(Ausubel et al., 1997). 10,000
cpm of radioactivity was loaded per lane onto a 1.2% agarose-formaldehyde gel.
RNA was transferred to Hybond-N+ membranes by vacuum blotting
(Amersham-Pharmacia), UV-crosslinked, and sprayed with En3hance
(NEN). The membrane was exposed to film for 4 days at -80°C.
PSY1968 and PSY1969 were grown at 30°C, and 108 cells were concentrated to a volume of 1 ml in met- media and treated with leptomycin B at a final concentration of 200 nM for 15 minutes. PSY580, PSY2090, PSY2092, and PSY2460 were grown at 25°C and shifted to 37°C for 1 hour. Cells were pulse labeled with 50 µCi of [methyl-3H]methionine for 1 minute and chased with 45 µl unlabeled methionine at a concentration of 20 mg/ml for 2, 4 or 10 minutes. Samples were processed as described above.
In separate experiments, PSY580 and PSY635 were grown at 25°C and
shifted to 37°C for 1 hour. PSY580 and PSY1772 were grown at 25°C,
shifted to 37°C for 3 hours, and then shifted back to 25°C for 0.5
hours. 109 cells were concentrated to a volume of 3 ml in
met- media, pulse labeled with 250 µCi of
[methyl-3H]methionine for 3 minutes, and chased with unlabeled
methionine at a final concentration of 1 mg/ml for 0, 3 or 10 minutes. Samples
were processed as previously described
(Moy and Silver, 1999).
Polysome profiles
Polysome profiles and ribosomal subunit profiles were performed as
described (Kressler et al.,
1997) with the following modifications. PSY2070 was covered by
pPS2098 (YRB2 TRP1 CEN) or pPS327 (TRP CEN) and grown in
trp- dropout media at 30°C to a density of
2x107 cells/ml. The culture was diluted and shifted to
15°C for 24 hours. For the polysome profiles, lysate (4 A260
units) was layered onto 10 ml linear 7-49% sucrose gradients. Samples were
centrifuged in a Beckman SW41Ti rotor for 2 hours at 261,000 g
at 4°C (Beckman Instruments, Fullerton, CA). For the ribosomal subunit
profiles, lysate (2 A260 units) was layered onto 10 ml linear
10-35% sucrose gradients and centrifuged in a Beckman SW41Ti rotor for 4 hours
at 261,000 g at 4°C. A Beckman fraction recovery system
was used to pass the gradients through a Pharmacia UV-1 monitor to measure
A254.
ts- mutant screening
The yeast strain FY23 was disrupted in XRN1
(Moy and Silver, 1999). The
xrn1
strain was mutagenized with ethyl methanesulfonate (EMS)
as described (Ausubel et al.,
1997
) to produce 50% cell death. 160 temperature-sensitive mutants
were isolated, essentially as described
(Amberg et al., 1992
).
Approximately 800 XRN1+ ts- mutants,
previously used in the screen for mRNA
trafficking mutants (RAT), were kindly provided by C. N.
Cole (Dartmouth Medical School, Hanover, NH)
(Amberg et al., 1992
). Yeast
strains were grown on YPD plates at 25°C for 3-4 days. The strains were
transferred to 2 ml YPD at a density of 107 cells/ml and cultured
for 1 hour at 25°C to allow cells to resume growth and stimulate ribosome
biogenesis. The cultures were shifted to 37°C for 1 hour. Half of the
culture was fixed with 100 µl 37% formaldehyde for 2 hours at 25°C. The
other half of the culture was shifted back to 25°C for 1 hour before
fixation. 5' ITS RNA was localized as previously described
(Moy and Silver, 1999
).
Complementation group analysis was used to determine which mutants are
defective in known nuclear transport factors. Mutants that were not identified
by complementation grouping were backcrossed to the FY23 or FY86 strains three
times (Winston et al., 1995
).
Then, the mutations were cloned by complementation of their
temperature-sensitivity with a genomic library
(Rose et al., 1987
). The
mutations were verified by complementation group analysis. The mutated gene
was PCR amplified, and the PCR product was subjected to DNA sequencing.
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Results |
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The assay for the localization of 5' ITS1 can also be carried out in
XRN1+ wild-type yeast cells. Although the loss of
cytoplasmic 5' ITS1 fluorescent signal is an easily identifiable change
in xrn1 mutants, we determined that the excess 5' ITS1
RNA in xrn1
strains can obscure a mild nuclear export defect.
In addition, we wished to use the assay to screen existing mutant collections,
which are XRN1-positive. Consequently, we have used
XRN1+ strains to examine small ribosomal subunit nuclear
export. Wild-type XRN1+ strains primarily localize
5' ITS1 RNA to the nucleolus and a very small amount to the cytoplasm
(Fig. 1d). However, in a mutant
blocked in export, accumulation of 5' ITS1 can be observed in both the
nucleolus and nucleoplasm (Fig.
1e).
Crm1 and its co-factor, Yrb2, affect the export of the small
ribosomal subunit
Cells bearing a leptomycin-B-sensitive (LMB) allele of crm1
display a block in the export of the small ribosomal subunit dependent on drug
treatment. Wild-type S. cerevisiae are insensitive to LMB because LMB
does not interact with wild-type Crm1 protein. However, the
crm1(T539C) mutant is inhibited by LMB, resulting in the nuclear
export defect of leucine-rich NES proteins
(Neville and Rosbash, 1999).
We performed the small ribosomal subunit export assay on strains treated with
LMB. LMB does not affect the localization of 5' ITS1 RNA in wild-type
(XRN1+) cells (Fig.
2Aa-c); 5' ITS1 remains localized to the nucleolus as
indicated by arrowheads, and it does not significantly accumulate in the
regions of the nucleus occupied by DAPI-stained chromatin as indicated by
arrows. However, LMB treatment causes 5' ITS1 to accumulate in the
entire nucleoplasm in crm1(T539C) cells as indicated by arrows and
arrowheads (Fig. 2Ad-f).
Mislocalization of 5' ITS1 in these cells occurs 5-15 minutes after
addition of LMB.
|
Cells deleted for YRB2, which is involved in Crm1-mediated export,
also show a defect in export of the small subunit. yrb2 cells
are cold-sensitive for growth, and export of NES-containing proteins is
blocked more so at the non-permissive temperature. While 5' ITS1 RNA is
confined to the nucleolus of wild-type cells
(Fig. 2Ba,b,e,f), 5' ITS1
localizes throughout the nucleoplasm in yrb2
cells
(Fig. 2Bc,d,g,h). The
mislocalization of 5' ITS1 in yrb2
cells is mildly
detectable at the permissive temperature of 30°C
(Fig. 2Bc,d), and the
mislocalization is more evident after the shift to the restrictive temperature
of 15°C (Fig. 2Bg,h). Since
YRB2 is not essential for growth in yeast, yrb2
cells
cannot be completely blocked in small subunit export, but this mutant may be
less efficient in this process.
In order to estimate the rate of small subunit export in
yrb2 cells, we examined pre-rRNA processing. Strains were
shifted to 15°C, pulse-labeled with [3H-methyl]-methionine for
five minutes, and then chased with excess unlabeled methionine for up to 60
minutes. In wild-type cells, 35S pre-rRNA is quickly converted to 27S and 20S
pre-rRNAs, and then these pre-rRNAs are matured into the 25S and 18S rRNAs
(Fig. 2C, lanes 1-6)
(Kressler et al., 1999
). At
15°C, wild-type cells convert 20S pre-rRNA to 18S rRNA after 10-20 minutes
of chase (Fig. 2C, lanes 1-6).
However, in yrb2
cells, this conversion is delayed and occurs
after 20-40 minutes of chase (Fig.
2C, lanes 7-12). Furthermore, the levels of 18S rRNA in
yrb2
cells at the 40-60 minute timepoints are significantly
lower than the levels of 25S rRNA. In contrast, wild-type cells produce
equimolar quantities of the 18S and 25S rRNAs since both rRNA species are
transcribed together in the 35S pre-rRNA. Therefore, in cells lacking
YRB2, the nascent small ribosomal subunit appears to be less stable
and its nuclear export is delayed.
To determine the effect of the yrb2 mutation on ribosome
activity, we examined polysome profiles. When cell lysates were subjected to
sucrose gradient centrifugation, the ribosome migrates as an 80S particle and
as polysome peaks, which contain multiple 80S ribosomes. In wild-type cells,
approximately 10% of the ribosomal subunits are not assembled into ribosomes,
and these subunits migrate as 40S or 60S peaks
(Fig. 3A, left profile). In
yrb2
cells, the amount of free 40S subunit is decreased while
the amount of free 60S ribosomal subunit is dramatically increased
(Fig. 3A, right profile). The
amount of polysomes in yrb2
is marginally decreased, and these
results suggest that the deficiency of the small ribosomal subunits is
limiting mRNA translation. We examined the relative concentration of 60S to
40S ribosomal subunits by disassociating the ribosome and separating the
subunits by sucrose gradient centrifugation. Wild-type cells contain an equal
molar ratio of 60S to 40S ribosomal subunits and the ratio of 60S:40S subunits
is 2.1:1 when measured by A254 because the 60S subunit contains
twice as much RNA as the 40S subunit (Fig.
3B, left profile). In yrb2
, the ratio of 60S:40S
is increased to 2.5:1 (t-test P<0.05,
Fig. 3B, right profile).
Therefore, the concentration of 40S ribosomal subunits is decreased by 10-20%
relative to the 60S ribosomal subunit in yrb2
cells.
|
Overexpression of YRB2 also causes a defect in small subunit
export similar to the effect on NES-containing proteins
(Taura et al., 1998).
YRB2 was expressed at high levels from a galatose-inducible promoter
in wild-type yeast cells. After a 1 hour induction with galactose, 5'
ITS1 accumulates in the nucleoplasm of cells expressing YRB2
(Fig. 4Ac-d). Overexpression of
YRB2 also causes the accumulation of 20S pre-rRNA (data not shown).
5' ITS1 localization and pre-rRNA processing was not affected in
galactose-treated cells containing the empty vector
(Fig. 4Aa-b). While expression
of YRB2 from its own promoter on a high copy plasmid is not toxic to
wild-type cells, high copy expression of YRB2 impairs the growth of
rat2ts-/nup120, a nucleoporin mutant defective in small
ribosomal subunit export (see below), at the permissive temperature of
25°C (Fig. 4B). Only
full-length YRB2 had this effect as N terminal, C terminal, or FG
repeat YRB2 truncation mutants were not toxic to rat2-1
cells (Fig. 4B)
(Taura et al., 1997
). The
percentage of rat2ts cells mislocalizing 5' ITS1 at
the permissive temperature was mildly increased in the presence of high copy
YRB2. The synergistic alteration in ribosome export in these cells
may explain their impaired growth rate. In any case, these data indicate that
proper levels of Yrb2 are required for the efficient nuclear export of the
small ribosomal subunit.
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The large ribosomal subunit appears to be exported out of the nucleus via a
Crm1-dependent mechanism (Gadal et al.,
2001; Ho et al.,
2000b
; Stage-Zimmermann et
al., 2000
). Therefore, we expected that the decreased efficiency
of NES export in yrb2
would also cause defects in 60S
ribosomal subunit export. However, we do not see mislocalization of the 60S
nuclear export reporter proteins Rp11 1b-GFP, Rp125-GFP, or
GALpro-Rp125-GFP in yrb2
cells (data not shown). It
may be that the 60S export assays are not sensitive enough to detect subtle
export defects in yrb2
cells. Alternatively, Yrb2 is not
involved in 60S export.
Identification of nucleoporin and Ran regulator mutants that are
defective in small ribosomal subunit nuclear export
In order to further characterize small ribosomal subunit export, we
performed the small ribosomal subunit export assay on libraries of
ts- mutants, and we examined the involvement of known
nuclear transport factors by analyzing the corresponding mutants in greater
detail. In addition to examining ts- mutants at the
restrictive temperature, our screens also used a shift-back protocol in which
cells at 37°C were transferred back to 25°C. This shift-back step is
useful when examining mutants that are defective in ribosome assembly at
restrictive temperatures. Ribosome assembly defects prevent the detection of
ribosome export defects. The shift-back protocol allows cells to resume
ribosome assembly, and ribosome export defects become detectable if the rate
of ribosome assembly exceeds the rate of ribosome export
(Hurt et al., 1999).
The utility of the shift-back protocol is illustrated with the nucleoporin
mutant nic96-1 that lacks the Xrn1 exonuclease
(Zabel et al., 1996). At
permissive temperatures, 5' ITS1 RNA is localized throughout the
cytoplasm in these cells similar to NIC96+
xrn1
cells (data not shown). At the restrictive temperature,
5' ITS1 accumulates in the nucleolus in the nic96-1 xrn1
strain, indicating a defect in ribosome assembly
(Fig. 5Ae-f). Upon shift-back
to permissive temperatures, the localization of 5' ITS1 RNA expands to
fill the entire nucleus (Fig.
5Ag,h). In contrast, the localization of 5' ITS1 does not
change in NIC96+ xrn1
cells at any
temperature (Fig. 5Aa-d). Upon
shifting back to permissive temperatures, the nic96-1
XRN1+ strain is less efficient than wildtype at converting 20S
pre-rRNA to 18S rRNA (Fig. 5B,
lanes 10-12). nic96-1 cells are still able to export the small
ribosomal subunit export, at a reduced rate, since 18S rRNA is produced in
these cells, but these results demonstrate that mild nuclear export defects
can be detected using this shift-back protocol.
|
The shift-back protocol is also useful in identifying mutants that affect
ribosome assembly. We previously reported that kap104-16 xrn1
and nmd5
xrn1
strains do not mislocalize 5' ITS1
RNA at 25°C or at 37°C (Moy and
Silver, 1999
). Using the shift-back protocol, we show that a small
amount of 5' ITS1 accumulates in the nucleolus of these cells
(Fig. 5Ak,l). Mammalian
orthologues of KAP104 and NMD5 have been shown to be
importers of ribosomal proteins (Jakel and
Görlich, 1998
). We interpret the defect of kap104-16
and nmd5
to be a result of decreased import of ribosomal
proteins.
In an attempt to identify additional factors important for export of the small ribosomal subunit, we screened 960 ts- mutants with the small ribosomal subunit export assay. Twelve mutants were found to mislocalize 5' ITS1 RNA. We determined that nine of these mutants are defective in the RAT2/NUP120, RAT3/NUP133, RAT7/NUP159, NSP1, GLE2/NUP40, MTR4/DOB1, PRP20 and YRB1 genes (Table 2). Two independent rat3ts- mutants were identified by this screen.
|
The RAT2, RAT3, and RAT7 (mRNA
trafficking) genes encode nucleoporins that are required for mRNA
export (Gorsch et al., 1995;
Heath et al., 1995
;
Li et al., 1995
). Both the
rat2-1 and the rat2-2 alleles display strong small ribosomal
subunit assembly and export defects. 5' ITS1 accumulated in the
nucleolus in rat2-1 xrn1
cells shifted to the restrictive
temperature (Moy and Silver,
1999
), and 5' ITS1 accumulated to the entire nucleoplasm
after shifting back to the permissive temperature (data not shown). In
rat2-1 XRN1+ cells, 5' ITS1 accumulated in the
nucleoplasm at both permissive temperatures
(Fig. 6Ab,i) and upon shift
back. These data suggest that RAT2/NUP120 is involved in both
ribosome assembly and nuclear export.
|
The rat3-1 and rat7-1 mutants have milder defects in
small ribosomal subunit nuclear export; the conditions in which these mutants
mislocalize 5' ITS1 are more limited. In rat3-1 cells,
nucleoplasmic accumulation of 5' ITS1 is most noticeable when these
cells are grown to stationary phase and then transferred to fresh media. These
conditions allow cells to resume growth which stimulates ribosome biogenesis
and facilitates the detection of ribosome export defects
(Hurt et al., 1999). In
rat7-1 cells, nucleoplasmic accumulation of 5' ITS1 was
detectable only when the mutant contained the Xrn1 exonuclease; rat7-1
xrn1
cells did not mislocalize 5' ITS1 at restrictive
temperatures or upon shift-back (Moy and
Silver, 1999
). rat7-1 XRN1+ cells accumulate
nucleoplasmic 5' ITS1 at all temperatures
(Fig. 6Ac,j;
Table 2). Furthermore, the
conversion of 20S pre-rRNA to 18S rRNA was defective in rat7-1 cells
(Fig. 6B, lanes 4-6). Similar
to the yrb2
mutant, the rat7-1 mutant appears to be
less efficient in the export of the small ribosomal subunit.
In our screen of the ts- mutant libraries, we also
identified a novel allele of the nucleoporin NSP1. Previously, we
have shown that the nsp1(10A) xrn1 strain is defective in
ribosome assembly in that it accumulated 5' ITS1 to the nucleolus at the
restrictive temperature (Moy and Silver,
1999
). Here, we identify a nsp1 mutant that encodes a
protein with a single amino acid change in which leucine 697 is altered to a
proline. The nsp1(L697P) mutant accumulates 5' ITS1 to the
entire nucleoplasm at all temperatures
(Fig. 6Ad,k;
Table 2). Addition of a plasmid
containing wild-type NSP1 rescues the mislocalization defect of the
nsp1(L697P) mutant (data not shown). At 37°C,
nsp1(L697P) is delayed in processing the 35S, 32S and 20S pre-rRNAs
(Fig. 6B, lanes 10-12). We
conclude that Nsp1 affects both assembly and nuclear export of the small
ribosomal subunit.
We identified a gle2 mutant that accumulates 5' ITS to the
nucleoplasm (Fig. 6Ae,l) and
has a mild delay in 20S pre-rRNA processing at the restrictive temperature
(Fig. 6B, lanes 13-15). This
gle2 mutant encodes a protein containing two amino acid changes in
which asparagine 273 is altered to a lysine and aspartate 290 is altered to an
asparagine. Previously, we reported that the gle2-1 xrn1
mutant does not mislocalize 5' ITS1 at permissive or restrictive
temperatures (Moy and Silver,
1999
; Murphy et al.,
1999
). The xrn1
mutation appears to have obscured
the ribosome export defect because the gle2-1 XRN1+ mutant
accumulates 5' ITS1 at the restrictive temperature (data not shown).
In addition to the nucleoporin mutants identified by our
ts- screen, we examined previously characterized
nucleoporin mutants to determine whether they affect small ribosomal subunit
export. Similar to the nic96-1 xrn1 mutant, nup49-313
xrn1
and nup116-5 xrn1
mutants accumulate 5'
ITS1 to the nucleus after shifting the cells back to their permissive
temperature. Furthermore, the nup49-313 mutant has a delay in 20S
pre-rRNA processing after the shift back in temperature (data not shown;
Table 2).
Finally, we identified two novel alleles of genes encoding regulators of the Ran GTPase: the RanGEF, PRP20, and the RanGAP accessory factor, YRB1. The yrb1 mutant encodes a protein in which phenyalanine 191 is mutated to a serine. This allele is similar to the previously identified yrb1-1 allele in that the majority of cells, when shifted to the restrictive temperature, accumulate nucleoplasmic 5' ITS1 (Fig. 6Af,m) and 20S pre-rRNA (Fig. 6B, lanes 16-18).
The novel prp20 allele is different from the much-studied
prp20-1 allele. The prp20 allele that we identified encodes
a protein in which serine 297 is converted into an asparagine. This results in
a weak allele of PRP20 with regard to nuclear transport defects.
Although this prp20(S297N) strain does not grow at 37°C, at this
restrictive temperature only a small fraction of cells (10-20%) exhibit
nucleoplasmic accumulation of 5' ITS1
(Fig. 6Ag,n; data not shown).
In contrast, greater than 95% of prp20-1 cells accumulate 5'
ITS1 at restrictive temperatures (Moy and
Silver, 1999). Furthermore, we cannot detect an mRNA export defect
in prp20(S297N) while the prp20-1 mutant has a strong mRNA
export defect (Amberg et al.,
1993
). Interestingly, the prp20(S297N) mutant was
previously identified from the same ts- mutant library in
a screen for mutants that mislocalize Np13
(Corbett and Silver, 1996
).
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Discussion |
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In previous studies, we showed that small subunit export is inhibited in
the temperature-sensitive mutant xpol-1. However, it remained a
formal possibility that this was an indirect effect since the xpol-1
mutant mislocalizes the RanGAP Rna1 to the nucleus at the restrictive
temperature resulting in the disruption of the Ran gradient
(Feng et al., 1999).
Consequently, when xpol-1 cells are shifted to the restrictive
temperature, they immediately show a defect in mRNA export
(Stade et al., 1997
), which
can be suppressed by overexpression of DBP5, an RNA helicase
essential for mRNA export (Hodge et al.,
1999
). In contrast, upon leptomycin B addition, the
crm1(T539C) mutant accumulates the NES reporter in the nucleus within
5 minutes, but the crm1(T539C) strain does not accumulate mRNA in the
nucleus until 1 hour after treatment, suggesting that the primary defect is
indeed in NES-dependent export (Neville
and Rosbash, 1999
). We now show that LMB-treated
crm1(T539C) cells accumulate the small ribosomal subunit in the
nucleus with timing similar to that of NES accumulation. Therefore, the
involvement of Crm1 in small ribosomal subunit export is likely to be
direct.
Yrb2 is a member of the Ran binding protein family and is required for the
efficient export of Crm1-mediated cargo
(Taura et al., 1998). Here, we
show that the export of the small ribosomal subunit is delayed in
yrb2
cells. Initially, we could not detect mislocalization of
5' ITS1 in yrb2
xrn1
cells because the
accumulation of cytoplasmic 5' ITS1 fragment conceals the small
ribosomal subunit export defect. However, in yrb2
XRN1+ cells we could detect nuclear accumulation of 5'
ITS1 and the delay in processing 20S pre-rRNA.
The exact function of Yrb2 in Crm1-mediated export is still not clear. In
vitro, Yrb2 disassociates the Crm1/NES/Ran-GTP export complex so Yrb2 may
function in the terminal release step in Crm1-mediated export
(Maurer et al., 2001). In
contrast, the mammalian orthologue of Yrb2, RanBP3, stimulates formation of
the export complex (Lindsay et al.,
2001
). These contrasting activities of Yrb2 and RanBP3 may stem
from different experimental conditions. When RanBP3 is at sub-stoichiometric
concentrations, the Crm1/NES/Ran-GTP complex formation is increased while
higher concentrations of RanBP3 inhibit complex formation
(Englmeier et al., 2001
).
Importantly, RanBP3 has variable effects on Crm1 export complex formation
depending on the cargo substrate. When Snurportin and the leucine-rich NES
cargos are mixed with Crm1 and RanGTP, the Crm1-Snurportin-Ran-GTP complex is
favored over the Crm1-NES-Ran-GTP complex because Snurportin forms a higher
affinity complex (Englmeier et al.,
2001
; Paraskeva et al.,
1999
). However, when RanBP3 is added to this mixture, the
Crm1/NES/RanGTP complex is efficiently formed and the complex formation of
Crm1/Snurportin/RanGTP is decreased
(Englmeier et al., 2001
).
The differential effects of RanBP3 on Crm1 function may help to explain why
loss of Yrb2 in yeast affects the biogenesis of the small ribosomal subunit
more than the biogenesis of the large ribosomal subunit. Both ribosomal
subunits appear to use a Crm1-dependent export pathway
(Gadal et al., 2001;
Ho et al., 2000b
). If Yrb2
functions similarly to RanBP3, Yrb2 may favor the nuclear export of the small
ribosomal subunit over the export of the large ribosomal subunit. The
decreased abundance of the small subunit in yrb2
cells could
be a result of the degradation of unexported small subunits. Nuclear export of
60S subunits is required for their stability
(Ho and Johnson, 1999
;
Ho et al., 2000a
).
The question remains as to how Crm1 and Yrb2 promote export. One possibility is that Crm1 binds directly to the small subunit. However, despite extensive attempts we could not detect Crm1 bound to small subunits and could not reconstitute Crm1 binding to purified ribosomal subunits. An attractive alternative is that there is an adaptor protein that promotes binding of Crm1 to the small subunit. Such an adaptor would be analogous to the role of Nmd3 in promoting binding of Crm1 to the large subunit. Yrb2 could be such an adaptor. However, YRB2 is not essential for normal growth while one might expect such an adaptor to be essential if it is a critical part of the small subunit export pathway. Further experiments will be required to identify such an adaptor.
In order to identify additional trans-acting factors involved in
small ribosomal subunit export, we screened 960 ts-
mutants with the 5' ITS1 localization assay. We identified 5 nucleoporin
mutants and 2 Ran regulator mutants that are defective in small subunit
export. Interestingly, no novel factors were identified. This could indicate
that it may be difficult to generate conditional alleles of such factors.
Alternatively, there may be inherent limitations to the assay presented here
as screens of the same collection have yielded large numbers of mutants
defective in assembly and export of the large ribosomal subunit
(Bassler et al., 2001;
Gadal et al., 2001
;
Milkereit et al., 2001
). From
our ts- screen, we identified three mutants in which the
ts- mutation was not linked to the 5' ITS1
mislocalization phenotype. After separation from the ts-
mutation, the 5' ITS1 mislocalizing mutants grew at normal rates at all
temperatures (data not shown). Since the characteristics of these mutants do
not match the phenotypes of known nuclear transport factors, these mutants may
correspond to novel factors involved in small ribosomal subunit export.
In total, we examined 17 nucleoporin mutants with the 5' ITS1
localization assay. Twelve of these mutants are defective in the assembly of
the small ribosomal subunit (Moy and
Silver, 1999). These assembly defects could be caused by
alterations in the nuclear export of mRNAs encoding ribosomal proteins or by
defects in the nuclear import of ribosomal proteins, assembly factors, or
ribosome biogenesis regulators. Seven of these nucleoporin mutants are also
defective in the nuclear export of the small ribosomal subunit
(Table 2). Two nucleoporin
mutants are defective in small ribosomal subunit export, but do not have
detectable defects in ribosome assembly. Overall, these results emphasize the
multiple transport functions of nucleoporins and the importance of the NPC in
ribosome biogenesis.
It is interesting to speculate on the role of certain nucleoporins in ribosome export. It could be that certain nucleoporins define docking sites on either side of the NPC that are critical for binding and/or release. They could also define contact sites within the NPC that the ribosome makes as it passes through the channel. The manner in which a large particle such as a ribosomal subunit passes through the NPC remains one of the outstanding questions in cell biology and further analysis of these and other transport mutants in combination with biochemical assays will help to further elucidate the process.
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References |
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