1 Department of Molecular Biology and Functional Genomics, Stockholm University,
SE-10691 Stockholm, Sweden
2 Department of Zoological Cell Biology, The Wenner-Gren Institute, Stockholm
University, SE-10691 Stockholm, Sweden
Author for correspondence (e-mail:
neus.visa{at}molbio.su.se)
Accepted 28 May 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: RNA-binding protein, Chironomus tentans, Oligomerization, Yeast two-hybrid, Transfection, Nuclear import
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the salivary glands of C. tentans, Hrp65 is located in
non-chromatin nucleoplasmic fibers, referred to as connecting fibers (CFs), as
shown by immuno-electron microscopy studies using an antibody that recognizes
all the Hrp65 isoforms. The CFs interact with pre-mRNP particles in the cell
nucleus, which suggests that Hrp65 is involved in mRNA biogenesis at the
post-transcriptional level (Miralles et
al., 2000). But recent studies have shown that the Hrp65-2 isoform
plays a role in transcription by RNA polymerase II
(Percipalle et al., 2003
).
Hrp65 is evolutionary conserved in metazoans and has high sequence
similarity to a group of RNA-binding proteins that includes the mammalian
proteins paraspeckle protein 1 (PSP1), polypyrimidine tract-binding
protein-associated splicing factor (PSF) and p54nrb/NonO, and the
Drosophila protein NonA/Bj6 (Fox
et al., 2002; Patton et al.,
1993
; Dong et al.,
1993
; Yang et al.,
1993
; von Besser et al.,
1990
; Jones and Rubin,
1990
; Shav-Tal and Zipori,
2002
). These proteins, together with Hrp65, share a conserved
central domain of 320 amino acids referred to as the DBHS domain, for
Drosophila behaviour and human
splicing (Dong et al.,
1993
). The DBHS domain consists of two RRMs followed by a
downstream element of approximately 100 amino acids recently identified as a
protein-protein interaction domain [(Peng
et al., 2002
) and this study].
The DBHS proteins appear to play several different roles in gene
expression. The most studied DBHS protein, the human PSF, was first
characterized as a splicing factor (Patton
et al., 1993). PSF is often found in a complex with another DBHS
protein, p54nrb/NonO, and several studies have implied a
function for the PSF/p54nrb complex in transcriptional
regulation (Basu et al., 1997
;
Yang et al., 1997
;
Mathur et al., 2001
;
Sewer et al., 2002
;
Emili et al., 2002
), splicing
(Peng et al., 2002
) and
nuclear retention of viral RNA (Zhang and
Carmichael, 2001
). In Drosophila, mutations of the NonA
gene cause defects in the central nervous system that affect vision and
behavior in the fly (Jones and Rubin,
1990
; Rendahl et al.,
1992
), but the function of NonA at the molecular level is
unknown.
In mammalian cells, PSF and p54nrb/NonO have been found to be stably associated with each other, which suggests that dimerization among proteins of the DBHS group is functionally important. In a yeast two-hybrid screening that we report here, we have found that Hrp65 can interact with itself. We have confirmed the Hrp65 self-interaction both in vivo and in vitro, shown that the different hrp65 isoforms can interact with each other, mapped the Hrp65 self-interaction domain to the downstream element of the DBHS domain, and found that Hrp65 can not only dimerize but also oligomerize into complexes of at least 400 kDa. Moreover, we have analyzed the functional significance of the Hrp65 self-interaction and found that the NLS of Hrp65-1 is required for the nuclear location of Hrp65-2 and Hrp65-3, which suggests that the interaction between different isoforms is necessary for the nuclear import of those isoforms that lack a NLS.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmid constructions for expression in Escherichia
coli
The three Hrp65 isoforms were expressed in bacteria with an N-terminal T7
tag and a C-terminal 6xHis tag by cloning their cDNAs into pET21b
(Novagen). A DNA fragment containing the complete ORF of Hrp65-2 was subcloned
from pEGFP-C3-Hrp65-2 (Miralles and Visa,
2001) into the BamHI-SalI site of pET21b to
generate T7-Hrp65-2-His. This construct was subsequently digested with
EcoRI and SalI, releasing the C-terminus of Hrp65-2. The
backbone was ligated with EcoRI-SalI and
EcoRI-XhoI fragments obtained from pCR2.1-Hrp65-1 and
pCR2.1-Hrp65-3 (Miralles and Visa,
2001
), to obtain T7-Hrp65-1-His and T7-Hrp65-3-His,
respectively.
Plasmid constructions for expression in yeast
The bait construct DBD-Hrp65 was generated by inserting a PCR fragment
encoding the complete ORF of Hrp65-1 into the SalI site of the
pBDGAL4 vector (Stratagene), generating pBDGAL4-Hrp65. PCR fragments encoding
Hrp65-1 sequences corresponding to amino acids 1-95, 1-267 and 416-535 were
inserted into the EcoRI-SalI sites of pBDGAL4 to obtain
pBDGAL4-Hrp65(1-95), pBDGAL4-Hrp65(1-267) and pBDGAL4-Hrp65(416-535).
PCR fragments encoding either the complete ORF of Hrp65-3 or amino acids
89-273 and 259-415 were each cloned into the BamHI-XhoI
sites of the pADGAL4 phagemid vector (Stratagene), generating pADGAL4-Hrp65,
pADGAL4-Hrp65(89-273) and pADGAL4-Hrp65(259-415). For the construction of
pADGAL4-Hrp65(259-415), cloning of the deletion construct was first
made in a mammalian expression vector. A PCR fragment encoding Hrp65 amino
acids 1-258, N-terminally fused to a Flag-epitope, was amplified from
pEGFP-C3-Hrp65-2 using the 65Bgl-Flag oligonucleotide and a 3'
oligonucleotide that gives amplification from Hrp65 amino acid 258. Another
PCR fragment encoding the Hrp65 amino acids 416-517 was amplified from the
same template using specific oligonucleotides. The Hrp65(1-258) and
Hrp65-2(416-517) fragments were joined in-frame by a two-step cloning of the
fragments into the mammalian vector, generating pCS2-Hrp65-2(
259-415).
From this plasmid, a new PCR fragment encoding the full sequence of Hrp65-2
with the 259-415 deletion (amino acids 1-258/416-517) was amplified with
oligonucleotides 65Bgl-Flag and 652-R-X. This fragment was
inserted into the BamHI-XhoI site of the isolated pADGAL4
vector to obtain pAD-Hrp65-2(
259-415).
To generate DBD-NonA(448-603) and DBD-PSF(443-601) fusion constructs, total RNA was isolated from Drosophila S2 Schneider cells or human HeLa cells, respectively, and used as templates for reverse transcriptase-polymerase chain reaction (RT-PCR). PCR fragments corresponding to amino acids 448-603 of NonA and 443-601 of PSF were amplified using specific oligonucleotides and inserted into the EcoRI-PstI sites of pBDGAL4 to generate pBDGAL4-NonA(448-603) and pBDGAL4-PSF(443-601).
Yeast two-hybrid system
The interaction screen was performed essentially according to the
supplier's instructions (Stratagene). A HYBRI-ZAP cDNA library from C.
tentans tissue culture cells was constructed and cloned into the
EcoRI and XhoI sites of the pAD-GAL4-2.1 phagemid vector.
The cDNA library was transformed into the yeast strain AH109 (Clontech) that
had been pre-transformed with DBD-Hrp65. The expression of the fusion proteins
encoded in the GAL4 DBD- and AD-fusion constructs was assayed by western
blotting before assessment of protein-protein interactions. The fusion
proteins were also expressed one by one in yeast to rule out reporter gene
activation.
Multipex PCR
DNA from isolated library plasmids was used as template in a multiplex PCR
reaction with a sense 65F5 oligonucleotide that is common to all
known Hrp65 isoforms plus two antisense oligonucleotides 65S,
specific for Hrp65-2 cDNA, and 65L, which anneals both Hrp65-1 and
Hrp65-3 cDNAs (Miralles and Visa,
2001).
In vitro binding assay
Plasmids encoding T7-Hrp65-1-His, T7-Hrp65-2-His or T7-Hrp65-3-His were
introduced into E. coli Bal21(DE3). After induction with
isopropyl-ß-D-thiogalactopyranoside (IPTG), bacterial extracts were
prepared in 50 mM MOPS at pH 7.0, 300 mM NaCl, 15 mM imidazole, and the
recombinant proteins were purified on Ni-NTA-agarose (Qiagen). The rabbit
reticulocyte coupled transcription/translation system (TnT; Promega) was used
for the expression of 35S-methionine-labeled proteins in vitro.
Ni-NTA-agarose containing purified Hrp65-1, -2 or -3 was washed in
radioimmunoprecipitation (RIPA) buffer (1% NP40, 0.5% sodium deoxycholate,
0.1% sodium dodecyl sulphate (SDS), 0.1 mg/ml PMSF in PBS) and incubated with
translation mixtures (35S-methionine-labeled Hrp65-1, -2 or -3) in
RIPA buffer containing 50 mM imidazole. After extensive washing in RIPA buffer
containing 50 mM imidazole, proteins were eluted, separated by SDS-PAGE and
analyzed by autoradiography.
Gel filtration
Recombinant His-Hrp65-1 protein was expressed in E. coli as
described for the in vitro binding assay and purified on Ni-NTA-agarose
(Qiagen). The purified protein was concentrated using a Nanosep device (Pall)
and stored in 50 mM MOPS, 300 mM NaCl, 250 mM imidazole. The protein was
fractionated on a Superose HR6 column (Amersham Biosciences) at a flow rate of
0.2 ml/minute, and fractions of 0.5 ml were collected. The proteins in the
fractions were precipitated with acetone, separated on 12% SDS-PAGE and
detected by Coomassie staining.
C. tentans protein extracts
Cytoplasmic and nuclear protein extracts were prepared as previously
described (Miralles et al.,
2000).
Transfection of HeLa cells
HeLa cells plated onto coverslips in 35 mm dishes were transiently
transfected with pEGFP-C3-Hrp65-1, pEGFP-C3-Hrp65(1-499)
(Miralles and Visa, 2001) or
pCS2-Flag-Hrp65-2 using the Lipofectamine reagent (Invitrogen). In the same
experiment, cells were cotransfected with pEGFP-C3-Hrp65-1 and
pCS2-Flag-Hrp65-2 or with pEGFP-C3-Hrp65(1-499) and pCS2-Flag-Hrp65-2.
Transfection medium was removed after 5 hours and cells were cultured in
serum-containing medium for 12 hours before immunoflourescence. Transfection
experiments were repeated at least three times with reproducible results.
Immunofluorescence
Transfected HeLa cells were treated for fluorescence using standard
procedures. Briefly, cells were fixed for 15 minutes in 3.7% formaldehyde/PBS
and permeabilized for 5 minutes in 0.5% Triton X-100 in PBS. Flag-tagged
proteins were visualized using an anti-Flag monoclonal antibody (Sigma) and a
Texas Red-conjugated secondary antibody (ICN/Cappel). Fluorescence was
observed in a Zeiss Axioplan-2 microscope. Cells transfected with a single
construct were also analyzed using both filters to rule out possible overlap
between the GFP and the Texas Red signals.
Salivary glands from fourth instar larvae of C. tentans were fixed
in PBS containing 4% paraformaldehyde for 60 minutes at room temperature,
cryoprotected with 2.3 M sucrose and frozen by immersion in liquid nitrogen.
Semi-thin sections were obtained in a cryoultramicrotome (Ultracut S/FC S,
Reichert) and mounted onto glass slides as previously described
(Visa et al., 1996). Indirect
immunofluorescence was performed according to standard procedures using a
FITC-conjugated secondary antibody.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously described three isoforms of the Hrp65 protein that are
generated by alternative splicing of one pre-mRNA
(Miralles and Visa, 2001). The
three isoforms share amino acids 1-499 but vary in their C-termini
(Fig. 1A). To determine the
isoform-specificity of the Hrp65-encoding clones obtained from the two-hybrid
screening, plasmid DNA isolated from individual cDNA clones was subjected to a
multiplex PCR reaction with oligonucleotides designed to amplify the distinct
Hrp65 cDNAs. All three Hrp65 isoforms were found in approximately equal
proportions (Fig. 1B), which
showed that Hrp65-1 could interact with all three Hrp65 isoforms.
|
Sequence analysis revealed that all the cDNAs encoding Hrp65 isolated in the screening were N-terminally truncated, as illustrated in Fig. 1C. Isolation of clones lacking amino acids 1-182 or 1-234 indicated that the Hrp65 self-interaction is mediated by a sequence downstream of the first RRM.
To verify whether the full-length Hrp65 can also self-interact, we co-expressed DBD-Hrp65 with a construct encoding the full ORF of Hrp65 fused to the GAL4 activation domain (AD-Hrp65). Yeast cells expressing both DBD-Hrp65 and AD-Hrp65 were able to grow in -His -Ade selective medium, which confirmed the ability of full-length Hrp65 to interact with itself.
To confirm the results of the yeast two-hybrid screening, we assayed the interaction of Hrp65-1 with the three Hrp65 isoforms in vitro. Recombinant Hrp65-1 was expressed with a C-terminal 6xHis-tag (His-Hrp65-1) and immobilized on Ni-NTA agarose. The immobilized His-Hrp65-1 was then incubated in a high stringency RIPA buffer with 35S-labeled Hrp65-1, -2 or -3 obtained by in vitro translation. In parallel, 35S-labeled proteins were incubated with Ni-NTA agarose without His-Hrp65 as a negative control. The 35S-Hrp65-1, -2 and -3 bound with similar efficiencies to His-Hrp65-1 immobilized on Ni-NTA (Fig. 2, lanes 7-9), but showed very little or no binding to the Ni-NTA agarose alone (Fig. 2, lanes 4-6). A control 35S-chloramphenicol acetyltransferase (CAT) protein did not bind to His-Hrp65 proteins immobilized on Ni-NTA agarose (not shown).
|
To investigate whether Hrp65-2 and Hrp65-3 can also interact with themselves and/or each other, we expressed recombinant His-tagged Hrp65-2 and -3 and assayed the interaction of these isoforms with the 35S-Hrp65 isoforms. Similarly to His-Hrp65-1, His-Hrp65-2 and -3 bound all three Hrp65 isoforms to the same extent (Fig. 2, lanes 10-15). These observations confirm that the in vitro interaction of Hrp65 is isoform-independent, in agreement with the results from the yeast two-hybrid screening reported above (Fig. 1C).
Hrp65 self-interaction is mediated by the downstream element of the
conserved DBHS domain
To identify the region of Hrp65 that mediates Hrp65-Hrp65 interaction, we
expressed truncated Hrp65 mutants and assayed their ability to interact with
full-length Hrp65 in the two-hybrid system. The results from these experiments
are shown in Fig. 3A. Yeast
transformed with two full-length Hrp65 constructs could grow readily under
-His as well as -His -Ade selection (Fig.
3A, top). As expected from the isolation of N-terminally truncated
clones in the two-hybrid screening, a truncation construct encoding the
N-terminal domain of Hrp65, 65(1-95), showed no interaction with the
full-length protein. A construct containing the C-terminal domain,
65(416-535), did not show any interaction either. We then tested two
constructs corresponding to the central DBHS element, one containing the two
RRMs, 65(89-273), and another containing the downstream region of the DBHS
domain, 65(259-415). The transformants of the 65(89-273) construct showed poor
growth on double-selective medium, but the transformants of the 65(259-415)
construct were able to grow on -His -Ade medium, which showed that the
downstream DBHS element interacted strongly with Hrp65. As no growth on
double-selective medium could be detected with a construct comprising the
entire N-terminal domain plus the two RRMs, 65(1-267), we concluded that the
259-415 region alone is responsible for mediating Hrp65-Hrp65 interaction.
Moreover, removal of the same region (259-415) totally abolished the
interaction, showing that the 259-415 region is not only sufficient but
necessary for mediating interaction between Hrp65 proteins.
|
In view of the present results, the DBHS domain can be described as a compound domain that contains two functional elements: an upstream element with two juxtaposed RRMs, and a downstream element that mediates protein self-interaction. We refer to this latter element as the protein binding domain, or PBD.
Other proteins of the DBHS group have been found to be associated with each other (see Discussion), and sequence comparisons show that the PBD is evolutionarily conserved among proteins of the DBHS group (Fig. 3B). The PBD of Hrp65 is 57% and 38% identical to the PBDs of Drosophila NonA and human PSF, respectively. To analyze the ability of the PBD to mediate interactions between DBHS proteins, we expressed the PBDs of the NonA (amino acids 448-603) and the PSF (amino acids 443-601) in the yeast two-hybrid system and analyzed their interaction with the full-length Hrp65. We found that both PSF(443-601) and NonA(448-603) interact with Hrp65 (Fig. 3C and data not shown). This result shows that the PBD is a conserved self-interaction domain for DBHS-proteins, capable of mediating both homodimerization and heterodimerization.
Hrp65 oligomerization in vitro
The observed Hrp65 self-interaction raised the possibility that the Hrp65
could form oligomers, which could be important for the formation of CFs in
vivo. To learn more about the Hrp65-Hrp65 association, we analyzed
oligomerization of recombinant Hrp65 in vitro. Electrophoretic analysis of
purified Hrp65 by SDS-PAGE in the absence of reducing agents revealed the
formation of high-molecular-weight complexes that were not affected by RNAse A
digestion (data not shown). To analyze the molecular mass of the detected
Hrp65 complexes, we purified recombinant Hrp65 and fractionated it on a
size-exclusion Superose HR6TM chromatography column. The proteins
in each fraction were collected and separated by SDS-PAGE. Most of the protein
was detected in fractions 29-33, corresponding to molecular masses in the
200-400 kDa range (Fig. 4).
This range corresponds to Hrp65 complexes between the size of a trimer and a
hexamer. Thus, we conclude that in our experimental conditions most of the
Hrp65 protein is in complexes that consist of three to six Hrp65
molecules.
|
Hrp isoforms that lack NLS can enter the nucleus by interacting with
Hrp65-1
We have previously shown by transient transfection assays that the variant
C-terminal sequences of the Hrp65 proteins are relevant for the subcellular
location of each isoform (Miralles and
Visa, 2001). When expressed in human and Drosophila
cells, GFP-Hrp65-2 and GFP-Hrp65-3 locate mainly to the cytoplasm, while
GFP-Hrp65-1 is efficiently imported to the nucleus due to the presence of a
NLS in its C-terminus (Miralles and Visa,
2001
). To analyze the cellular distribution of endogenous Hrp65-2
in C. tentans cells, we used a rabbit antibody against the C-terminal
sequence of Hrp65-2 (Percipalle et al., in press). This antibody labeled a
protein of approximately 65 kDa in both cytoplasmic and nuclear extracts of
C. tentans tissue culture cells
(Fig. 5A). We also used the
anti-Hrp65-2 antibody for immunoflourescent labeling of C. tentans
salivary gland cells. The Hrp65-2 antibody labeled chromosomes and
nucleoplasm, and also gave a faint spotted staining in the cytoplasm
(Fig. 5B). Thus, a significant
fraction of the endogenous Hrp65-2 is present in the nuclei of C.
tentans cells, despite the absence of an NLS in Hrp65-2. This suggested
that in vivo nuclear import of Hrp65-2 is mediated by interaction with another
protein. Given the strong interaction observed between Hrp65 isoforms, we
decided to investigate the possibility that Hrp65-2 enters the nucleus by
binding to Hrp65-1. For this purpose, we analyzed the simultaneous expression
of different Hrp65 isoforms. Owing to the absence of methods for transfecting
C. tentans cells, we performed transient transfections of Hrp65
constructs in HeLa cells. To be able to analyze co-expression of Hrp65-1 and
Hrp65-2, we expressed Hrp65-1 as a fusion with GFP and Hrp65-2 with a Flag
tag. Transfection of the Flag-Hrp65-2 construct showed that Flag-Hrp65-2
localizes mainly to the cytoplasm (Fig.
6A), which agrees with the previously reported localization of
GFP-Hrp65-2 (Miralles and Visa,
2001
). GFP-Hrp65-1 localizes to the nucleus, while a truncation of
the C-terminal sequence of Hrp65-1 that contains the NLS [GFP-Hrp65-1(1-499)]
renders the protein cytoplasmic. We then proceeded to cotransfection
experiments to analyze the distribution of Hrp65-2 in the presence of Hrp65-1.
Interestingly, when co-expressed with GFP-Hrp65-1, the distribution of
Flag-Hrp65-2 became mainly nuclear (Fig.
6B). Moreover, when Flag-Hrp65-2 was co-expressed with
GFP-Hrp65-1(1-499) instead of GFP-Hrp65-1, both GFP-Hrp65-1(1-499) and
Flag-Hrp65-2 remained in the cytoplasm. Similar results were obtained for
Hrp65-1 and Hrp65-3: Hrp65-3 was cytoplasmic when expressed alone, but nuclear
when co-expressed with Hrp65-1 (not shown). Our experiments show that Hrp65-2
and -3 can enter the nucleus by association with Hrp65-1.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The DBHS domain contains a protein-binding domain
We have mapped the sequences responsible for Hrp65 self-interaction to a
part of the protein that we refer to as the PBD located next to the RRMs in
the DBHS domain. The PBD of Hrp65 is conserved in all the members of the DBHS
group and the degree of similarity in this region is comparable to that
observed in the RRMs.
In mammalian cells, PSF and p54nrb have been found to
be associated with each other (e.g. Mathur
et al., 2001; Zhang and
Carmichael, 2001
), and deletions covering the PBD region of
p54nrb have been shown to disrupt the
p54nrb-PSF association
(Peng et al., 2002
). We have
now shown that the PBDs of NonA and PSF are sufficient to mediate the
interaction with full-length Hrp65 in vivo. Altogether, these observations
indicate that the PBD is an evolutionary conserved domain able to mediate not
only self-interaction but also heterodimerization between different proteins
of the DBHS group.
The DBHS proteins appear to be able to interact with a large number of
proteins in different physiological situations. For instance, PSF has been
associated with several pre-mRNA splicing factors
(Patton et al., 1993;
Lutz et al., 1998
;
Peng et al., 2002
). PSF and
p54nrb, in a complex with matrin 3, have been implicated
in nuclear retention of inosine-rich RNA, as part of a cellular defense
mechanism aimed at preventing expression of viral genomes
(Zhang and Carmichael, 2001
).
PSF has been found associated with nuclear structures such as the nuclear
envelope (Otto et al., 2001
)
and the nucleolus (Fox et al.,
2002
). PSF and p54nrb have also been found
associated with the RNA polymerase II
(Emili et al., 2002
), and the
p54nrb-PSF complex has been implicated in transcriptional
regulation through interactions with nuclear hormone receptors and Sin3A
(Mathur et al., 2001
;
Sewer et al., 2002
). In C.
tentans, Hrp65 has been found to be associated, directly or indirectly,
with a large number of proteins, including the hnRNP protein hrp36
(Wurtz et al., 1996
), the
putative transcription factor p2D10 (Sabri
et al., 2002
), and actin
(Percipalle et al., 2003
).
These many different interactions and the multiple functions attributed to the
DBHS proteins, suggest that a general role of this family of proteins is to
serve as platforms for protein-protein interactions in different cellular
processes. In this context, the dimerization or oligomerization of the DBHS
proteins may constitute a mechanism to mediate long-range intermolecular
interactions.
A role for Hrp65 in the formation of connecting fibers?
Hrp65 was identified in the salivary gland cells of C. tentans as
a protein located in connecting fibers (CFs), thin fibers associated with
pre-mRNPs in transit from the gene to the nuclear envelope
(Miralles et al., 2000).
Whether hrp65 is a structural component of the CFs or a protein bound to the
CFs remains to be established. However, our present finding that Hrp65 can
build oligomeric complexes suggests that this protein could be a structural
component of the CFs. On the basis of the dimensions of the CFs, characterized
by a diameter of approximately 5-7 nm and a variable length in the 50-100 nm
range, it can be estimated that a typical CF is composed of 50-100 protein
molecules of average size. Thus, the molecular mass of the oligomers detected
in our in vitro assay is not sufficient to explain the formation of CFs as it
occurs in vivo. Perhaps Hrp65 contributes only to the proximal part of the CF,
in contact with the pre-mRNP, as suggested by previous immuno-electron
microscopy experiments showing that Hrp65 is not located along the full length
of the CFs but only in the proximal part
(Miralles et al., 2000
). It is
also likely that other proteins contribute to the architecture of CFs.
We attempted to study the functional significance of Hrp65 self-interaction in CF formation by microinjecting recombinant PBD into live salivary gland cells. However, such experiments failed beause we could not prevent the recombinant PBD from polymerizing in vitro under the physiological conditions required for microinjection.
It is also important to notice that both C. tentans Hrp65 and recombinant Hrp65 produced in E. coli are prone to form large insoluble aggregates under native conditions. The buffers used in our protocol for Hrp65 purification were optimized to keep the recombinant Hrp65 in solution (see Materials and Methods), and by doing so we may have impaired the formation of larger Hrp65 multimers. In summary, our results indicate that the Hrp65 protein has the ability to form oligomeric complexes, but the molecular mass of Hrp65 oligomers in vivo and their contribution to the formation of CFs must be further investigated.
The Hrp65-Hrp65 interaction is required for intracellular
localization of Hrp65 isoforms
Our present results reveal that the Hrp65-Hrp65 interaction is required for
the localization of the Hrp65 isoforms inside the cell. The nuclear import of
Hrp65-1 is mediated by an NLS located in the C-terminus of the protein,
whereas Hrp65-2 and Hrp65-3 lack this NLS and are cytoplasmic when transiently
expressed in heterologous systems
(Miralles and Visa, 2001). We
have now reported that the PBD of PSF can interact with Hrp65, which suggests
that the endogenous PSF could potentially mediate nuclear import of Hrp65
isoforms in transfected mammalian cells. Indeed, in single transfection
experiments, a certain increase in nuclear fluorescence was observed after
long post-transfection times for both Hrp65-2 and Hrp65-3. However, in the
conditions of our experiments, Hrp65-2 and Hrp65-3 appeared as mainly
cytoplasmic due to the high overexpression levels.
Immunoblot and immunofluorescence analysis with an antibody specific for
Hrp65-2 showed that the endogenous Hrp65-2 isoform is not restricted to the
cytoplasm but is also present in the nucleus of C. tentans cells, in
agreement with the reported location of Hrp65-2 at transcription sites on
C. tentans polytene chromosomes
(Percipalle et al., 2003). Our
cotransfection experiments indicate that the import of Hrp65-2 and Hrp65-3 is
dependent on the presence of Hrp65-1 and requires the C-terminal NLS of
Hrp65-1. Thus, we conclude that Hrp65-2 and Hrp65-3 are imported into the
nucleus associated with Hrp65-1. Given the role of Hrp65-2 in transcription
(Percipalle et al., 2003
), it
is tempting to speculate that this heterodimerization-dependent import could
be regulated and could influence other intranuclear events.
NonA, PSP1 and PSF also exist in several isoforms with variable C-terminal
sequences, and in each case one of the isoforms is similar to Hrp65-1.
Moreover, at least in the case of PSF, one isoform lacks the C-terminal NLS
(Dye and Patton, 2001), as does
Hrp65-2. The present results about Hrp65, together with the conservation of
isoform structure among DBHS proteins, suggest that the intracellular
localization of other DBHS proteins is also based on
dimerization/oligomerization mechanisms.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Basu, A., Dong, B., Krainer, A. R. and Howe, C. C. (1997). The intracisternal A-particle proximal enhancer-binding protein activates transcription and is identical to the RNA- and DNA-binding protein p54nrb/NonO. Mol. Cell. Biol. 17, 677-686.[Abstract]
Daneholt, B. (2001). Assembly and transport of
a premessenger RNP particle. Proc. Natl. Acad. Sci.
USA 98,
7012-7017.
Dong, B., Horowitz, D. S., Kobayashi, R. and Krainer, A. R. (1993). Purification and cDNA cloning of HeLa cell p54nrb, a nuclear protein with two RNA recognition motifs and extensive homology to human splicing factor PSF and Drosophila NONA/BJ6. Nucleic Acids Res. 21, 4085-4092.[Abstract]
Dye, B. T. and Patton, J. G. (2001). An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles. Exp. Cell Res. 263, 131-144.[CrossRef][Medline]
Emili, A., Shales, M., McCracken, S., Xie, W., Tucker, P. W.,
Kobayashi, R., Blencowe, B. J. and Ingles, C. J. (2002).
Splicing and transcription-associated proteins PSF and p54nrb/nonO bind to the
RNA polymerase II CTD. Rna
8,
1102-1111.
Fox, A. H., Lam, Y. W., Leung, A. K., Lyon, C. E., Andersen, J., Mann, M. and Lamond, A. I. (2002). Paraspeckles: a novel nuclear domain. Curr. Biol. 12, 13-25.[CrossRef][Medline]
Jones, K. R. and Rubin, G. M. (1990). Molecular analysis of no-on-transient A, a gene required for normal vision in Drosophila. Neuron 4, 711-723.[Medline]
Lutz, C. S., Cooke, C., O'Connor, J. P., Kobayashi, R. and
Alwine, J. C. (1998). The snRNP-free U1A (SF-A) complex(es):
identification of the largest subunit as PSF, the polypyrimidine-tract binding
protein-associated splicing factor. Rna
4,
1493-1499.
Mathur, M., Tucker, P. W. and Samuels, H. H.
(2001). PSF is a novel corepressor that mediates its effect
through Sin3A and the DNA binding domain of nuclear hormone receptors.
Mol. Cell. Biol. 21,
2298-2311.
Miralles, F. and Visa, N. (2001). Molecular characterization of Ct-hrp65: identification of two novel isoforms originated by alternative splicing. Exp. Cell Res. 264, 284-295.[CrossRef][Medline]
Miralles, F., Öfverstedt, L. G., Sabri, N., Aissouni, Y.,
Hellman, U., Skoglund, U. and Visa, N. (2000). Electron
tomography reveals posttranscriptional binding of pre-mRNPs to specific fibers
in the nucleoplasm. J. Cell Biol.
148,
271-282.
Otto, H., Dreger, M., Bengtsson, L. and Hucho, F.
(2001). Identification of tyrosine-phosphorylated proteins
associated with the nuclear envelope. Eur. J. Biochem.
268,
420-428.
Patton, J. G., Porro, E. B., Galceran, J., Tempst, P. and Nadal-Ginard, B. (1993). Cloning and characterization of PSF, a novel pre-mRNA splicing factor. Genes Dev. 7, 393-406.[Abstract]
Peng, R., Dye, B. T., Perez, I., Barnard, D. C., Thompson, A. B. and Patton, J. G. (2002). PSF and p54nrb bind a conserved stem in U5 snRNA. RNA 10, 1334-1347.[CrossRef]
Percipalle, P., Fomproix, N., Kylberg, K., Miralles, F.,
Björkroth, B., Daneholt, B. and Visa, N. (2003). An
actin-ribonucleoprotein interaction is involved in transcription by RNA
polymerase II. Proc. Natl. Acad. Sci. USA
100,
6475-6480.
Rendahl, K. G., Jones, K. R., Kulkarni, S. J., Bagully, S. H. and Hall, J. C. (1992). The dissonance mutation at the no-on-transient-A locus of D. melanogaster: genetic control of courtship song and visual behaviors by a protein with putative RNA-binding motifs. J. Neurosci. 12, 390-407.[Abstract]
Sabri, N., Östlund Farrants, A. K., Hellman, U. and Visa,
N. (2002). Evidence for a Posttranscriptional Role of a
TFIIIC-like Protein in Chironomus tentans. Mol. Biol.
Cell 13,
1765-1777.
Sewer, M. B., Nguyen, V. Q., Huang, C. J., Tucker, P. W.,
Kagawa, N. and Waterman, M. R. (2002). Transcriptional
activation of human CYP17 in H295R adrenocortical cells depends on complex
formation among p54(nrb)/NonO, protein-associated splicing factor, and SF-1, a
complex that also participates in repression of transcription.
Endocrinology 143,
1280-1290.
Shav-Tal, Y. and Zipori, D. (2002). PSF and p54(nrb)/NonO-multi-functional nuclear proteins. FEBS Lett. 531, 109-114.[CrossRef][Medline]
Visa, N., Alzhanova-Ericsson, A. T., Sun, X., Kiseleva, E., Björkroth, B., Wurtz, T. and Daneholt, B. (1996). A pre-mRNA-binding protein accompanies the RNA from the gene through the nuclear pores and into polysomes. Cell 84, 253-264.[Medline]
von Besser, H., Schnabel, P., Wieland, C., Fritz, E., Stanewsky, R. and Saumweber, H. (1990). The puff-specific Drosophila protein Bj6, encoded by the gene no-on transient A, shows homology to RNA-binding proteins. Chromosoma 100, 37-47.[Medline]
Wurtz, T., Kiseleva, E., Nacheva, G., Alzhanova-Ericcson, A., Rosen, A. and Daneholt, B. (1996). Identification of two RNA-binding proteins in Balbiani ring premessenger ribonucleoprotein granules and presence of these proteins in specific subsets of heterogeneous nuclear ribonucleoprotein particles. Mol. Cell. Biol. 16, 1425-1435.[Abstract]
Yang, Y. S., Hanke, J. H., Carayannopoulos, L., Craft, C. M., Capra, J. D. and Tucker, P. W. (1993). NonO, a non-POU-domain-containing, octamer-binding protein, is the mammalian homolog of Drosophila nonAdiss. Mol. Cell. Biol. 13, 5593-5603.[Abstract]
Yang, Y. S., Yang, M. C., Tucker, P. W. and Capra, J. D.
(1997). NonO enhances the association of many DNA-binding
proteins to their targets. Nucleic Acids Res.
25,
2284-2292.
Zhang, Z. and Carmichael, G. G. (2001). The fate of dsRNA in the nucleus: a p54(nrb)-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs. Cell 106, 465-475.[Medline]