Molecular Biology of the Cell
Vol. 20, 2265–2275, April 15, 2009
Nuclear Shuttling of She2p Couples ASH1 mRNA
Localization to its Translational Repression by Recruiting
Loc1p and Puf6p
Zhifa Shen, Nicolas Paquin,* Ame ´lie Forget, and Pascal Chartrand
De ´partement de Biochimie, Universite ´ de Montre ´al, Montre ´al, QC, H3C 3J7 Canada
Submitted November 26, 2008; Revised February 10, 2009; Accepted February 12, 2009
Monitoring Editor: Marvin P. Wickens
The transport and localization of mRNAs results in the asymmetric synthesis of specific proteins. In yeast, the nucleo-
cytoplasmic shuttling protein She2 binds the ASH1 mRNA and targets it for localization at the bud tip by recruiting the
She3p–Myo4p complex. Although the cytoplasmic role of She2p in mRNA localization is well characterized, its nuclear
function is still unclear. Here, we show that She2p contains a nonclassical nuclear localization signal (NLS) that is
essential for its nuclear import via the importin ? Srp1p. Exclusion of She2p from the nucleus by mutagenesis of its NLS
leads to defective ASH1 mRNA localization and Ash1p sorting. Interestingly, these phenotypes mimic knockouts of LOC1
and PUF6, which encode for nuclear RNA-binding proteins that bind the ASH1 mRNA and control its translation. We find
that She2p interacts with both Loc1p and Puf6p and that excluding She2p from the nucleus decreases this interaction.
Absence of nuclear She2p disrupts the binding of Loc1p and Puf6p to the ASH1 mRNA, suggesting that nuclear import
of She2p is necessary to recruit both factors to the ASH1 transcript. This study reveals that a direct coupling between
localization and translation regulation factors in the nucleus is required for proper cytoplasmic localization of mRNAs.
The cytoplasmic transport and localization of mRNAs is
used by several eukaryotic organisms to control, in space
and time, the expression of proteins involved in cell fate
determination, cellular polarity, or asymmetric cell division
(St. Johnston, 2005; Du et al., 2007). With more than 30
mRNAs localized at the bud tip, the budding yeast Saccha-
romyces cerevisiae is an excellent model system for studying
the mechanisms behind mRNA transport and localization
(Chartrand et al., 2001; Darzacq et al., 2003). One of these
transcripts is the ASH1 mRNA, which is localized at the
distal tip of daughter cells during late anaphase (Long et al.,
1997; Takizawa et al., 1997). This localization promotes the
asymmetric sorting of Ash1p to the daughter cell nucleus
and results in the inhibition of mating-type switching in the
daughter cell (Jansen et al., 1996; Sil and Herskowitz, 1996).
The RNA-binding protein She2p is responsible for the rec-
ognition of bud-localized mRNAs, via its interaction with
specific localization elements in these transcripts (Olivier
et al., 2005). She2p also interacts with the transport machin-
ery, constituted of the type V myosin Myo4p, via the bridg-
ing protein She3p (Bohl et al., 2000; Long et al., 2000;
Takizawa and Vale, 2000). During its transport to the bud
tip, the ASH1 mRNA is translationally repressed by the
RNA-binding proteins Khd1p (Irie et al., 2002) and Puf6p
(Gu et al., 2004), and phosphorylation of these factors at the
bud tip promotes the local synthesis of the Ash1 protein
(Paquin et al., 2007; Deng et al., 2008).
Previous studies have shown that nuclear factors play an
important role in yeast mRNA localization. Puf6p and Loc1p
are predominantly nucleolar RNA-binding proteins that
bind directly the 3? untranslated region (UTR) of ASH1
mRNA in vitro and are involved in the translational repres-
sion of this transcript (Long et al., 2001; Gu et al., 2004; Komili
et al., 2007). Knockouts of these genes disrupt ASH1 mRNA
localization and Ash1p sorting to the daughter cell (Long
et al., 2001; Gu et al., 2004). She2p shuttles between the
cytoplasm and the nucleus (Kruse et al., 2002), and recent
evidence suggests that the nuclear transit of She2p is in-
volved in the translational regulation of ASH1 mRNA (Du
et al., 2008). However, the specific role of She2p in the
nucleus is still unclear.
In this study, we show that She2p contains a nonclassical
nuclear localization signal (NLS) that is essential for its
nuclear import by the importin ? Srp1p. Exclusion of She2p
from the nucleus by mutagenesis of its NLS leads to defec-
tive mRNA localization and Ash1p sorting. We find that
nuclear She2p is associated with Puf6p and Loc1p indepen-
dently of their interaction with RNA. Exclusion of She2p
from the nucleus decreases its interaction with Loc1p and
Puf6p and disrupts the binding of these factors to the ASH1
mRNA. This study leads us to suggest a mechanism where
She2p interacts with the translation regulation factors Puf6p
and Loc1p in the nucleus and recruits these factors to the
ASH1 mRNA, thereby promoting the localization of this
transcript at the bud tip. This coordinated recruitment of a
localization factor and translational repressors to ASH1 tran-
scripts in the nucleus suggests that mRNA transport and
translational control machineries are coupled and that this
coupling in the nucleus is required for proper cytoplasmic
localization and local translation of this transcript.
This article was published online ahead of print in MBC in Press
on February 25, 2009.
* Present address: Department of Biology, MIT, Cambridge, MA 02139.
Abbreviations used: NLS: nuclear localization signal.
© 2009 by The American Society for Cell Biology2265
MATERIALS AND METHODS
Growth Media and Yeast Strains
Yeast cells were grown in either synthetic growth media lacking the nutrients
indicated or rich media (Rose et al., 1990). Transformation was performed
according to the protocol of Schiestl and Giestz (1989). Yeast gene disruption
cassette was created by PCR amplification of the loxP-KAN-loxP construct in
plasmid pUG6 and primers specifics for the gene of interest (Guldener et al.,
1996). Specific disruption was confirmed by PCR analysis of genomic DNA.
Yeasts strains and plasmids used in this study are described in Supplemen-
tary Tables S1 and S2, respectively.
Immunoprecipitation and Reverse Transcription-PCR
Fifty milliliters of yeast cells were grown to early log phase (OD600? 1) at
30°C in the appropriate medium. Formaldehyde was added to a final con-
centration of 1%, and cells were incubated at room temperature (RT) for 20
min. Glycine was added to a final concentration of 300 mM. Cells were
washed twice in 1? PBS, harvested by centrifugation, and resuspended at an
OD600of 100 in the extraction buffer (25 mM HEPES-KOH, pH 7.5, 150 mM
KCl, 2 mM MgCl2, 0.1% IGEPALCA-630, 1 mM dithiothreitol, 87.5 ?g/ml
phenylmethylsulfonyl fluoride, 0.5 ?g/ml pepstatin, 0.5 ?g/ml leupeptin, 0.5
?g/ml aprotinin, and 23 U/ml RNAguard). The cells were broken with glass
beads, vortexed five times for 30 s, on ice with a 1-min pause between each
vortex. The supernatant was used for immunoprecipitation and Western blot.
For the immunoprecipitation of myc-tagged She2p, 10 ?g of anti-myc anti-
body (9E10) was added to 500 ?l of supernatant and incubated at 4°C with
agitation for 1 h; 50 ?l of protein A-Sepharose beads was then added, and the
incubation at 4°C was continued for 2 h. For immunoprecipitation of tandem
affinity purification (TAP)-tagged proteins, 50 ?l of IgG-agarose beads was
added to 500 ?l of supernatant. The beads were washed four times for 3 min
at 4°C with a wash buffer (25 mM HEPES-KOH, pH 7.5, 150 mM KCl, and 2
mM MgCl2). The RNA was eluted from the beads with 200 ?l of 50 mM
Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, and 1% SDS by incubating 10
min at 65°C, followed by a phenol-chloroform extraction and ethanol precip-
itation. For the reverse transcription, 2 ?l of RNA was incubated at 70°C for
5 min in the presence of 0.5 ?g of pd(N)6 and quickly chilled on ice. The
reverse transcription reaction was performed according to indications in a 1?
buffer (50 mM Tris-HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, and 10 mM
dithiothreitol) containing 10 mM dNTPs and 20 U of RNAguard, with 100 U
reverse transcriptase for 1 h at 42°C. The cDNAs were then amplified by PCR
using primers in the ASH1 sequence.
Fluorescence In Situ Hybridization and
Yeast cells were processed for fluorescence in situ hybridization (FISH) and
immunofluorescence according to the protocols described in Chartrand et al.
(2000). For in situ hybridization, yeast spheroplasts were hybridized with a
pool of Cy3-conjugated ASH1 DNA oligonucleotide probes. For immunoflu-
orescence, a 1:50 dilution of a mouse anti-myc 9E10 antibody (Oncogene
Science, Cambridge, MA) was used as primary antibody. For the secondary
antibody, a 1:1000 dilution of a anti-mouse Cy3-conjugated antibody (Jackson
ImmunoResearch Laboratories, West Grove, PA) was used.
Protein Expression and Purification
Recombinant protein GST-She2, GST-She2-M2, and GST-Srp1 were overpro-
duced in Escherichia coli BL21 transformed with pGEX-6P1-She2, pGEX-6P1-
She2-M2, and pGEX-5x3-Srp1. The cells were harvested 3 h after induction
with 1 mM IPTG at 30°C, resuspended in 0.1% PBS-Triton X-100, 1 M NaCl,
and 1 mg/ml lysozyme and antiproteases cocktail (PMSF ? pepstatin ?
leupeptin ? aprotinin) for 30 min on ice and sonicated. The lysate was cleared
by centrifugation for 15 min at 15,000 ? g at 4°C, to yield the supernatant with
the overexpressed soluble protein. The glutathione S-transferase (GST) fusion
proteins were purified by affinity chromatography with glutathione-Sepha-
rose 4B (GE Healthcare, Waukesha, WI) and eluted with 10 mM reduced
glutathione in PBS. The recombinant protein fractions were dialyzed over-
night in PBS and concentrated using a 10-kDa molecular-weight cutoff filter
unit (Centricon-Millipore, Bedford, MA). For the elution of Srp1p, the GST tag
was cleaved with Factor Xa overnight at room temperature.
GST Pulldown Assays
For the recombinant protein interactions of Srp1p with She2p and She2-M2,
purified Srp1p was incubated with 5 ?g of GST-She2p (wild type or mutant)
bound to glutathione-Sepharose 4B (GE Healthcare). The binding was per-
formed at room temperature for 3 h in 500 ?l of binding buffer (50 mM
HEPES-KOH, pH 7.3, 20 mM potassium acetate, 2 mM EDTA, 0.1% Triton
X-100, and 5% glycerol). The matrix was recovered by centrifugation and
washed four times with 500 ?l of binding buffer. The bound proteins were
eluted by boiling in Laemmli buffer and separated on a 10% SDS-PAGE. For
the interactions between recombinant GST-Srp1p and endogenous She2p-
myc, She2-M2-myc, She2-M2-M5A-myc, and She2-M5A-myc, 5 ?g of recom-
binant GST-Srp1p was bound to glutathione-Sepharose 4B and incubated
with yeast extract for 2.5 h at 18°C. The matrix was recovered by centrifuga-
tion and washed four times with 500 ?l of binding buffer. The bound proteins
were eluted with preheated SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2%
SDS, 10% glycerol, 1% ?-mercaptoethanol, 12.5 mM EDTA, and 0.02% bro-
mophenol blue). Eluted proteins were analyzed by Western blot.
Monomeric She2p Interacts Directly with the Importin ?
Srp1p in Order to Enter the Nucleus
Previous work has shown that She2p transits through the
nucleus (Kruse et al., 2002; Du et al., 2008). Because of its size
(26 kDa), which is below the nuclear pore diffusion limit (40
kDa in yeast), a She2p monomer may enter passively
through the nuclear pores. However, because the functional
structure of She2p is that of a homodimer of more than 50
kDa (Niessing et al., 2004), it is possible that active nuclear
import is required. To determine if She2p shuttles between
the nucleus and the cytoplasm actively or passively, a yeast
genetic assay was used (Rhee et al., 2000). In this assay, the
protein of interest is fused to a chimera made of a modified
LexA protein (mLexA), containing a disrupted NLS, and of
the GAL4 activation domain (mLexA-GAL4AD). If the pro-
tein of interest contains a NLS, it promotes the nuclear
import of the mLexA-GAL4AD chimera, which activates the
expression of reporter genes (LacZ and HIS3). In this assay,
a fusion of She2p with the mLexA-GAL4AD resulted in the
activation of LacZ, whereas the mLexA-GAL4AD itself in-
duced little ?-galactosidase activity, suggesting that She2p
promotes the nuclear import of the fusion protein (Figure
1A). It was possible that the She2p-mLexA-GAL4AD fusion
protein could diffuse passively through the nuclear pores.
To eliminate this possibility, the 70-kDa protein VirE2, from
Agrobacterium tumefaciens, was added to the She2p-mLexA-
GAL4AD in order to increase the size of the fusion protein
(Rhee et al., 2000). Even this large fusion protein was actively
imported in the nucleus in a She2p-dependent manner (Fig-
ure 1A), suggesting that She2p contains an active NLS.
The main nuclear import pathway in yeast depends on the
importin ? Srp1p (Lange et al., 2007). A previous large scale
two-hybrid screen in yeast found that Srp1p interacts with
She2p (Ito et al., 2001), suggesting that nuclear import of
She2p may depend on Srp1p. To explore this possibility, the
interaction between She2p and Srp1p was tested in a yeast
two-hybrid assay. However, no interaction between these
two proteins could be detected in this assay (Figure 1B).
Although She2p forms a homodimer and dimerization is im-
portant for its RNA-binding capacity (Niessing et al., 2004), it is
possible that Srp1p interacts only with the She2p monomer.
Two mutants of She2p that have been shown to disrupt She2p
dimerization, She2p-M1 and She2p-M2, containing the muta-
tions Cys683Tyr and Ser1203Tyr, respectively, were gener-
ated (Niessing et al., 2004; and data not shown). When these
monomeric mutants were tested in the yeast two-hybrid assay,
both interacted strongly with Srp1p (Figure 1B), suggesting
that it is indeed the She2p monomer which interacts with
Srp1p. To confirm these results, recombinant Srp1p, GST-
She2p, and GST-She2p-M2 were purified from bacteria and the
direct interaction between Srp1p and the She2p variants was
tested by GST pulldown. As shown in Figure 1C, although the
GST-She2 protein was unable to pull down Srp1p, GST-
She2p-M2 and Srp1p did bind in this assay, suggesting a direct
interaction between Srp1p and a She2p monomer.
The observation that only monomeric She2p interacted
with Srp1p raised the possibility that a fraction of endoge-
nous yeast She2p may be able to interact with Srp1p. To
investigate this question, wild-type She2p and mutant M2
Z. Shen et al.
Molecular Biology of the Cell2266
tagged with 9xMyc were expressed at endogenous levels in
a she2 yeast strain. Recombinant GST-Srp1p was used to pull
down the She2p-myc variants from yeast protein extracts.
Interestingly, using this GST pulldown assay, both wild-
type She2p-myc and She2p-M2-myc from yeast extracts in-
teracted with GST-Srp1p at similar levels (Figure 1D). Al-
though one would have expected that more She2p-M2-myc
than wild-type She2p-myc would be pulled down by GST-
Srp1p, equal amount of both proteins were repeatedly
pulled down. This is possibly due to the saturation of GST-
Srp1p by the numerous NLS-containing proteins in the ex-
tract, so that only a small but equal amount of She2p wild-
type and M2 mutant may be pulled down by GST-Srp1p.
Nevertheless, these results suggest that a significant fraction
of She2p-myc can interact with Srp1p in vivo.
Because the monomeric She2p interacts with Srp1p better
than the She2p dimer, this raises the possibility that the
monomeric protein may accumulate in the nucleus. The
myc-tagged She2 wild-type, M1 and M2 proteins were ex-
pressed at endogenous levels in a she2 yeast strain and their
intracellular distribution was determined by immunofluo-
rescence. As shown in Figure 2A, although the wild-type
She2p-myc was present in both cytoplasm and nucleus of
yeast cells, She2p-M1-myc and She2p-M2-myc accumulated
only in the nucleus. To determine if this nuclear accumula-
tion of the monomeric She2p depends on Srp1p, She2p-M2-
myc was expressed in a temperature-sensitive mutant strain
of SRP1, the srp1-31 strain (Loeb et al., 1995), and its distri-
bution at permissive (25°C) and restrictive (37°C) tempera-
tures was measured. Although She2p-M2-myc was mostly
nuclear in the srp1-31 strain at 25°C, its nuclear import was
impaired when the strain was shifted to nonpermissive tem-
perature for 2 h (Figure 2B). Altogether, these data suggest
that the nuclear import of She2p depends on Srp1p.
A Nonclassical NLS Promotes the Nuclear Import of She2p
As mentioned previously, no NLS had yet been identified in
She2p. To map this NLS, we used the genetic nuclear import
assay. Plasmids expressing the chimeric protein mLexA/GAL4AD alone or in fusion with She2p, VirE2, or She2p?VirE2 were transformed
in L40 yeast strain, and their nuclear import efficiency was determined by measuring ?-galactosidase activity. (B) Yeast two-hybrid assay.
Srp1p was used as bait, and coexpressed with She2 wild-type or the mutants She2-M1 and She2-M2 in pJ69-4A strain. Protein interactions
were determined by measuring ?-galactosidase activity. (C) GST pulldown assay to detect interaction between Srp1p and She2p in vitro.
Recombinant proteins GST, Srp1p, GST-She2p, and GST-She2-M2 expressed and purified from bacteria were loaded on gel for input levels.
Equal amount of Srp1p were loaded on glutathione beads bound with GST, GST-She2p, or GST-She2-M2. After washes, proteins on the beads
were eluted by boiling, loaded on SDS-PAGE gel, and detected by Coomassie Blue staining. (D) GST pulldown assay to detect interaction
between Srp1p and She2p-myc or She2p-M2-myc from yeast extracts. Input: total She2p-myc or She2p-M2-myc in yeast extracts; GST: She2p
from yeast extracts interacting with GST alone; GST-Srp1p: She2p from yeast extracts interacting with GST-Srp1p.
Monomeric She2p interacts directly with the importin ? Srp1p and is actively imported into the nucleus. (A) Yeast nuclear import
She2p Links Puf6p and Loc1p to ASH1 mRNA
Vol. 20, April 15, 20092267
and yeast two-hybrid assays described above. Five different
deletions were generated in She2p (She2p-H1 to -H5, Figure
3A), fused to the mLexA-GAL4AD protein and tested in the
nuclear import assay. As shown in Figure 3A, only the
deletions containing the last 61 amino acids of She2p
(She2p-H2 and -H4) were able to activate the expression of
LacZ. In the yeast two-hybrid assay using Srp1p as bait, only
the deletion that contains the C-terminal end of She2p
(She2p-H2) interacted with Srp1p (Figure 3B), confirming
the results from the nuclear import assay. To define the
She2p NLS more precisely, successive deletions of 16 amino
acids were performed from the C-terminal end of the
She2p-M2 protein (She2p-?16 to -?64) and tested for their
interaction with Srp1p in the yeast two-hybrid assay. As
shown in Figure 3C, whereas a deletion of the last 16 amino
acids of She2p still interacted with Srp1p (She2-?16), dele-
tion of the last 32 amino acids completely disrupted this
interaction (mutant She2-?32), suggesting that part of the
NLS lies between amino acids 214 and 230 of She2p.
Surprisingly, the amino acid sequence of this region of
She2p is very poor in basic amino acids (arginine and ly-
sine), which are commonly found in classical NLS sequences
(Lange et al., 2007). An alignment of amino acids 200–230 of
She2p from Saccharomyces sensu stricto species showed the
presence of only one highly conserved lysine (position 222)
and no arginine (Figure 4A). To determine if this region of
She2p can independently act as a NLS, a 30-amino acid
peptide encompassing positions 200–230 of She2p was fused
at the amino-terminal end of green fluorescent protein (GFP;
She2NLS-GFP), and the distribution of this fusion protein
was determined by epifluorescence microscopy. As shown
in Figure 4B, GFP alone was distributed in both the cyto-
plasm and the nucleus of yeast cells, as a She2p-GFP fusion
protein (which shuttles between the nucleus and cytoplasm).
However, She2NLS-GFP accumulated in the nucleus of
yeasts. This She2NLS peptide was also found to interact
with Srp1p in the yeast two-hybrid assay (Figure 4C), sug-
gesting that this peptide has the properties of a true NLS.
To better define the NLS in this peptide, the K222residue
and four other highly conserved amino acids surrounding
this lysine (W215, I219, L220, and L223) were mutated to ala-
nine in She2p to generate the mutant She2-M5A (Figure 4A).
When tested in the nuclear import assay, the She2-M5A
protein failed to promote the nuclear accumulation of the
mLexA-GAL4AD fusion protein and to activate the expres-
sion of LacZ (Figure 5A). A myc-tagged She2-M5A protein
was expressed at endogenous levels in a she2 strain and its
interaction with Srp1p was tested using a GST pulldown
assay. Mutation of these five residues in the NLS disrupted
the interaction between She2p and GST-Srp1p (Figure 5B).
To eliminate the possibility that the M5A mutation favors
the dimeric conformation of She2p over the monomeric form
and therefore inhibits its interaction with Srp1p, the
Ser1203Tyr mutation, which produces a She2p monomer,
was introduced in She2p-M5A. The resulting protein, She2p-
M2-M5A-myc, was still unable to bind GST-Srp1p (Figure
5B), suggesting that the M5A mutation disrupts the binding
interface between She2p and Srp1p. Finally, the distribution
of this mutant She2p was determined by immunofluores-
cence. As shown in Figure 5C, although the wild-type
pends on Srp1p. (A) Immunofluorescence detection
of wild-type She2p-myc (WT) and mutants She2p-
M1-myc (SHE2-M1) and She2p-M2-myc (SHE2-M2)
in yeast cells. Percentage of cells with the displayed
phenotype is indicated. Scale, 2 ?m. (B) Immunoflu-
orescence on She2p-M2-myc in srp1-31 strain at per-
missive (top panels) or restrictive (bottom panels)
temperature. Percentage of cells with the displayed
phenotype is indicated. Scale, 2 ?m.
Nuclear import of monomeric She2p de-
Z. Shen et al.
Molecular Biology of the Cell2268
She2p-myc was found in both cytoplasm and nucleus, the
She2-M5A-myc protein was excluded from the yeast nu-
cleus. Altogether, these results show that the NLS of She2p
is present between amino acids 214–222 at the C-terminal
end of this protein and that mutation of five specific residues
in this NLS disrupts the nuclear targeting of She2p.
Nuclear Import of She2p Is Required for Proper
Localization of the ASH1 mRNA at the Bud Tip and for
the Sorting of Ash1p
The generation of a mutant She2 protein that cannot be
targeted to the nucleus opened the possibility of exploring
the nuclear function of She2p in terms of ASH1 mRNA
localization and Ash1p asymmetric distribution. First, the
She2p-M5A mutant was tested to determine if the point
mutations in the NLS had any effect on its RNA-binding
capacity or its interaction with She3p. Expression levels of
She2p-myc and She2p-M5A-myc in yeast were similar, as
shown by Western blot (Figure 6A). Immunoprecipitation of
the myc-tagged She2p variants, followed by RT-PCR ampli-
fication of the ASH1 mRNA, showed no significant differ-
ence in ASH1 mRNA binding between the wild-type and the
M5A mutant in vivo (Figure 6B). Because only a small
amount of ASH1 mRNA is present in the nucleus, this result
this assay (H1–H5). She2p deletions were fused to the LexA/GAL4AD chimera, and their nuclear import efficiency was determined by
measuring ?-galactosidase activity. Vector: plasmid expressing LexA/GAL4AD chimera alone. (B) Yeast two-hybrid assay between Srp1p
and She2p-M2 deletions. Diagram of the She2p-M2 deletions used in this assay (H1–H3). Interaction between Srp1p and a She2p-M2 deletion
was determined by measuring ?-galactosidase activity. AD: plasmid expressing Gal4 activation domain only. (C) Yeast two-hybrid assay
between Srp1p and She2p-M2 deletions at its C-terminus. Diagram of the She2p-M2 deletions used in this assay (?16–?64). Interaction
between Srp1p and a She2p-M2 deletion was determined by measuring ?-galactosidase activity. AD, plasmid expressing Gal4 activation
Identification of an NLS at the C-terminal end of She2p. (A) Yeast nuclear import assay. Diagram of the She2p deletions used in
She2p Links Puf6p and Loc1p to ASH1 mRNA
Vol. 20, April 15, 20092269
shows that even when She2p is excluded from the nucleus,
it can still efficiently interact with this transcript in the
cytoplasm. This was confirmed in a yeast three-hybrid assay
that measures the interaction between She2p and the ASH1
mRNA zip codes (Long et al., 2000; Olivier et al., 2005). In this
assay, one of the ASH1 zip code (the domain 1 of element E2B
or D1), was used as bait for the interaction with She2p. No
observed in binding of this ASH1 localization element (Figure
6C). Finally, to determine if this mutation cause any disruption
myosin Myo4p, a yeast two-hybrid assay was used (Long et al.,
2000). In this assay, She2p-M5A showed no defect in its inter-
action with the C-terminal domain of She3p compared with
wild-type She2p (Figure 6D).
To determine the effect of the nuclear exclusion of She2p
on ASH1 mRNA localization, myc-tagged She2p or She2p-
M5A were expressed in a she2 yeast strain and the localiza-
tion of ASH1 mRNA in these strains was visualized by FISH.
As shown in Figure 7A, although the wild-type She2p pro-
moted the localization of the ASH1 mRNA at the bud tip of
cells in anaphase, the She2p-M5A had a reduced efficiency in
ASH1 mRNA localization, as it accumulated in the whole
bud of the cells. Indeed, although more than 90% of late-
anaphase cells expressing wild-type She2p had bud tip lo-
calization of the ASH1 mRNA, this percentage dropped to
below 10% in yeasts expressing She2p-M5A (Figure 7B). The
effect of the mutation of the She2p NLS on the asymmetric
distribution of the Ash1 protein and HO promoter activity
was determined using the yeast strain K5547, in which the
ADE2 gene is under the control of the HO promoter and
which contains a deletion of the SHE2 gene (Jansen et al.,
1996). In this strain, symmetric distribution of Ash1p leads
to repression of the ADE2 gene and absence of growth on
plate lacking adenine (?Ade). If She2p function is restored,
Ash1p accumulates in the daughter cell, so expression of the
ADE2 gene in the mother cell allows growth on ?Ade plates.
When transformed with a plasmid expressing the wild-type
She2p, the K5547 strain grew on ?Ade plates, whereas the
same strain transformed with the empty vector did not grow
(Figure 7C). When transformed with a plasmid expressing
She2p-M5A, a slower growth on ?Ade plates was observed
compared with the strain expressing wild-type She2p, suggest-
ing that the M5A mutation partially disrupt the asymmetric
distribution of Ash1p. However, expression of a She2-M5A
protein containing the SV40 NLS (She2p-M5A?SV40NLS),
which restores the nuclear localization of She2p-M5A and
maintains its interaction with ASH1 mRNA in vivo (Figure 6B),
resulted in the same growth on ?Ade plates as the strain
expressing wild-type She2p (Figure 7C). This suggests that it
was the defective nuclear import of She2p-M5A that disrupted
the asymmetric localization of Ash1p. Altogether, these results
show that the nuclear import of She2p is required for the
of She2p. (A) Sequence alignment of a 30-amino acid
peptide containing the NLS of She2p. Sequences from
She2p homologues from S. cerevisiae (Scer), S. paradoxus
(Spar), S. mikatae (Smik), S. bayanus (Sbay), S. kuderii
(Skud), and S. casei (Scas) are shown. Amino acids mu-
tated are underlined in gray. (B) Epifluorescence mi-
croscopy on yeast cells expressing She2-GFP (top pan-
els), GFP alone (bottom panels), or GFP fused to a
30-amino acid NLS of She2p (middle panels). Scale, 2
?m. (C) Srp1p interacts with the 30-amino acid NLS
peptide from She2p in a yeast two-hybrid assay. Inter-
action between Srp1p and She2p-M2 protein or the
She2NLS peptide was determined by measuring ?-ga-
lactosidase activity. AD, plasmid expressing Gal4 acti-
vation domain only.
A nonclassical NLS mediates nuclear import
Z. Shen et al.
Molecular Biology of the Cell2270
localization of the ASH1 mRNA at the bud tip and the sorting
of the Ash1 protein to the daughter cell nucleus.
Nuclear Import of She2p Is Essential for the Recruitment
of Loc1p and Puf6p to the ASH1 mRNA
The work presented so far shows that the nuclear import of
She2p is important for its function in cytoplasmic mRNA
localization, but the reason is not clear. Two other nuclear
RNA-binding proteins are known to play a role in ASH1
mRNA localization and Ash1p sorting: Puf6p and Loc1p.
Puf6p is predominantly nucleolar, binds the localization
element E3 in the 3?UTR of ASH1 and is involved in the
translational repression of this transcript (Gu et al., 2004;
Deng et al., 2008). Its deletion results in defects in both ASH1
mRNA localization and Ash1p asymmetric distribution.
Loc1p is also a nucleolar protein, and it binds the 3?UTR of
the ASH1 mRNA (Long et al., 2001). Its deletion disrupts
ASH1 mRNA localization, and recent data suggest that it is
also involved in the translational regulation of this transcript
(Long et al., 2001; Komili et al., 2007). Because the She2p-
M5A mutant displayed phenotypes similar to the PUF6 and
LOC1 deletions (whole bud accumulation of ASH1 mRNA,
partial asymmetric distribution of Ash1p), we raised the
hypothesis that nuclear She2p might be involved in the
as functional as the wild-type She2p. (A) Ex-
pression levels of wild-type She2p-myc and
Western blot. Expression levels were normal-
ized with Pgk1 protein. (B) Immunoprecipi-
tation of She2p-myc wild-type (WT), M5A
mutant (M5A), or M5A mutant?SV40NLS,
followed by reverse transcription and PCR
amplification of endogenous ASH1 mRNA. In-
put: ASH1 mRNA from total yeast extract;
IP?RT-PCR: ASH1 mRNA from immunopre-
cipitate, reverse-transcribed, and amplified by
PCR; IP ? PCR (?RT): ASH1 mRNA from
immunoprecipitate, amplified by PCR without
reverse transcriptase. (C) Three-hybrid assay
using yeast strain YBZ1 she2 transformed with
plasmid expressing either wild-type (She2WT)
or NLS mutant (She2M5A) of She2p. This
strain also expressed the GAL4 activation do-
main fused with the C-terminal domain of
The NLS-mutated She2 protein is
She3p, along with a plasmid expressing a chimeric RNA containing the localization E2B-D1 fused to the MS2 stem-loop (D1). Controls: empty
vector (pIIIA/MS2-2) and an inactive fragment of the localization element E2B (D2; Olivier et al., 2005). (D) Interaction between the C-terminal
domain of She3p and wild-type (She2WT) or NLS-mutated (She2M5A) She2 proteins in a yeast two-hybrid assay. AD, plasmid expressing
Gal4 activation domain only.
action with Srp1p and nuclear import of this factor.
(A) Yeast nuclear import assay. She2p wild-type
(WT) or NLS mutant (She2p-M5A) were fused to the
LexA/GAL4AD chimera and their nuclear import
efficiency was determined by measuring ?-galacto-
sidase activity. Vector: plasmid expressing LexA/
GAL4AD chimera alone. (B) GST pulldown assay to
detect interaction between Srp1p and She2p-myc,
M5A-myc from yeast extracts. Input: total She2p-
myc, She2p-M2-myc, She2p-M5A-myc or She2p-M2-
M5A-myc in yeast extracts; GST: She2p from yeast
extracts interacting with GST alone; GST-Srp1p:
She2p from yeast extracts interacting with GST-
Srp1p. (C) Immunofluorescence on yeast cells ex-
pressing wild-type She2p-myc (top panels) or
She2p-M5A-myc (bottom panels). White arrows
point to low She2p-myc level in the nuclei, and
numbers reflect extent of the phenotype observed.
Scale, 2 ?m.
Mutations in NLS of She2p impair inter-
She2p Links Puf6p and Loc1p to ASH1 mRNA
Vol. 20, April 15, 20092271
binding of Puf6p and Loc1p to the ASH1 mRNA. Hence, the
absence of She2p in the nucleus might disrupt the recruit-
ment of Puf6p and Loc1p to the ASH1 mRNA, leading to
phenotypes similar to PUF6 and LOC1 knockouts.
To explore this possibility, She2p and She2p-M5A were
expressed at endogenous levels in strains deleted of the
endogenous SHE2 gene and containing a TAP-tag integra-
tion at the C-terminus of either PUF6 or LOC1 open reading
frames. Expression of She2p-M5A had no effect on Loc1p-
TAP and Puf6p-TAP expression levels (data not shown). The
interaction between the ASH1 mRNA and the Puf6-TAP and
Loc1-TAP proteins in vivo was determined by RNA immu-
noprecipitation. In this assay, the RNP complexes were
cross-linked in vivo with formaldehyde, followed by immu-
noprecipitation of the Puf6-TAP and Loc1-TAP proteins.
After de-crosslinking, the associated mRNAs were purified
and reverse-transcribed, and the ASH1 cDNA was detected
by PCR amplification. In a yeast strain expressing the wild-
type She2p, both Puf6p-TAP and Loc1p-TAP interacted with
the ASH1 mRNA in vivo (Figure 8A), but not with ACT1
mRNA (Figure 8B). However, when She2p-M5A was ex-
pressed, no ASH1 mRNA was found associated with Puf6p-
TAP and very little with Loc1p-TAP (Figure 8A), suggesting
for proper ASH1 mRNA localization and Ash1p
sorting. (A) Fluorescent in situ hybridization on
ASH1 mRNA in yeast cells expressing either
wild-type She2p-myc (top panels) or She2p-
M5A-myc (bottom panels). Scale, 2 ?m. (B)
Scores on mRNA localization phenotypes from
A. (C) Yeast genetic assay for Ash1p asymmetric
distribution. Tenfold dilutions of exponentially
growing K5547 (HO-ADE2, she2) transformed
either with the empty YCPlac22 plasmid (vec-
tor), YCP22-She2-myc (SHE2), YCP22-She2-
were spotted on plates lacking tryptophan
(?Trp) or lacking tryptophan and adenine
(?Trp ?Ade) and incubated at 30°C.
Nuclear import of She2p is required
strains expressing either She2p-myc wild-type (WT) or She2-M5A-myc (M5A), followed by RNA purification and RT-PCR amplification of
ASH1 mRNA. Input: ASH1 mRNA from total yeast extract; IP?RT-PCR: ASH1 mRNA from immunoprecipitate, reverse-transcribed, and
amplified by PCR; IP?PCR (?RT): ASH1 mRNA from immunoprecipitate, amplified by PCR without reverse transcriptase; Beads: mRNA
from yeast extract incubated with protein A-Sepharose beads, without antibody. (B) Detection of ACT1 mRNA from immunoprecipitated
Puf6p-TAP and Loc1p-TAP. Input: ACT1 mRNA from total yeast extract; IP?RT-PCR: ACT1 mRNA from immunoprecipitate, reverse-
transcribed, and amplified by PCR. These results are representative of three independent experiments. (C) She2p-myc coimmunoprecipitates
with Loc1p-TAP and Puf6p-TAP. No TAP: yeast strain without TAP-tagged Loc1p or Puf6p; Input: She2p-myc from total yeast extract; IP:
immunoprecipitated She2p-myc; IP?RNAse: immunoprecipitated She2p-myc after treatment with RNAse A. The asterisk corresponds to the
IgG heavy chain. (D) Nuclear import of She2p is required for its interaction with Loc1p and Puf6p. Immunoprecipitation of Loc1p-TAP and
Puf6p-TAP from strains expressing either wild-type She2p-myc (WT), She2-M5A-myc (M5A), or She2-M5A?SV40NLS (M5A?SV40NLS).
Input: wild-type She2p-myc from total yeast extract. IP, immunoprecipitation. The asterisk corresponds to the IgG heavy chain.
Nuclear She2p recruits Puf6p and Loc1p on the ASH1 mRNA. (A) Immunoprecipitation of Loc1p-TAP and Puf6p-TAP from
Z. Shen et al.
Molecular Biology of the Cell2272
that the presence of She2p in the nucleus is essential for
Puf6p and Loc1p to bind the ASH1 mRNA in vivo.
These results raise the possibility that She2p interacts with
Puf6p and Loc1p, and recruit them to the ASH1 mRNA.
Therefore, interaction between She2p, Puf6p, and Loc1p in
vivo was explored using coimmunoprecipitation. Pulldown
of both TAP-tagged Puf6p and Loc1p resulted in the coim-
munoprecipitation of She2p-myc (Figure 8C), suggesting an
interaction between these factors in vivo. This interaction
was independent of RNA because treatment of yeast extracts
with RNAse A before immunoprecipitation still resulted in
an efficient pulldown of She2p-myc by both Puf6p-TAP and
Loc1p-TAP (Figure 8C). Finally, to determine if the presence
of She2p in the nucleus is important for its interaction with
Puf6p and Loc1p, the coimmunoprecipitation was repeated
with She2p-M5A-myc mutant. In this experiment, cross-
linking of protein complexes with formaldehyde before im-
munoprecipitation was performed in order to avoid recon-
stitution of complexes in the yeast extract after breaking the
cells. As shown in Figure 8D, a clear reduction in the amount
of She2p-M5A-myc that coimmunoprecipitated with either
Puf6p-TAP or Loc1p-TAP was observed compared with
wild-type She2p-myc. Wild-type levels of interaction were
recovered when the SV40 NLS was fused to the She2p-M5A
protein (Figure 8D), suggesting that nuclear import of She2p
is required for its interaction with Loc1p and Puf6p. Alto-
gether, these results suggest that nuclear import of She2p is
required for its interaction with Puf6p and Loc1p and for
their recruitment to the ASH1 mRNA.
A Nonclassical NLS Promotes the Nuclear Import of
She2p by Binding Importin ?
In this work, we show that She2p is actively imported into
the nucleus via its interaction with the importin ? Srp1p.
Our data suggest that She2p is not imported as a native
dimer, which is the conformation that binds RNA (Niessing
et al., 2004), because the She2p dimer was unable to interact
with Srp1p in vitro. She2p from yeast extracts was able to
interact with Srp1p, suggesting that a fraction of She2p in
vivo may adopt a conformation that is different from the
native dimer. However, it is still unclear if this population of
She2p corresponds to monomers. A possibility is that this
population of She2p may contain posttranslational modifi-
cations. Phosphorylation is known to regulate the nuclear
import of proteins, such as Gln3p in yeast (Carvalho et al.,
2001), and She2p has been reported to be a phosphoprotein
in vivo (Gonsalvez et al., 2003). Once in the nucleus, She2p
adopts a dimeric conformation because it can bind RNA.
Intriguingly, wild-type She2p-myc was not excluded from
the nucleus of the srp1-31 strain at nonpermissive tempera-
ture (data not shown). This is possibly due to the rapid
shutdown of transcription when this strain is shifted at 37°C
(Liu et al., 1999). Because She2p nuclear export depends on
the nuclear export of newly synthesized mRNAs (Kruse
et al., 2002), the shutdown of transcription would explain the
absence of nuclear depletion of wild-type She2p in the
Using the interaction between Srp1p and She2p, a 30-
amino acid sequence with NLS properties was identified.
This NLS promotes the nuclear import of GFP and interacts
with Srp1p. Interestingly, its sequence is very divergent
from classical monopartite and bipartite NLS because it
contains only one lysine and is rich in hydrophobic residues.
To our knowledge, all the currently reported NLS that bind
directly importin ? contain at least two essential basic amino
acids (Chen et al., 2005; Lange et al., 2007), suggesting that
the repertoire of nuclear localization signals may be larger
than suggested. Mutation of five conserved residues in this
NLS disrupted the nuclear targeting of She2p and its inter-
action with Srp1p, confirming its role as a nuclear localiza-
tion sequence. The defective ASH1 mRNA localization and
poor asymmetric sorting of Ash1p seen in the She2p-M5A
mutant strain seem to result from the nuclear exclusion of
this protein and not from secondary effects of this mutation.
Indeed, the RNA-binding capacity of the NLS-mutated
She2p was similar to wild-type She2p, as was its interaction
with She3p. More important, adding an heterologous clas-
sical NLS (like the SV40 NLS) to the She2p-M5A completely
restored the function of this protein in vivo.
Nuclear She2p Couples mRNA Localization and
Disrupting the nuclear import of She2p affects the local-
ization of the ASH1 transcript and the asymmetric distri-
bution of Ash1p. We provide evidence that this localiza-
tion defect is linked to the disrupted interaction between
Puf6p and Loc1p with the ASH1 mRNA because 1) She2p
interacts in vivo with both Puf6p and Loc1p; 2) the pres-
ence of She2p in the nucleus is important for this interac-
tion; 3) exclusion of She2p from the nucleus disrupts the
binding of Loc1p and Puf6p to the ASH1 mRNA, and 4)
nuclear exclusion of She2p phenocopies the knockouts of
LOC1 and PUF6 in term of ASH1 mRNA localization and
Altogether, these data suggest a direct coupling between
the mRNA transport and translational control machineries.
A role of nuclear She2p in translational control is indeed
supported by recent data from the Jansen lab, which showed
that nuclear exclusion of She2p accelerates Ash1p synthesis
(Du et al., 2008). Intriguingly, Du et al. did not report any
defect in ASH1 mRNA localization when She2p was ex-
cluded from the nucleus, unlike what we observed (see
Figure 7). They used a She2 protein fused to the Myo4p-
binding domain of She3p, which resulted in a fusion protein
that remained anchored on the actin cytoskeleton via its
binding to Myo4p and is excluded from the nucleus. How-
ever, it is possible that such tight association of She2p, and
of the ASH1 mRNA, to the localization machinery and to the
actin cytoskeleton suppresses the localization defects caused
by the nuclear exclusion of She2p.
Because She2p binds several localization elements within
the coding sequence of the ASH1 mRNA (Chartrand et al.,
2002), this coupling may reduce the possibility that elongat-
ing ribosomes could displace She2p from this transcript.
Such coupling is supported by the finding that She2p coim-
munoprecipitates with Puf6p and Loc1p, suggesting an in-
teraction between these proteins in vivo. The mechanism by
which She2p promotes the recruitment of Loc1p and
Puf6p on the ASH1 mRNA in vivo is not known, because
all three proteins can bind the 3?UTR of this transcript
independently in vitro (Bohl et al., 2000; Long et al., 2001;
Gu et al., 2004). One possibility is that, being in the nu-
cleolus, Puf6p and Loc1p are spatially restricted from
polyA?mRNAs. Because She2p has been recently shown
to transit through the nucleolus (Du et al., 2008), it may
either bring the ASH1 mRNA in the nucleolus, where
Puf6p and Loc1p can bind this transcript, or She2p may
recruit these two factors in the nucleolus and bring them
to the ASH1 mRNA in the nucleoplasm.
She2p Links Puf6p and Loc1p to ASH1 mRNA
Vol. 20, April 15, 20092273
Roles of Nuclear Proteins in Cytoplasmic mRNA
The importance of nuclear events in cytoplasmic mRNA
localization is a well-described phenomenon. Several RNA-
binding proteins implicated in cytoplasmic mRNA localiza-
tion are known to be exclusive residents of the nucleus or to
shuttle between the cytoplasm and the nucleus (Farina and
Singer, 2002). Reports from several model systems have
shown that mRNA processing in the nucleus affects its cy-
toplasmic fate. For instance, proper splicing of the oskar
mRNA is required for its localization at the posterior pole of
the Drosophila embryo (Hachet and Ephrussi, 2004). In this
case, members of the exon junction complex, such as Y14-
Mago and eIFIIIA, are assembled on the oskar mRNA in the
nucleus and are involved in the cytoplasmic localization of
this transcript. In Xenopus oocyte, the nucleocytoplasmic
shuttling proteins hnRNP I and Vg1RBP/Vera initiate a
localization complex with the Vg1 mRNA in the nucleus
(Cote et al., 1999; Kress et al., 2004). Remodeling of this
ribonucleoprotein complex has been shown to occur after its
nuclear export (Kress et al., 2004). In fibroblasts, both ZBP1
and ZBP2/KSRP proteins can bind the ?-actin mRNA in the
nucleus (Gu et al., 2002; Oleynikov and Singer, 2003). A
handover mechanism has been proposed where the predom-
inantly nuclear ZBP2 binds the nascent ?-actin transcript
and facilitates the subsequent recruitment of ZBP1, the fac-
tor involved in the cytoplasmic localization of the ?-actin
mRNA (Pan et al., 2007).
Our study reveals another function for the nucleo-cyto-
plasmic shuttling of RNA-binding proteins involved in
mRNA localization. By promoting the recruitment of Puf6p
and Loc1p on the nuclear ASH1 mRNA, She2p initiates the
translational repression of the localized mRNA before its
export in the cytoplasm and prevents premature translation
of this transcript. Coupling mRNA localization and transla-
tional control constitutes an efficient way to ensure that the
translation of transcripts targeted for localization will be
properly regulated. This raises the possibility that other
transcripts that are localized at the bud tip of yeasts may also
be translationally repressed by Puf6p and/or Loc1p via their
recruitment with She2p.
We thank Drs. Gerry Fink (Whitehead Institute) and Michael Culbertson (Uni-
versity of Wisconsin–Madison) for reagents and strains. We also thank Emman-
uelle Querido for critical reading of the manuscript. This work was supported by
a grant from the Canadian Institutes for Health Research. P.C. is a Chercheur-
Junior 2 fellow from the Fond de Recherche en Sante ´ du Que ´bec.
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Vol. 20, April 15, 20092275