Full text of DOI:10.1016/S1097-2765(00)00065-4

Molecular Cell, Vol. 6, 673–682, September, 2000, Copyright 2000 by Cell Press
Pre-mRNA Splicing Imprints mRNA in the Nucleus
with a Novel RNA-Binding Protein
that Persists in the Cytoplasm
have been identified as effectors of mRNA export (Go¨ r-
lich and Kutay, 1999; Nakielny and Dreyfuss, 1999). The
mechanism of HIV-1 Rev-mediated RNA export has
been studied extensively (Pollard and Malim, 1998). Rev
binds to unspliced or partially spliced viral mRNAs via
the Rev-responsive element (RRE), and the export factor
CRM1, a member of nuclear transport receptor (NTR)
family (Go¨ rlich and Kutay, 1999; Nakielny and Dreyfuss,
1999), binds to the leucine-rich nuclear export signals
(NES) of Rev and mediates export of this complex in a
RanGTP-dependent manner (Pollard and Malim, 1998;
Naoyuki Kataoka, Jeongsik Yong, V. Narry Kim,
Francisco Velazquez,† Robert A. Perkinson,
Fan Wang, and Gideon Dreyfuss*
Howard Hughes Medical Institute and
Department of Biochemistry and Biophysics
University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania 19104
Summary
Gorlich and Kutay, 1999; Nakielny and Dreyfuss, 1999).
¨
We describe a novel RNA binding protein, Y14, a pre-
dominantly nuclear nucleocytoplasmic shuttling pro-
tein. Interestingly, Y14 associates preferentially with
mRNAs produced by splicing but not with pre-mRNAs,
introns, or mRNAs produced from intronless cDNAs.
Y14 associates with both nuclear mRNAs and newly
exported cytoplasmic mRNAs. Splicing of a single in-
tron is sufficient for Y14 association. Y14-containing
nuclear complexes are different from general hnRNP
complexes. They contain hnRNP proteins and several
unique proteins including the mRNA export factor TAP.
Thus, Y14 defines novel intermediates in the pathway
of gene expression, postsplicing nuclear preexport
mRNPs, and newly exported cytoplasmic mRNPs,
whose composition is established by splicing. These
findings suggest that pre-mRNA splicing imprints
mRNA with a unique set of proteins that persists in
the cytoplasm and thereby communicates the history
of the transcript.
This allows intron-containing transcripts to bypass
splicing and export efficiently. Another cis-acting ele-
ment in the simian type D retroviruses, the constitutive
transport element (CTE), has also been reported to simi-
larly facilitate export of unspliced viral RNAs (Bray et
al., 1994; Zolotukhin et al., 1994). A host factor, TAP,
has been identified as a mediator of CTE-dependent
RNA export (Gruter et al., 1998). TAP is the vertebrate
¨
homolog of S. cerevisiae Mex67p, which is an essential
mRNA export factor in yeast (Segref et al., 1997). More-
over, an excess amount of CTE RNA prevents cellular
mRNA export (Gruter et al., 1998), and TAP can be cross-
¨
linked to poly(A)ϩ RNA in vivo (Katahira et al., 1999). It
is therefore likely that TAP participates in mRNA export
in mammalian cells.
Despite considerable efforts, the events that take
place in the nucleus between the time mRNA forms by
pre-mRNA splicing and its engagement with the nuclear
pore complexes through which mRNAs are exported to
the cytoplasm are not known. This is due, in large part,
to the fact that postsplicing, preexport nuclear mRNP
complexes have not been identified or isolated. Here,
we have identified a novel mRNA binding protein that
was initially isolated as part of a two-hybrid screening
effort to identify cargoes of RanBP5, a member of the
NTR family (Deane et al., 1997). This protein, termed
Y14, is preferentially associated with mRNAs that are
produced by splicing both in vitro and in vivo but not
with pre-mRNAs or intronless mRNAs. Y14 could not be
detected in general hnRNP complexes but is present in
mRNP complexes that contain the putative mRNA ex-
port factor TAP. Comparative immunoprecipitations us-
ing antibodies to general hnRNP complexes and to Y14
reveal that they isolate complexes that have distinct
compositions. Thus, Y14 defines and makes possible
the isolation of postsplicing mRNP complexes. These
findings demonstrate that pre-mRNA splicing trans-
forms the composition of the transcript and generates an
mRNP complex that is endowed with specific proteins
that are most likely needed for proper postsplicing events.
Introduction
As nascent transcripts, pre-mRNAs become associated
with hnRNP proteins and with components of the splic-
ing machinery including snRNPs (Dreyfuss et al., 1993;
Krecic and Swanson, 1999). While snRNPs dissociate
from the spliced mRNA product at the completion of the
splicing reaction, many of the hnRNP proteins remain
associated with the mRNA, and several of them accom-
pany the mRNA to the cytoplasm (Pin˜ ol-Roma and Drey-
fuss, 1992, 1993; Visa et al., 1996). This suggested that
in addition to their nuclear functions, hnRNP proteins
also play important roles in mRNA export and in the
metabolism of mRNA in the cytoplasm. Consistent with
this, numerous examples of specific functions for
hnRNP proteins in both nuclear events such as splice
site selection (Mayeda and Krainer, 1992; Ca´ ceres et
al., 1994; Yang et al., 1994; Bai et al., 1999) as well as
in mRNA translation, stability, and localization in the
cytoplasm have been reported (Dreyfuss et al., 1993;
Svitkin et al., 1996; Ostareck et al., 1997; Hoek et al.,
1998; Lall et al., 1999; Ostareck-Lederer et al., 1998;
Krecic and Swanson, 1999; Kwon et al., 1999).
Results
Identification of Y14, a Novel Single RBD RNA
Binding Protein
In addition to hnRNP proteins, several other proteins
Several of the NTRs, including importin␤, transportin1,
importin7 (also referred to as Ran binding protein 7),
and Ran binding protein 5 (RanBP5) have been shown
to have the capacity to import ribosomal proteins (Rout
*To whom correspondence should be addressed (e-mail: gdreyfuss@
hhmi.upenn.edu).
Present address: Lankenau Medical Hospital, Wynnewood, Penn-
†
sylvania 19096.
et al., 1997; Jakel and Gorlich, 1998). However, unlike
¨ ¨

Molecular Cell
674
Figure 1. Amino Acid Sequence of Y14 and Alignment with Putative Homologs in Several Species
Y14 and proteins from mouse (AL022712), Xenopus laevis, D. melanogaster (AC006074), C.elegans (Z32683), and S. pombe (AL021813) were
aligned using ClustalW alignment. Identical residues are indicated by dark shading, and similar residues are shown by light shading. The
positions of RNP2 and RNP1 consensus motifs of the RNA binding domain are indicated by bars.
other receptors, specific cargoes other than ribosomal
proteins have not been identified for RanBP5. We carried
out yeast two-hybrid screening on a HeLa cell cDNA
library to identify potential cargoes for RanBP5. We used
RanBP5 lacking the amino-terminal 395 amino acids as
bait, because the carboxyl-terminal region of NTR family
members contains the cargo binding domain (Go¨ rlich
and Kutay, 1999; Nakielny and Dreyfuss, 1999). We ana-
lyzed 17 positive clones, 14 of which turned out to be
identical and encode a putative RNA binding protein,
designated here as Y14. Database searches revealed
Y14 to be identical to RNA binding motif protein 8, a
protein of unknown function isolated by sequencing
mRNAs from HeLa cells (GenBank accession number
AF127761). Y14 is a 174 amino acid protein that contains
a single RNA binding domain (RBD) in the middle of the
protein (Figure 1). The carboxyl-terminal domain con-
tains many basic residues and two arginine-serine di-
peptide repeats that are characteristic of SR splicing
factors (Fu, 1995). The amino-terminal domain is rich in
arginines and lysines (Figure 1). By screening a Xenopus
cDNA library with human Y14 cDNA, we isolated a puta-
tive Y14 homolog from Xenopus laevis. The Xenopus cDNA
encodes a 174 amino acid protein that shows 90% identity
and 95% similarity to human Y14 protein (Figure 1). Data-
base searches revealed other putative homologs in
mouse, Drosophila melanogaster, Caenorhabditis ele-
gans, and the fission yeast Schizosaccharomyces pombe
(Figure 1). Direct binding experiments (N. K. and G. D.,
unpublished data) showed that Y14 binds to RanBP5 in
a RanGTP-sensitive manner, suggesting that this NTR
is a nuclear import receptor for Y14.
reacted with a single band corresponding to 24 kDa on
a Western blot of HeLa whole cell lysate (Figure 2A) and
specifically immunoprecipitated in vitro translated Y14
(Figure 2B). Immunofluorescence microscopy using 4C4
on HeLa cells showed that Y14 is localized predomi-
nantly in the nucleoplasm and in nuclear speckles. Low
but significant cytoplasmic staining was also observed
(Figure 2C).
Y14 Is Preferentially Associated with mRNAs
Produced by Splicing
Because several splicing factors, such as SR proteins,
localize to speckles (Fu, 1995; Lamond and Earnshaw,
1998; Misteli and Spector, 1998), we sought to determine
if Y14 has a role in pre-mRNA splicing. We found that
anti-Y14 antibodies did not inhibit pre-mRNA splicing
in vitro (data not shown). To examine whether Y14 is
present in spliceosomes, we carried out immunoprecipi-
tations from in vitro splicing reactions. We first used pre-
mRNA derived from the adenovirus major late promoter
(Ad2) (Pellizzoni et al., 1998) and performed the immuno-
precipitations after 40 min incubations from the splicing
reaction. Antibodies against the hnRNP A1 and C pro-
teins used in parallel preferentially immunoprecipitated
pre-mRNA and lariat intron (Figure 3A). In contrast, the
anti-Y14 antibody 4C4 preferentially and efficiently im-
munoprecipitated the mRNA product (Figure 3A). This
pattern is somewhat similar to that observed with ASF/
SF2 antibody (Hanamura et al., 1998); however, anti-Y14
immunoprecipitates spliced mRNA much more effi-
ciently (Figure 3A). 4C4 did not efficiently immunoprecip-
itate the same mRNA produced by transcription in vitro
and incubated in the extract under the same experimen-
tal conditions (Figure 3B). To determine whether this
effect is general or is unique to the mRNA produced from
the Ad2 pre-mRNA, we carried out similar experiments
Y14 Is Localized Mainly in Nucleus and Shows
a Speckled Pattern
To further characterize Y14, we produced monoclonal
antibodies to it. One of the monoclonal antibodies, 4C4,

Y14 Defines Postsplicing mRNP Complexes
675
Figure 2. Specificity of Anti-Y14 Antibodies and Immunolocalization of the Y14 Protein
(A) Western blotting on HeLa cell total lysate. The band corresponding to Y14 is indicated by an arrow.
(B) The anti-Y14 antibody can specifically precipitate Y14. Anti-hnRNP C1 (4F4), anti-Y14 (4C4), and control antibody (SP2/0) were immobilized
on protein G sepharose and incubated with in vitro translated 35S-labeled hnRNP C1 and Y14. An aliquot equivalent to 10% of the amount of
hnRNP C1 and Y14 included in the reaction is shown in the lanes marked translation.
(C) Subcellular localization of Y14 in HeLa cells. Hela cells grown on the coverslips were fixed, permeabilized, and stained with anti-Y14, 4C4.
using another splicing substrate derived from chicken
␦-crystallin pre-mRNA (Ohno and Shimura, 1996), CDC.
As shown in Figures 3C and 3D, anti-Y14 also efficiently
immunoprecipitated spliced mRNA derived from CDC
pre-mRNA but did not efficiently immunoprecipitate
CDC mRNA that was transcribed in vitro and incubated
under the same conditions. We also used in vitro tran-
scribed dihydrofolate reductase (DHFR) mRNA, which
is naturally intronless, for similar immunoprecipitation
experiments. Anti-Y14 did not efficiently immunoprecip-
itate the intronless DHFR mRNA, while antibodies
against hnRNP A1 and C proteins did (Figure 3E). These
results demonstrate that Y14 binds efficiently only to
mRNAs that are produced by pre-mRNA splicing.
To examine these observations in vivo, we microin-
jected RNAs into Xenopus laevis oocytes and analyzed
the RNAs that were immunoprecipitated with 4C4 from
the nucleus and cytoplasm. The 4C4 antibody also spe-
cifically reacts (by immunoprecipitation and Western
blotting) with Xenopus oocyte Y14 (data not shown). As
shown in Figure 4A, the anti-Y14 antibody efficiently and
preferentially immunoprecipitated spliced mRNA from
the nuclear fraction. In contrast, anti-Xenopus hnRNP
C1 antibody, 10C1, preferentially immunoprecipitated
the lariat intron (Figure 4A). Because the anti-Y14 anti-
body did not immunoprecipitate intronless mRNA in
vitro (Figures 3B, 3D, and 3E), we tested its ability to
immunoprecipitate mRNA that was not produced by
splicing in the oocyte nucleus. Adenovirus-derived and
DHFR mRNAs produced by in vitro transcription were
microinjected into oocyte nuclei, and immunoprecipita-
tions were carried out 1.5 hr later from the nucleus and
cytoplasm. As was the case in vitro, the Y14 antibody
did not efficiently immunoprecipitate in vitro transcribed
mRNA from either the nucleus or the cytoplasm (Figure
4B). These results strongly suggest that efficient associ-
ation of Y14 with mRNA requires that the mRNAs be
produced by pre-mRNA splicing. Interestingly, spliced
mRNA that was exported to the cytoplasm was also
efficiently immunoprecipitated by anti-Y14 antibody
(Figure 4A). To address the possibility that cytoplasmic
Y14 binds to spliced mRNA in the cytoplasm after mRNA
export rather than being exported with it from the nu-
cleus, we microinjected both pre-mRNA and mRNA pro-
duced by in vitro transcription into oocyte cytoplasm.
After a 1.5 hr incubation, immunoprecipitations were
carried out from the cytoplasmic fraction with the anti-
bodies indicated. As shown in Figure 4C, only trace
amounts of these RNAs could be immunoprecipitated
with anti-Y14 antibody. These results suggest that Y14
binds to spliced mRNA in the nucleus, stays on it during
export, and remains associated with the newly exported
mRNA in the cytoplasm.
Y14 Becomes Associated with pre-mRNAs
after Splicing of a Single Intron
To determine whether Y14 binding is directly coupled
to splicing or is simply part of a postsplicing process
that occurs after the processing of the mRNA has been
completed, we examined the binding of Y14 to splicing
intermediates of pre-mRNAs that contain more than one
intron. For these experiments, we used a 661 nucleotide
pre-mRNA derived from the chicken ␦-crystallin pre-
mRNA containing exons 13, 14, and 15, and the introns
that separate them in the native pre-mRNA (Ohno et
al., 1987). This construct, (CDC2), has been extensively
used and characterized in in vitro splicing reactions
(Ohno et al., 1987). Figure 5 shows that this pre-mRNA
is spliced in the oocyte nucleus, and the resulting mRNA
is exported to the cytoplasm. Within the time frame of
this experiment (1 hr incubation), the pre-mRNA con-
taining both introns is no longer detected, but in addition
to the final mRNA product and the intron lariats a splicing

Molecular Cell
676
Figure 3. Immunoprecipitation of RNAs from In Vitro Splicing Reactions
(A–E) The indicated 32P-labeled pre-mRNAs and mRNAs were incubated with HeLa cell nuclear extracts under splicing conditions, followed
by immunoprecipitation with the indicated antibodies. Recovered RNAs were separated by polyacrylamide gel electrophoresis. The structure
of immunoprecipitated RNAs is schematically shown at the right of each panel. Boxes and line represent exons and intron, respectively. Lanes
marked total contain 10% of input RNA.
intermediate containing one remaining intron is still de-
tectable. Interestingly, the anti-Y14 antibody immuno-
precipitates the intermediate that still contains one in-
tron indicating that the splicing of a single intron is
sufficient for Y14 association. Thus, rather than requiring
complete processing of the pre-mRNA, Y14 association
is directly coupled to the splicing process itself. Two
additional observations from this experiment are note-
worthy. First, removal of a single intron appears to be
sufficient for export in this system. This suggests that
the presence of an intron is not, at least in the context
of this pre-mRNA, sufficient to retain the transcript in
the nucleus. Rather, once the first intron is removed
(and Y14 associates with the pre-mRNA) a competition
takes places between export and splicing of the re-
maining intron(s). This suggests that Y14 association is
a marker, if not an outright effector, of an RNP that is
directed toward export. Second, the hnRNP C proteins
preferentially associate with intron lariats. This appears
to be the case for all of the lariats we have tested both
in vivo and in vitro, and it suggests that there is a specific
high-affinity interaction between these proteins and ei-
ther intronic sequences or with components that are
bound to the intron lariats. Further experiments to define
this interaction will be of interest.
Y14-Containing RNP Complexes Are Distinct
from General hnRNP Complexes
In light of the results described above, we asked if Y14
is present in hnRNP complexes. To do so, we compared
hnRNP complexes and Y14-containing complexes by
immunoprecipitating from [35S]methionine-labeled HeLa
cell nucleoplasm with antibodies to hnRNP A1 and Y14,
respectively. As shown in Figure 6A, the anti-hnRNP A1
antibody coimmunoprecipiated the hnRNP proteins as
reported previously (Pin˜ ol-Roma et al., 1988). The Y14
antibody coimmunoprecipitated several proteins in ad-
dition to Y14 (Figure 6A). Although the pattern of the
Y14-containing immunoprecipate is similar to that of the
hnRNP complex, several bands are either specific to or
enriched in Y14 complexes, while others are specific to

Y14 Defines Postsplicing mRNP Complexes
677
Figure 4. Immunoprecipitation of RNAs from
Xenopus Oocyte Nucleus and Cytoplasm
Immunoprecipitations and RNA analyses
were performed as described in Figure 3. N
and C represent nucleus and cytoplasm, re-
spectively.
(A) 32P-labeled Ad2 pre-mRNA was injected
in oocyte nuclei. RNA was immunoprecipi-
tated from the nuclear and the cytoplasmic
fractions with anti-Y14 (4C4), anti-Xeno-
pus (x)hnRNP C1 (10C1), and SP2/0. (B)
32P-labeled Ad2 and DHFR mRNAs were in-
jected into oocyte nuclei, and following incu-
bation RNAs were immunoprecipitated as de-
scribed in (A).
(C) Ad2 pre-mRNA, Ad2 mRNA, and DHFR
mRNA was injected into oocyte cytoplasm,
and following incubation the cytoplasmic
fraction was used for RNA immunoprecipi-
tation.
or enriched in hnRNP complexes (Figure 6A). The Y14
protein itself is not intensely labeled presumably be-
cause it only contains three methionines (Figure 1).
To further examine the difference between hnRNP and
Y14-containing complexes, we performed immunopre-
cipitation followed by Western blotting analyses. As ex-
pected, hnRNP A1 was efficiently co-immunoprecipi-
ated with anti-hnRNP C proteins antibodies (Figure 6B).
However, Y14 could not be detected in the anti-hnRNP
C or anti-hnRNP A1 immunoprecipitated complexes
(Figure 6B; data not shown). This demonstrates that Y14
is not present in general hnRNP complexes or is a very
minor component of hnRNP complexes. Similar immu-
noprecipitation experiments were carried out using the
Y14 antibody. Both hnRNP A1 and C proteins were coim-
munoprecipitated with Y14 (Figure 6B). However, the
amount of these proteins in Y14-containing complexes
was considerably lower than that in hnRNP complexes
(Figure 6B). Interestingly, TAP was detected in Y14 com-
plexes but not in hnRNP complexes (Figure 6B). TAP is
directly involved in export of viral RNAs and has been
suggested to be an mRNA export factor (Gruter et al.,
¨
1998; Bear et al., 1999; Braun et al., 1999; Kang and
Cullen, 1999; Katahira et al., 1999; Bachi et al., 2000).
These results suggest that there are at least two classes
of hnRNP-containing complexes in the nucleus. General
hnRNP complexes contain predominantly pre-mRNAs
and Y14 complexes most likely contain predominantly
nuclear postsplicing, pretransport mRNPs.
Y14 Shuttles between the Nucleus and the Cytoplasm
Although Y14 is present predominantly in the nucleus,
a clearly detectable signal is also found in the cytoplasm.
This, together with the observation that Y14 is bound to
mRNAs both in the nucleus as well as to newly exported

Molecular Cell
678
shuttling, cells were fixed and stained with anti-myc
antibodies after only 1 hr of incubation. Heterokaryons
were formed by PEG-mediated fusions of HeLa cells
that had been previously transfected with the indicated
constructs and mouse 3T3 cells. Staining of the cells
with the DNA dye Hoechst allows definitive distinction
between the HeLa and the 3T3 nuclei. Fusions and sub-
sequent short-term incubations were carried out in the
presence of cycloheximide to ensure that the observed
signals result from proteins in the HeLa nucleus and not
from newly synthesized proteins in the cytoplasm (Pin˜ ol-
Roma and Dreyfuss, 1992; Michael et al., 1995, 1997;
Nakielny and Dreyfuss, 1996). Constructs containing the
nonshuttling hnRNP C1 protein and the SV40 large T
antigen NLS served as negative controls. Using a similar
fusion to the rapid shuttler hnRNP A1 as a positive con-
trol, the data, presented in Figure 7, show that Y14 shut-
tles between the nucleus and the cytoplasm. We con-
clude that Y14 has a functional NLS (that is probably
recognized by and is imported by RanBP5) and a func-
tional NES and that it shuttles rapidly between the nu-
cleus and the cytoplasm.
Discussion
In this report, we have identified a novel nuclear RNA
binding protein, Y14. The amino acid sequence of Y14
is highly conserved among divergent organisms, sug-
gesting that it has an important cellular function. Al-
though a likely Y14 ortholog is found in S. pombe, no
proteins with significant homology are apparent in Sac-
charomyces cerevisiae. Because it contains an RNP mo-
tif (Burd and Dreyfuss, 1994), it is likely that Y14 binds
to RNA. Antibodies to Y14 preferentially immunoprecipi-
tate spliced mRNAs from both in vitro splicing reac-
tions and from Xenopus oocytes nuclei. In contrast, the
same antibodies could not efficiently immunoprecipitate
mRNAs synthesized by in vitro transcription, although
the sequences of these mRNAs are identical to those
of the mRNAs produced by splicing. This indicates that
Y14 becomes associated with mRNA only as a conse-
quence of the splicing process. Although it is possible
that the preferential immunoprecipitation of mRNPs with
the anti-Y14 antibody (4C4) results simply from epitope
accessibility (i.e., that Y14 is in fact present also on pre-
mRNAs and lariat introns), we find this unlikely because
the same results were obtained using several mono-
clonal antibodies to different parts of Y14 (N. K. and
G. D., unpublished data) and because there is no detect-
able Y14 in hnRNP complexes isolated with antibodies
to hnRNP A1 and C1/C2 proteins.
Y14 was not detected in general hnRNP complexes
but rather only in complexes purified with anti-Y14.
These novel complexes contain many of the major
known hnRNP proteins including A1 and C proteins, but
the amount of these proteins in Y14-containing com-
plexes is significantly reduced compared to that found
in hnRNP complexes. Most of the anti-Y14 coimmuno-
precipitated proteins are no longer detected after RNase
treatment (data not shown), supporting the conclusion
that Y14 is a component of RNA-containing complexes
presumably mRNAs. Notably, the Y14 complexes but
not hnRNP complexes contain the mRNA export factor
Figure 5. Y14 Associates with pre-mRNAs or mRNAs after Removal
of a Single Intron
Immunoprecipitations and RNA analyses were performed as de-
scribed in Figures 3 and 4. N and C represent nucleus and cyto-
plasm, respectively. 32P-labeled CDC-derived pre-mRNA comprising
exons 13–15 and the intervening sequences as found in the native
pre-mRNA was injected into oocyte nuclei. RNA was immunoprecip-
itated from the nuclear and the cytoplasmic fractions with anti-Y14
(4C4), anti-Xenopus (x)hnRNP C1 (10C1), and SP2/0. The designa-
tion of the products and the splicing intermediates is based on
previous characterization (Ohno et al. 1987).
mRNA in the cytoplasm, suggested that Y14 shuttles
between the nucleus and the cytoplasm. Consistent with
this, microinjections of Y14 produced by in vitro tran-
scription/translation into oocyte cytoplasm or nucleus
showed that it is readily imported and exported from
the nucleus (data not shown). However, because the
molecular mass of Y14 is considerably smaller than the
diffusion limit through nuclear pores we could not con-
clude on the basis of these experiments that Y14 is a
shuttling protein. To circumvent these difficulties and
address this directly, we constructed chimeric proteins
comprised of Y14 fused to the nucleoplasmin core do-
main and containing a nuclear localization signal from
the SV40 large T antigen. The nucleoplasmin core do-
main forms highly stable large oligomers that remain in
the nucleus unless coupled to a strong NES, and such
constructs have been previously used in conjunction
with heterokaryons to determine if proteins contain
NESs (Michael et al., 1995, 1997; Nakielny and Dreyfuss,
1996). A myc-tagged epitope was fused at the amino
terminus of all constructs to provide for equivalent de-
tection of all the chimeric proteins. To monitor for rapid
TAP (Gruter et al., 1998; Braun et al., 1999; Kang and
¨
Cullen, 1999; Katahira et al., 1999; Bachi et al., 2000).
These results indicate that there are at least two distinct
hnRNP protein-containing complexes in the nucleus,

Y14 Defines Postsplicing mRNP Complexes
679
Figure 6. Y14 Is in a Novel RNP Complex Dif-
ferent from General hnRNP Complexes
(A) Immunoprecipitations were performed
from 35S-labeled HeLa cell nucleoplasm with
anti-hnRNP A1 (4B10), anti-hnRNP (4F4), and
anti-Y14 (4C4) antibodies followed by SDS-
PAGE. Proteins specific or enriched in Y14
complex are shown by closed circles. Both
hnRNP A1, C1, and Y14 are indicated.
(B) Immunoprecipitations were performed us-
ing the indicated antibodies and subjected to
Western blot analyses. The lane marked as
nucleoplasm contains 3% of input fraction.
SP2/0 was used as a negative control.
the general hnRNP complexes and the Y14-containing
mRNP complexes. Y14 is an abundant protein although
it is much less abundant (5- to 10-fold) than hnRNP A1
and the C proteins (data not shown). Based on these
results, we conclude that Y14 is a specific component
of a class of postsplicing mRNA-containing hnRNP com-
plexes that also contain several unidentified unique pro-
teins.
A plethora of observations accumulated over many
years strongly suggests that there is an intimate link
Figure 7. Y14 Shuttles Rapidly between the
Nucleus and the Cytoplasm
Expression vectors encoding hnRNP A1, C1,
and the given NPc fusion proteins were trans-
fected into HeLa cells. After expression of the
transfected cDNAs, the cells were fused with
mouse NIH3T3 cells to form heterokaryones
and incubated in media containing 100 ␮g/
ml cycloheximide for 1 hr. The cells were then
fixed and stained for immunofluorescence
microscopy with mAb 9E10 (anti-myc tag:
panel anti-myc) to localize the proteins, and
Hoechst 33258 (panel Hoechst), which differ-
entiates the human and mouse nuclei within
the heterokaryon (arrows identify the mouse
nuclei). The panels marked Phase show the
phase-contrast image of the hetrokaryons;
the cytoplasmic edge is highlighted by a bro-
ken line.

Molecular Cell
680
Figure 8. A Schematic Representation of
Two Different Nuclear RNP Complexes
The new Y14-containing nuclear RNP com-
plex and general hnRNP complex are dis-
cussed in the text.
between the various steps in the posttranscriptional
pathway of gene expression, particularly between splic-
ing and the downstream events including mRNA export,
translation, stability, and localization (Cheng et al., 1994;
Carter et al., 1996; Maquat, 1996; Li et al., 1997; Matsu-
moto et al., 1998; Zhang et al., 1998). Previous findings
have led to the conclusion that splicing promotes the for-
mation of an mRNA that is relieved of nuclear retention
elements and endowed with positively acting export fac-
tors (Legrain and Rosbash, 1989; Chang and Sharp,
1990; Stutz and Rosbash, 1998; Luo and Reed, 1999;
Nakielny and Dreyfuss, 1999; Cullen, 2000). Recent ex-
periments have shown that nuclear microinjection of
pre-mRNAs results in the efficient splicing and nuclear
export of spliced mRNAs, while nuclear injection of the
spliced forms of the same mRNAs results in less efficient
export (Luo and Reed, 1999). The RNPs formed on
mRNAs spliced in vitro have a slower electrophoretic
mobility on nondenaturing gels than those formed on
the corresponding intronless mRNAs synthesized by in
vitro transcription (Luo and Reed, 1999). Therefore, it is
likely that splicing can promote not only the removal of
nuclear retention factors but also the formation of more
efficient export complexes. The Y14 complex that forms
in the nucleoplasm is an excellent candidate for such
an export complex. We note, however, that the nuclear
microinjection experiments using the two-intron con-
taining CDC2 pre-mRNA argue that introns are not reten-
tion elements in themselves but rather that removal of
an intron (and the concomittant attachment of Y14 com-
plex) directs the transcript toward export.
metabolism such as mRNA stability, translation, detec-
tion of premature stop codons, or cytoplasmic local-
ization.
The antibodies to the hnRNP proteins A1 and C pro-
teins that we have used in these experiments efficiently
immunoprecipitated pre-mRNAs and intron lariats but
not the spliced mRNA product. However, this does not
imply that these proteins are not associated with spliced
mRNAs in vivo. In fact, immunoelectronmicroscopy has
clearly visualized these proteins (hnRNP A1-like pro-
teins) associated with mRNAs that are in the process
of export to the cytoplasm and on polysomes in the
cytoplasm (Visa et al., 1996). In order to facilitate analysis
of splicing intermediates and products by polyacryl-
amide gel electrophoresis, we and others have used
very small transcripts. It is likely that the lack of efficient
immunoprecipitation of the small mRNAs that are pro-
duced here by anti-hnRNP antibodies is simply a conse-
quence of their local displacement first by components
of the spliceosome and later by proteins of the Y14
complex.
Finally, our findings suggest that pre-mRNA splicing
is a process that serves not only to remove introns but
also to imprint the mRNA, through the binding of Y14
and other specific proteins. It is of significance that this
imprint, in the form of the Y14 complex, persists on the
mRNA even after its export in the cytoplasm. Because
a single splicing event is sufficient for Y14 association,
we consider it likely that each spliceosome deposits a
Y14 complex at or near the point at which the exons
joined. This would provide a mechanism for marking
and communicating to the cytoplasm the position on
the mRNA of where introns were present even though
they have been removed earlier in the nucleus. Such a
tagging mechanism would provide an efficient means
of coupling pre-mRNA splicing to postsplicing events.
Further characterization of the components of Y14-con-
taining complexes should shed light on this pathway.
Our view of Y14-containing complexes in relation to
the postsplicing pathway is depicted in Figure 8. The
presence of TAP in Y14 complexes is of considerable
interest as this protein appears to function as an mRNA
export factor. TAP contains domains that can mediate
binding to both the mRNP and nuclear pore complex
and is critical for the final step of mRNA export (Gruter
¨
et al., 1998; Katahira et al., 1999; Bachi et al., 2000). Y14
or other unique components of the Y14 complexes in
a postsplicing, preexport complex may serve to recruit
TAP and other factors that are required for mRNA export
(Figure 8). Further analysis of Y14 complexes may reveal
a bridge between two major nuclear events, pre-mRNA
splicing and mRNA export. Although the Y14-containing
mRNPs likely represent mRNPs that are competent for
and poised for nuclear export, the specific function, if
any, of Y14 in nuclear export is not known. It may in
fact be that Y14 does not have a direct function in mRNA
export but rather in other aspects of postsplicing mRNA
Experimental Procedures
Construction of Plasmids and Recombinant Protein Preparation
Full length human Y14 cDNA was PCR amplified with Pfu DNA
polymerase (STRATAGENE) from the two-hybrid plasmid and in-
serted into EcoRI and XhoI digested pGEX-5X-1 (Amersham Phar-
macia Biotech Inc.) and pET28a (Novagen). For construction of
pAd2m and pCDCm, spliced mRNAs were recovered from a poly-
acrylamide gel after being spliced from Ad2 and CDC (previously
called SP14-15) pre-mRNAs, respectively (Ohno and Shimura, 1996;
Pellizzoni et al., 1998). DNA fragments were synthesized from these
mRNAs using specific primers and reverse transcriptase (Behringer-
Mannheim), followed by PCR amplification with specific primers.

Y14 Defines Postsplicing mRNP Complexes
681
The resultant fragments were cloned into pCDNA3 (Invitrogen) and
pSP65 (Promega) and sequenced. pT7-DHFR was kindly provided
by Dr. Iain Mattaj. Proteins were overexpressed in the E. coli
BL21(DE3) and purified according to the manufacturer’s recommen-
dation. The plasmids myc-hnRNP A1, C1, and myc-NPc-T have
been previuously described (Michael et al., 1995, 1997; Nakielny
and Dreyfuss, 1996). To construct myc-NPc-T-Y14, the full-length
fragment of Y14 was PCR amplified and inserted between XhoI and
XbaI of myc-NPc-T.
grants from the National Institutes of Health and the Human Frontier
Science Program Organization. G. D. is an Investigator of the How-
ard Hughes Medical Institute.
Received April 5, 2000; revised July 7, 2000.
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