In mammalian cells, nascent RNAs associate with a wide
number of proteins, the most abundant being those known as
heterogeneous nuclear ribonucleoproteins (hnRNPs). The
human hnRNP family consists of about 20 abundant nuclear
RNA binding proteins involved at different stages of the
mRNA maturation pathway (Dreyfuss et al., 1993; Siomi and
Dreyfuss, 1997; Swanson, 1995; Weighardt et al., 1996). Some
of them, such as hnRNP A1, were shown to play a role during
pre-mRNA splicing (Caceres et al., 1994; Chabot, 1996;
Mayeda and Krainer, 1992; Mayeda et al., 1994), in mRNA
compartmentalization during embryogenesis (Matunis et al.,
1994), in the nucleo-cytoplasmic transport of mRNA
molecules (Izaurralde et al., 1997; Nakielny and Dreyfuss,
1997; Nakielny et al., 1997; Siomi et al., 1997; Visa et al.,
1996) and possibly in translation (Camacho-Vanegas et al.,
1997; Siomi and Dreyfuss, 1997). It is commonly accepted that
these proteins bind nascent RNA in a transcript-specific
assembly and that both protein-RNA and protein-protein
interactions govern the final outcome of the ribonucleoprotein
fiber, which forms the substrate of the ensuing processing
events (Bennet et al., 1992; Matunis et al., 1993).
The elucidation of the functional properties of hnRNP proteins
entails the identification of the molecular partners through which
they act. For instance, the nucleo-cytoplasmic shuttling property
of hnRNP A1, which was suggested to be involved in mature
mRNA export, was shown to rely on the interaction of hnRNP
A1 with transportin, a novel member of the importin β family
(Siomi et al., 1997). In order to gain further insight into the
physiological role of hnRNP proteins in RNA metabolism we
screened a human cDNA library by the two-hybrid approach in
our search for new hnRNP A1 interacting factors. We identified
a nuclear protein, termed HAP (hnRNP A1 associated protein)
that, as we show in this paper, is in fact a novel hnRNP protein.
Sequence analysis revealed that HAP is identical to two
apparently unrelated proteins: (1) the previously described
Scaffold Attachment Factor B (Renz and Fackelmayer, 1996), for
which a role in the association of chromatin with the
transcriptional and splicing machineries was recently proposed
(Nayler et al., 1998) and (2) HET, a transcriptional regulator of
Heat Shock Protein 27 (Hsp27) gene (Oesterreich et al., 1997).
Journal of Cell Science 112, 1465-1476 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
A two-hybrid screening in yeast for proteins interacting with
the human hnRNP A1, yielded a nuclear protein of 917
amino acids that we termed hnRNP A1 associated protein
(HAP). HAP contains an RNA binding domain (RBD)
flanked by a negatively charged domain and by an S/K-R/E-
rich region. In in vitro pull-down assays, HAP interacts with
hnRNP A1, through its S/K-R/E-rich region, and with
several other hnRNPs. HAP was found to be identical to the
previously described Scaffold Attachment Factor B (SAF-B)
and to HET, a transcriptional regulator of the Heat Shock
Protein 27 gene. We show that HAP is a bona fide hnRNP
protein, since anti-HAP antibodies immunoprecipitate from
HeLa cell nucleoplasm the complete set of hnRNP proteins.
Unlike most hnRNP proteins, the subnuclear distribution of
HAP is profoundly modified in heat-shocked HeLa cells.
Heat-shock treatment at 42°C causes a transcription-
dependent recruitment of HAP to a few large nuclear
granules that exactly coincide with sites of accumulation of
Heat Shock Factor 1 (HSF1). The recruitment of HAP to the
granules is temporally delayed with respect to HSF1 and
persists for a longer time during recovery at 37°C. The
hnRNP complexes immunoprecipitated from nucleoplasm of
heat-shocked cells with anti-HAP antibodies have an altered
protein composition with respect to canonical complexes.
Altogether our results suggest an involvement of HAP in the
cellular response to heat shock, possibly at the RNA
Key words: hnRNP, RNA processing, Transcription, Heat shock,
Nuclear scaffold, HeLa cell
A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes
and relocates in nuclear granules in response to heat shock
Florian Weighardt1,2, Fabio Cobianchi1, Luca Cartegni1, Ilaria Chiodi1,2, Antonello Villa3, Silvano Riva1and
1Istituto di Genetica Biochimica ed Evoluzionistica, CNR Via Abbiategrasso 207, 27100 Pavia, Italy
2Dipartimento di Genetica e Microbiologia, Università di Pavia Via Abbiategrasso 209, 27100 Pavia, Italy
3MIA Department of Pharmacology, CNR and ‘B. Ceccarelli’ Centers and DIBIT Scientific Institute San Raffaele, Via Olgettina 60,
Università di Milano, 20132 Milano, Italy
*Author for correspondence (e-mail: email@example.com)
Accepted 13 March; published on WWW 22 April 1999
We found that the subcellular distribution of this hnRNP
protein changes in response to different cell treatments; in
particular, after heat shock, HAP relocates to a few nuclear
granules, thus providing a new opportunity to investigate the
response to stress treatments in mammalian cells.
Heat shock induces a number of structural and functional
modifications and is commonly considered as a model of gene
expression regulation. In addition to drastically perturbing the
RNA polymerase II transcriptional program, heat shock inhibits
RNA polymerase I activity, altering the structure and function
of the nucleolus, inhibits pre-mRNA splicing in both yeast and
metazoan cells, and almost abrogates nucleo-cytoplasmic
trafficking of macromolecules leading to poly(A)+RNA nuclear
accumulation (Liu et al., 1996; Saavedra et al., 1996; Spector
et al., 1991; Zu et al., 1998). Moreover, heat-shock treatment
causes the rapid and transient activation of genes encoding heat-
shock proteins (hsps), mediated by a family of heat-shock
transcription factors (HSF) (Lindquist, 1986). In vertebrates,
four members of the HSF gene family (HSF 1-4) have been
characterized. In mammalian cells, a key role in this response
is played by HSF1, which is normally present in an inert, non-
DNA-binding state and becomes activated to a DNA-binding,
transcriptionally active state upon heat shock (Wu, 1995). Heat
shock also induces remarkable changes in the subnuclear
structure, as exemplified by the accumulation of HSF1 in a few
large nuclear granules (Cotto et al., 1997; Jolly et al., 1997).
Little is known about the fate of hnRNP proteins and
complexes after heat shock. On the other hand a few hnRNP
proteins, allegedly associated with the nuclear matrix hnRNP
subcomplexes, were found to rapidly move to a population of
salt-resistant hnRNP complexes after heat shock (Mahl et al.,
1989), and under these conditions appear to be recruited to
HSF1-like granules, but there is no evidence that these granules
coincide with those containing HSF1 (Gattoni et al., 1996;
Mahe et al., 1997).
In this paper we show that HAP is recruited to HSF1
granules after heat shock and we identify several parameters
that control this phenomenon. In addition, we show that
relocalization occurs in parallel with the association of the
protein with a different class of hnRNP complexes. The
possible roles of HAP in the RNA metabolism during heat
shock and the subsequent recovery are discussed.
MATERIALS AND METHODS
Yeast two-hybrid screening and isolation of full-length
The human HeLa cDNA library, yeast strains and cloning vectors
were from Clontech (USA). All library screenings and yeast
manipulations were carried out according to the manufacturer. S.
cerevisiae HF7c was stably transformed with pGBT9-hnRNP A1,
which expresses the entire hnRNP A1 ORF fused to the activation
domain of GAL4 (Cartegni et al., 1996). The resulting strain was used
as a recipient to screen a HeLa cDNA library (Clontech, Cat.
HL4000AA). A total of 107transformants were plated onto 15 cm
plates of leu−, his−and trp−synthetic medium. About 100 his+
colonies were isolated and β-galactosidase filter assays were
performed by streaking the positives onto filters placed on leu−and
trp−synthetic medium plates. Seven of the his+clones were blue.
Plasmids were isolated from these colonies and retrotransformed to
confirm the interaction. Plasmids inserts were sequenced using the
Sequenase Version II kit (USB; USA). Six clones exactly matched the
hnRNP A1 sequence and only one, p371 (see Fig. 1C), was unrelated
to any hnRNP protein.
To isolate a full-length cDNA, the insert of p371 was labeled by
random priming and used to screen approximately 2×106 plaque-
forming units of a HeLa cell cDNA library in λgt11 vector (Clontech)
as previously described (Buvoli et al., 1990). Several clones were
plaque-purified and subcloned into pBluescript vector (Stratagene;
USA) for sequencing. By this approach, the HAP cDNA was extended
by 550 bp and plasmid p23.1 was obtained (see Fig. 1C). The 5′-end
of HAP cDNA was synthesized through two successive rapid
extensions of cDNA end (RACE) cycles from 1.5 µg of total HeLa
cell RNA, reverse transcribed in a 10 µl reaction with primers
RACE 2 (5′-CCTGTGCAAATGGCTGCTCC-3′) and BACK 3 (5′-
GGGCTTGGGGCTTCCGTG-3′), each at a final concentration of 0.2
µM in the assay, for the first and second cycles, respectively. Reverse
transcription was performed with 140 U of MMuLV reverse
transcriptase (RT) at 37°C for 2 hours in the buffer provided by the
supplier (Gibco-BRL; USA). A poly(A) tail was added to the RT
product using terminal deoxynucleotidyl transferase (TDT) in a 20 µl
reaction containing 0.2 mM dATP and 5 U of TDT in the buffer
provided by supplier (Promega; USA), and incubated for 5 minutes
at 37°C and 5 minutes at 65°C. Double-stranded cDNA was obtained
by PCR using primer (dT)17 RoRi and RACE 3 (5′-TGGCTCA-
GATTTACAAGTTTCCCC-3′), RACE 1 (5′-GTCTGCCTTGCTCG-
ACTGAGCGTG-3′) and BACK 8 (5′-GGAGCGGTTCCCTCGCA-
GG-3′) for the first and second cycles, respectively, as previously
described (Bione et al., 1994). PCR products were fractionated in a
1.5% agarose gel, cloned in pBluescript and sequenced. The resulting
plasmid was called pRACE. The full-length HAP clone (pHAP-full)
was assembled by combining the EcoRI/HincII fragment of pRACE,
the HincII/XhoI fragment of p23.1 and the XhoI/XhoI fragment of
p371 into EcoRI/XhoI digested pBluescript II SK (Stratagene; USA).
Full-length HAP carrying an N-terminal histidine tag was generated
by amplification of pHAP-full with the primer pairs BACK 9
(5′-CATGGAATT-CATGGCGGAGACTCTGTCA-3′) and RACE 1,
designed to remove the 5′-untranslated region of the cDNA. The PCR
product was digested with EcoRI and HincII and ligated into pHAP-
full digested with the same enzymes. Fragment BamHI/KpnI of
pHAP-full was excised and ligated into pTrcHisA (Invitrogen; USA),
digested with the same enzymes, to obtain plasmid pHis-HAP.
The insert of clone p23.1 (see Fig. 1C) was excised with EcoRI and
subcloned into the EcoRI site of the expression vector pGEX-4T-1
(Pharmacia; Sweden) to obtain clone GST-23, which was completely
sequenced. The bacterial GST-23 fusion protein was expressed,
purified according to the supplier’s instructions, and used to immunize
New Zealand rabbits following standard procedures. The polyclonal
antiserum was affinity-purified on recombinant His-tagged HAP,
encoded by plasmid pHis-HAP, coupled to Sepharose-4B (see below)
according to Harlow and Lane (1988); antibodies against glutathione
S-transferase (GST) were adsorbed out. Other antibodies used (kindly
provided by G. Dreyfuss) were: mAb 4B10 (anti-hnRNP A1); mAb
4F4 (anti-hnRNP C1/C2); mAb 7G12 (anti-hnRNP I); mAb 12G4
(anti-hnRNP K); mAb 3G6 (anti-hnRNP U). The rat anti-HSF1 mAb
10H8 was kindly provided by R. I. Morimoto. The previously
described polyclonal antibodies raised against recombinant hnRNP
A1 (Buvoli et al., 1992) and against cellular hnRNPs A1, A2 and I
(Valentini et al., 1985) were used in some experiments. Anti-Xpress
mAb and anti-GST mAb GST-19 were purchased from Invitrogen and
used as suggested.
Mapping of the hnRNP A1 determinants interacting with HAP was
performed by in vitro pull-down assays with the entire His-tagged
HAP protein. The protein was expressed in E. coli and purified by
affinity chromatography on a Ni2+-chelate resin as suggested by the
F. Weighardt and others
1467 hnRNPs and heat shock
manufacturer (Invitrogen). Purified protein was coupled to Sepharose-
4B (cyanogen bromide-activated; Pharmacia) according to standard
procedures and 20 µl of gel slurry (50 µg of protein) were used in
each assay. The ability of this matrix to retain different portions of
hnRNP A1 fused to GST was tested. Production and purification of
GST-hnRNP A1 fusions were as previously described (Cartegni et al.,
1996) and bacterial pellets from 1 ml cultures were used in each assay.
After lysis by sonication in binding buffer, the supernatant was treated
with nucleases (see above) for 15 minutes at 30°C and then incubated
with gel slurry in 0.5 ml of binding buffer by mixing for 120 minutes
at 4°C. Beads were then washed with five volumes of binding buffer,
recentrifuged and proteins eluted with five-volume washes at
increasing ionic strength. Fractions were resolved by 10% SDS-PAGE
and protein revealed in western blotting with mAb GST-19 specific
for glutathione S-transferase.
HAP domains involved in the interaction with hnRNP A1 were
identified by in vitro pull-down assays. The entire hnRNP A1 was
fused to GST and different portions of HAP were expressed as His-
tagged proteins (see Fig. 1C). Production of full-length hnRNP A1-
GST fusions and binding to glutathione-agarose beads were as
previously described (Cartegni et al., 1996). In each pull-down assay,
40 µl of gel slurry were used. Different portions of HAP cDNA were
subcloned into suitable pTrcHis vectors. His-tagged HAP fusions
were produced in E. coli DH5α according to the indications of the
manufacturer (Invitrogen) and an equivalent of 5 ml of culture was
used in each pull-down assay. Bacterial pellets were resuspended in
0.5 ml of binding buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2.5
mM MgCl2, 1 mM CaCl2, 1 mM 4-(2-aminoethyl)benzene sulfonyl
fluoride (AEBSF), 1 µg/ml pepstatin, 1 µg/ml aprotinin, 1 µg/ml
leupeptin), lysed by sonication and centrifuged. The supernatant was
incubated with 1 U micrococcal nuclease (Sigma; USA), 10 µg/ml
DNase I, RNase (A+T1; 200 and 2 µg/ml, respectively) and with 40
µl of glutathione-agarose beads bearing hnRNP A1 protein fused to
GST. After incubation for 1 hour at 4°C on a rotating platform, beads
were washed five times with 1 ml of binding buffer, boiled with 50 µl
of gel sample buffer, and eluted proteins were resolved by 10% SDS-
PAGE. Proteins were then transferred to nitrocellulose and analyzed
by western blotting with anti-Xpress mAb (Invitrogen), which
recognizes an epitope in the N-terminal leader peptide of the His-
HeLa cells were grown in DMEM (Dulbecco’s Modified Eagle
Medium; Sigma), 10% fetal calf serum, 50 µg/ml gentamicin, 2 mM
L-glutamine. Heat-shock experiments were performed at 42°C in
complete medium with 40 mM Hepes buffer (Sigma). Where required,
cycloheximide (Sigma), 5,6-dichlorobenzimidazole riboside (DRB)
(Sigma) or actinomycin D (Boehringer Mannheim, Germany) were
added to final concentrations of 20 µg/ml, 40 µg/ml and 5 µg/ml,
respectively. HeLa cell synchronization at the G1/S border was
achieved through two successive thymidine blocks (2 mM thymidine
in complete medium) as described (Stein and Stein, 1989).
When required, HeLa cells, grown on plates to subconfluent
densities, were labeled overnight in DMEM containing one tenth the
normal amount of methionine, supplemented with 5% dialyzed fetal
bovine serum (Sigma), 50 µg/ml gentamicin, 2 mM L-glutamine and
containing 20 µCi/ml [35S]methionine (Amersham, UK).
HeLa cell nuclear extracts were prepared as described (Piñol-Roma et
al., 1990). Briefly, cells grown on 10 cm tissue culture plates were
rinsed twice in cold phosphate-buffered saline (PBS). 1 ml of ice-cold
immunopurification (IP) buffer (10 mM Tris-HCl, pH 7.4, 100 mM
NaCl, 2.5 mM MgCl2, 0.5% v/v Triton X-100, 2 µg/ml aprotinin, 2
µg/ml pepstatin, 2 µg/ml leupeptin, 1 mM AEBSF) was then added to
each plate. Cells were scraped with a rubber policeman and lysed by
four passages through a 25-gauge needle. When requested, MgCl2 in
IP buffer was substituted with 5 mM EDTA, pH 8.0. When indicated,
IP buffer was adjusted to 10 mM vanadyl ribonucleoside complexes
(VdRN; Fluka, USA) to prevent endogenous RNase activities. Nuclei
and the cytoplasmic fraction were separated by brief centrifugation at
3000 g. Supernatant, corresponding to the cytoplasmic fraction, was
immediately frozen to be used in western blotting experiments. Nuclei
were resuspended in 500 µl IP buffer and disrupted by mild sonication.
The sonicated material was layered onto a 30% sucrose cushion (30%
sucrose w/v in IP buffer) and centrifuged at 4000 g for 15 minutes at
4°C. The material overlying the cushion at the end of the centrifugation
was the nucleoplasm, whereas the pellet contained the insoluble
fraction of nuclei (matrix, chromatin, nucleoli, etc.). The nuclear pellet
was resuspended in gel sample buffer for SDS-PAGE analysis and
western blotting. The nucleoplasm was either resuspended in sample
buffer for SDS-PAGE or for 2-dimensional non-equilibrium pH
gradient electrophoresis (2D-NEPHGE)/SDS-PAGE analysis, or used
directly in immunoprecipitation. In the latter case, the nucleoplasm was
cleared with a further 15 minutes centrifugation at 15000 g at 4°C. The
nucleoplasm, prepared as above, was also used in pull-down
experiments with His-tagged HAP coupled to Sepharose-4B. In this
case, the nucleoplasm was treated with 0.5 U/ml micrococcal nuclease
(Sigma) in the presence of 1 mM CaCl2 for 15 minutes at 30°C and
samples (500 µg total proteins) were incubated with the insoluble
matrix as described above. Interacting proteins were revealed by
western blotting using suitable antibodies.
10 mg of Protein A-Sepharose CL-4B (Pharmacia) were resuspended
in 500 µl of IP buffer (without Triton X-100) containing 1 mg/ml BSA
and rocked overnight at 4°C. Beads were then washed three times with
IP buffer before incubating with the antibody of interest for 1 hour at
4°C with rocking. After extensive washing with IP buffer, beads were
incubated for 2 hours at 4°C with rocking with 500 µl of nucleoplasm
prepared as detailed above. At the end of the incubation, beads were
layered onto a 30% sucrose cushion (30% sucrose w/v in IP buffer),
centrifuged at 4000 g for 15 minutes at 4°C and finally washed three
times with IP buffer. Beads were either resuspended in sample
buffer for SDS-PAGE separation or in sample buffer for 2×D-
NEPHGE/SDS-PAGE analyses that were performed as described
(Buvoli et al., 1990). After antibody decoration, immunoblots were
developed using a horseradish peroxidase-labelled anti-antibody and
the SuperSignalTMULTRA chemiluminescent substrate (Pierce;
USA). After 2D-NEPHGE/SDS-PAGE the gels containing 35S-
labeled proteins were fixed, dried and scanned with a PhosphorImager
445 SI apparatus (Molecular Dynamics, USA).
Cells grown on coverslips were washed once with PBS, fixed for 10
minutes in ice-cold methanol and immediately rehydrated in PBS
prior to the staining reactions. Primary antibodies were diluted to a
working concentration in PBS containing 2% skimmed milk
(DIFCO, USA) and then added to the coverslips. Primary antibodies
used were: affinity-purified rabbit anti-HAP polyclonal antibodies
and rat anti-HSF1 monoclonal antibody (mAb) 10H8. After 1 hour
at 37°C in a humid chamber, coverslips were washed three times
with PBS. Secondary antibodies used were: FITC-conjugated anti-
rabbit IgG swine antibodies (DAKO); FITC-conjugated anti-rat IgG
goat antibodies (Sigma); rhodamine-conjugated anti-rabbit IgG
sheep antibodies (Boehringer); rhodamine-conjugated anti-mouse
IgG sheep antibodies (Boehringer). Secondary antibodies were
diluted in PBS, containing 2% skimmed milk, at the final
concentration recommended by the supplier and added to each
coverslip. Coverslips were incubated for 1 hour at 37°C in a humid
chamber, washed three times with PBS, rinsed and mounted in 90%
glycerol in PBS. Microscopy was performed either with a Leitz
Orthoplan microscope or with a BioRad 1024 confocal laser
Two-hybrid screening for hnRNP A1 partners
The entire hnRNP A1 coding sequence, stably expressed in
yeast as a fusion with the GAL4 DNA binding domain, was
used in a two-hybrid screening of a HeLa cell cDNA library.
Seven positive clones were obtained, six of which exactly
matched the hnRNP A1 sequence. We focused on the only
clone (clone p371; Fig. 1C) unrelated in sequence to hnRNP
A1 and we found that the encoded protein interacted in the two-
hybrid assay with the gly-rich domain but not with the 2× RBD
(UP-1) portion of hnRNP A1 (not shown). The full-length
cDNA (isolated as described in Materials and methods)
contains an open reading frame (ORF) of 2754 bp encoding a
protein of 917 amino acids that was named HAP (hnRNP A1
associated protein). Sequence analysis revealed the presence in
the central part of the protein (residues 398-482) of an RNA
binding domain (RBD), a hallmark of RNA binding proteins
(Weighardt et al., 1996) (see Fig. 1B). The RBD is preceded
by an acidic domain of about 300 amino acids, and is followed
by an extended region rich in S/K and R/E dipeptides. The C-
terminal portion of the protein (last 150 residues) is basic and
gly-rich. The structural features of HAP, as predicted by the
primary sequence analysis, are depicted in Fig. 1A. The fact
that the original cDNA isolate (clone p371) lacked the first 450
residues, including most of the RBD, locates the hnRNP A1-
binding determinants within the C-terminal half of the protein.
During this work, a sequence became available in the EMBL
database (accession number: L43631) that turned out to be
identical to the HAP sequence. Later, this sequence was
reported to encode two apparently unrelated proteins: the
Scaffold Attachment Factor-B
Fackelmayer, 1996), and a protein termed HET (for Hsp27-
ERE-TATA-binding protein), a transcription regulator of
Hsp27 gene (Oesterreich et al., 1997).
(SAF-B) (Renz and
HAP interacts with hnRNP A1 in vitro
In order to rule out that the HAP/ hnRNP A1 interaction in the
two-hybrid assay could be mediated by other proteins or
nucleic acids, we asked whether the two purified proteins could
interact in vitro under nucleic acid-free conditions. Thus, the
entire HAP, covalently bound to Sepharose-4B beads, was
tested for its ability to pull down a GST-hnRNP A1 fusion (see
Materials and methods). As shown in Fig. 2a, the HAP/hnRNP
A1 interaction was stable up to 0.5 M NaCl. In contrast, the
2× RBD portion of hnRNP A1 (UP1), added to the same assay,
F. Weighardt and others
100200 300400 500600 700800 9000
α α α α α α α α α α α
EKGRSSCGRN FWVSGL -SSTTRATD L KNL F SKY- G K V --VG A KVVTNARSPGA RCYGFVTM STAEEATKC I NH L HKTELHGKMISVEKA
KTDPRSMNSR VFIGNL NTLVVKKSD V EAI F SKY- G K I -VGC S VH--------- KGFAFVQY VNERNARA- A V- A GEDGRMIAGQVLDIN
SPKEPEQLRK LFIGGL -SFETTDES L RSH F EQW- G T L TDCV V MRDPNTKRS-- RGFGFVTY ATVEEVDA- A MN A RPHKVDGRVVEPKRA
RPGAHLTVKK IFVGGI -KEDTEEHH L RDY F EQF- G K I EVIE I MTDRGSGKK-- KGFAFVTF DDHDSVDK- I V- I QKYHTVNGHNCEVRK
PYANPTKRYR AFITNI -PFDVKWQS L KDL V KEKV G E V TYVE L LMDAEGKS--- RGCAVVEF KMEESMKK- A AE V LNKHSLSGRPLKVKE
ALQAGRLGST VFVANL -DYKVGWKK L KEV F SMA- G V V VRAD I LEDKDGKS--- RGIGTVTF EQSIE---- A VQ A ISMFNGQLLFDRPMH
APGVARKACQ IFVRNL -PFDFTWKM L KDK F NEC- G H V LYAD I KMENGKS---- KGCGVVKF ESPEV---- A ER A CRMMNGMKLSGREID
---------- UxUxxL xxx[x0-6] Z [x]xxx F xxx G x U xx Z [x6-27-++] Ux VxF [x]xxxxxx Z xx A ---------------
U=Uncharged residues:L,I,V,A,G,F,W,Y,C,M Z=U+S,T x=Any aminoacid ++=Further expansion
100 200 300400500 600700 8009000
263 - 842 (p23.1)
263 - 757
263 - 552
451- 917 (p371)
Fig. 1. HAP structural features. (A) Schematic diagram highlighting structural domains of HAP as deduced from primary sequence analysis.
The predicted secondary folding is depicted. Two putative NLS (Nuclear Localization Signals) are shown. (B) Alignment of residues 398-482
of HAP with the RNA binding domain (RBD) of selected hnRNPs and with the RBD consensus sequence as defined by Birney et al. (1993).
The RNP1 and RNP2 consensus sequences are underlined along with the most conserved residues. (C) Mapping of the HAP protein domain
responsible for hnRNP A1 binding. The indicated His-tagged HAP fragments, expressed in E. coli, were subjected to pull-down experiments
with a GST-hnRNP A1 fusion bound to glutathione-agarose beads (see Materials and methods). The ability of each His-tagged polypeptide to
be bound was scored as + (interaction) or – (lack of interaction). The original two-hybrid isolate (p371) is indicated.
1469 hnRNPs and heat shock
was not bound, thus pointing to the gly-rich domain of hnRNP
A1 as the HAP binding determinant. In fact, a GST fusion
carrying the entire gly-rich domain was retained to the same
extent (Fig. 2b). Interestingly, both halves of the same domain
were also retained, although to a lower extent (Fig. 2c,d),
suggesting that, as in the case of hnRNP A1/hnRNP A1
homocomplexes, the protein motif involved is distributed along
the entire gly-rich domain (Cartegni et al., 1996).
To map the HAP domain involved in the interaction with
hnRNP A1, different portions of HAP were expressed in E. coli
as His-tagged fusions and assayed in pull-down experiments
with a GST-hnRNP A1 fusion bound to glutathione-agarose
beads. The results of this analysis are schematically shown in
Fig. 1C. Consistent with the result of the two-hybrid assay, a
His-tagged protein corresponding to the original p371 isolate,
which lacks the entire N-terminal acidic domain and most of
the RBD (residues 451-917), was efficiently bound. Binding
was also observed with a fragment spanning residues 263-842,
which lacks half of the C-terminal domain, or with a protein
deleted of the entire C-terminal domain (residues 263-757). In
contrast, no interaction was detected when the S/K-R/E region
was removed (residues 263-552), thus mapping the hnRNP A1-
binding determinant of HAP to the S/K-R/E region.
In order to assess whether HAP was able to bind other proteins
of the hnRNP fiber, we used the HAP-Sepharose-4B matrix in
pull-down experiments to select interacting proteins from a HeLa
cell nuclear extract. Nucleoplasm from exponentially growing
cells was incubated, under nucleic acid-free conditions, with
HAP-Sepharose-4B beads and proteins bound after extensive
washings at 0.2 M NaCl were resolved by 10% SDS-PAGE and
analyzed in western blotting with specific anti-hnRNP antibodies.
As shown in Fig. 3 (lanes 1-6), besides hnRNP A1, hnRNPs A2,
C1/C2, K, I and U were also efficiently selected.
A recent report (Nayler et al., 1998) seems to further expand
the number of HAP-interacting proteins. It was in fact shown
that SAF-B (HAP) can bind RNA polymerase II and a subset
of splicing factors of the SR family. Interestingly, in this case
the protein binding determinant was also mapped to the S/K-
R/E-rich region that mediates the HAP/ hnRNP A1 interaction.
HAP is a novel member of the hnRNP protein family
Our observation that HAP contains an RBD and interacts in
vitro with several hnRNP proteins suggests that it could be a
component of the hnRNP complexes, as it is the case for
hnRNP U, which was found to be identical to Scaffold
Attachment Factor (SAF-A) (Fackelmayer et al., 1994). To
verify that this was the case, we first tested whether HAP
cofractionated with hnRNP proteins in the soluble nucleoplasm
or else remained entirely associated with the insoluble nuclear
pellet. HeLa cells were biochemically fractionated into
cytoplasm, nucleoplasm and nuclear pellet under different
experimental conditions, designed to allow or to prevent
endogenous RNase or DNase activities (see Materials and
methods). The distribution of HAP in these fractions was
assessed by SDS-PAGE and western blotting using affinity-
purified anti-HAP antibodies, raised in rabbit against the
recombinant protein (see Materials and methods). These
antibodies specifically recognize in HeLa cell extract a single
band migrating with a molecular mass of 140-150 kDa versus
a calculated molecular mass for HAP of 102.8 kDa (Fig. 3,
left). This anomalous mobility has already been reported for
SAF-B (Renz and Fackelmayer, 1996) and is likely due to post-
translational modifications or else to the presence of highly
charged regions. As shown in Fig. 4A, HAP was partially
released in the nucleoplasm under conditions that either allow
(+Mg2+) or inhibit (+EDTA) endogenous DNase activities. In
contrast, all the protein remained in the nuclear pellet when
endogenous RNase activities were selectively inhibited
Fig. 2. Identification of the hnRNP A1
determinants involved in HAP binding.
HAP, covalently bound to Sepharose-4B
beads, was used in pull-down experiments
with the indicated GST fusions (see
Materials and methods for experimental
details). The recombinant 2× RBD domain
of hnRNP A1 (UP1) was tested unfused.
Samples of loaded (L), unbound (U) and
bound (B) fractions at the indicated salt
concentrations were fractionated by 10%
SDS-PAGE. Western blotting in (a) was
performed with an anti-hnRNP A1
polyclonal antibody recognizing both
hnRNP A1 and UP1 (Valentini et al., 1985).
Blots in (b-e) were probed with the anti-
GST mAb GST-19.
(+VdRN complexes +Mg2+). Thus, the association of HAP
with insoluble nuclear structures appears to depend on RNA
integrity, since HAP fractionated in the nucleoplasm when
limited (endogenous) RNase activity was allowed.
We next immunoprecipitated HAP from HeLa cell
nucleoplasm and searched for the presence of coprecipitated
hnRNPs. As shown in Fig. 4B (lanes 1), hnRNPs A1, C1/C2
and U were all coprecipitated, suggesting that HAP could be
associated to the hnRNP complexes. Since no hnRNP protein
was coprecipitated when RNase was added during the
immunoprecipitation (Fig. 4B, lanes 2) we concluded that the
presence of HAP in multiprotein complexes relies, as in the
case of the other hnRNPs, on RNA binding, most likely
mediated by the RBD motif.
To verify the association of HAP with hnRNP complexes,
we determined the protein composition of the complexes
immunopurified with anti-HAP antibodies. Nucleoplasm of
metabolically labeled HeLa cells was immunoprecipitated with
affinity-purified anti-HAP antibodies and the proteins
fractionated by 2D-NEPHGE/SDS-PAGE. As a control,
canonical hnRNP complexes were immunopurified, from the
same nucleoplasm, with the anti-hnRNP C mAb 4F4. As
shown in Fig. 5, the same set of polypeptides was selected with
anti-HAP (Fig. 5A) and with anti-hnRNP C (Fig. 5B)
antibodies, even though with differences in the relative
abundance of the single protein species. In both cases, when
extracts were treated with excess RNase, only the cognate
proteins were precipitated (not shown), as expected from the
fact that hnRNP complexes are held together by RNA
(Dreyfuss et al., 1993). In conclusion, HAP appears to be a
true, although minor, component of hnRNP complexes.
HAP marks a different class of hnRNP complexes in
SAF-B (HAP) was recently reported to be identical to HET
(Hsp27-ERE-TATA-binding protein) a transcriptional regulator
F. Weighardt and others
Fig. 3. HAP interacts in vitro with cellular hnRNP proteins. Nucleic
acid-free nucleoplasm, prepared from exponentially growing HeLa
cells (see Materials and methods), was challenged in pull-down
experiments with His-tagged HAP coupled to Sepharose-4B beads.
After extensive washing at 0.2 M NaCl, bound proteins were
separated by 10% SDS-PAGE and revealed in western blotting with
specific antibodies. Lane 1, mAb 4B10 (anti-hnRNP A1); lane 2,
polyclonal antibody that recognizes hnRNP A1, A2 and I (Valentini
et al., 1985); lane 3, mAb 4F4 (anti-hnRNP C1/C2); lane 4, mAb
12G4 (anti-hnRNP K); lane 5, mAb 7G12 (anti-hnRNP I); lane 6,
mAb 3G6 (anti-hnRNP U). (Left) SDS-PAGE separation of HeLa
cell extract probed in western blotting with the affinity-purified anti-
HAP rabbit polyclonal antibodies.
Fig. 4. HAP is associated to ribonucleoprotein complexes.
(A) Partial release of HAP in the nucleoplasm by
endogenous RNase activity. HeLa cells were fractionated
into cytoplasm (Cy), nucleoplasm (Nu) and insoluble
nuclear pellet (Np) as described in Materials and methods.
The experiment was performed in triplicate under conditions
of endogenous nuclease digestion (+2.5 mM MgCl2); DNase
inhibition (+5 mM EDTA) and RNase inhibition (+2.5 mM
MgCl2and 10 mM VdRN complexes). Proteins were
separated by SDS-PAGE and HAP was revealed by western
blots with anti-HAP antibodies and chemiluminescent
substrate. (B) Anti-HAP antibodies coimmunoprecipitate
hnRNP proteins from nucleoplasm. Immunoprecipitations of
HAP from HeLa cell nucleoplasm, prepared as described in
Materials and methods, were performed either in presence of
RNase inhibitor (0.5 U/µl RNasin; Sigma) (lanes 1 and 3) or
in presence of an excess of RNase A+T1 (200 µg/ml; 2
µg/ml) (lanes 2). The immunoprecipitates with affinity-
purified anti-HAP antibodies (lanes 1 and 2) or with an
unrelated antibody (lanes 3) were fractionated by SDS-
PAGE and probed in western blots with anti-HAP antibodies
or with mAbs against the indicated hnRNP proteins. The
immunoprecipitated protein bands are indicated by arrows.
1471 hnRNPs and heat shock
of the small heat-shock protein Hsp27 (Oesterreich et al., 1997),
suggesting an involvement in the heat-shock response. Heat
shock drastically perturbs both nuclear structure and nucleic
acid metabolism; in particular it is alleged to inhibit both
transcription and splicing and mRNA export (Bond, 1988;
Lindquist, 1986; Spector et al., 1991; Stutz and Rosbash, 1998).
Therefore, although little is known about the heat-shock effect
on hnRNP complexes, it seems likely that the hnRNP structure
could also be affected. In this regard, it is worth noticing that
two hnRNP proteins (hnRNP M and 2H9) were previously
reported to dissociate from ribonucleoprotein complexes, upon
heat shock, to associate to the nuclear matrix and to change their
subnuclear distribution (Gattoni et al., 1996; Mahe et al., 1997).
On the basis of these considerations, we investigated
whether after heat shock HAP was still associated to the
hnRNP complexes. Anti-HAP antibodies were used to
immunopurify hnRNP complexes from nucleoplasm prepared
from cells heat shocked for 1.5 hours at 42°C. As shown in
Fig. 5C, under these conditions HAP-containing multiprotein
complexes are precipitated but their protein moiety is
significantly altered, both in composition and relative
abundance with respect to canonical complexes, being enriched
in F, H, K, M, N and S proteins. In contrast, many polypeptides
are absent or, as in the case of ‘core’ hnRNP proteins (types
A, B and C), under-represented. No significant difference in
protein composition was
immunopurified from the same nucleoplasm with anti hnRNP
C antibodies (not shown). In conclusion, upon heat shock, HAP
appears to label a new and different class of hnRNP complexes.
observed in complexes
HAP is recruited to a few sub-nuclear sites in heat-
Immunostaining of exponentially growing HeLa cells with
affinity-purified anti-HAP polyclonal antibodies revealed a
punctuate distribution consisting of numerous small granules
scattered throughout the nuclear volume excluding the nucleoli
(Fig. 6a) with a few brighter granules, often close to nucleoli,
visible in most cells. No colocalization with sites of SC35
accumulation, the so-called ‘speckles’, was observed by
confocal laser microscopy (not shown). This is in contrast to the
recent report that the C-terminal half of SAF-B, fused to green
fluorescent protein (GFP), colocalizes with splicing factor SC35
in speckles (Nayler et al., 1998; our unpublished results). Such
a discrepancy might indicate that the N-terminal domain of the
protein, and notably the RNA binding domain, plays a critical
role in the subcellular distribution of the entire protein. On the
basis of the results in the previous section, we asked whether
heat shock could perturb HAP subcellular distribution, as
reported for hnRNP M (Gattoni et al., 1996). HeLa cells were
incubated for 45 minutes, 1 hour and 1.5 hours at 42°C and
immunostained with anti-HAP antibodies. As shown in Fig. 6b-
d, this treatment caused the recruitment of HAP to a small
Fig. 5. Protein composition of HAP-containing complexes.
Nucleoplasm was prepared from HeLa cells metabolically labeled
with [35S]methionine (see Materials and methods). Proteins
immunoprecipitated with affinity-purified anti-HAP antibody (A) or
with anti-hnRNP C mAb 4F4 (B) were fractionated by 2D-
NEPHGE/SDS-PAGE electrophoresis and visualized by
PhosphorImager scanning. HAP and the major hnRNP protein spots
are identified by letterheads. The position of the HAP spot was
independently confirmed by western blotting analysis of 2D-
NEPHGE/SDS-PAGE separation of nucleoplasm (not shown).
(C) Same experiment as in A except that nucleoplasm was prepared
from cells heat shocked for 1.5 hours at 42°C. The pI scale was
interpolated on the basis of the migration of IEF standards (BioRad).
The positions of molecular mass markers are shown.
number of large nuclear granules. Notably, no such a
redistribution was observed with hnRNP A1, C, I and U (not
shown). The fraction of cells in which such phenomena occurred
increased with the length of treatment from about 10-20% (at 45
minutes) to 80-90% (at 1.5 hours). Interestingly, if a 1 hour heat
shock was followed by a 3 hour incubation at 37°C, HAP
relocalization took place in virtually all cells (see Fig. 6e), and
under these conditions most of the protein was recruited to a few
nuclear sites, leaving the rest of the nucleus almost empty. HAP
relocalization was almost completely reversed after a 6 hour
recovery period at 37°C (Fig. 6f). Such a reversal is energy-
dependent, as it was inhibited by incubation at 4°C (Fig. 6g,h).
HAP relocalization does not require de novo protein synthesis
since it was not affected by cycloheximide treatment (Fig. 6i,j).
Moreover, western blot analysis of total cell extract prepared
from untreated and heat-shocked HeLa cells, grown either in
absence or in presence of cycloheximide, showed that HAP
levels remained constant during this treatment (not shown), thus
indicating that recruitment to and release from the subnuclear
granules is not attributable to de novo synthesis or degradation.
We next asked whether RNA synthesis, which is known to
be perturbed by heat shock, is required for the observed
relocalization to occur, and we examined the effect of DRB, a
reversible inhibitor of RNA polymerase II activity, on HAP
subnuclear distribution. We first checked if DRB could perturb
HAP localization in non-heat shocked cells. As shown in Fig.
7a, a 3 hour treatment with DRB caused a redistribution of
HAP towards the inner nuclear envelope and the perinucleolar
rims. Incidentally, no such redistribution was detectable with
hnRNPs A1, C1/C2 and I (not shown; Nakielny and Dreyfuss,
1996; Piñol-Roma and Dreyfuss, 1991). Moreover, contrary to
shuttling hnRNPs (Piñol-Roma and Dreyfuss, 1991; Weighardt
et al., 1995), HAP did not migrate to the cytoplasm under these
We then determined the effect of a 3 hour treatment with
DRB in cells previously heat shocked for 1 hour at 42°C. It
should be reminded that, as shown in Fig. 6e, a 3 hour
incubation at 37°C following 1 hour heat shock at 42°C caused
the appearance of HAP granules in virtually all the cells. As
shown in Fig. 7b, DRB treatment abrogated the progression of
heat shock-induced relocalization of HAP. Identical results
were obtained when actinomycin D was used in place of DRB
(not shown). Overall these results suggest that in both untreated
and heat-shocked cells, ongoing transcription has a key role in
HAP subnuclear distribution.
The cell population heterogeneity in the heat shock response
(Fig. 6b-d) suggests that the kinetics of HAP recruitment to
F. Weighardt and others
Fig. 6. Effect of heat shock on HAP subnuclear distribution. After
the indicated treatments, HeLa cells were fixed with methanol and
stained with affinity-purified anti-HAP antibodies; protein
localization was revealed by indirect immunofluorescence with
FITC-conjugated anti-rabbit IgG swine antibodies. (a) untreated
cells. (b-d) Cells heat shocked at 42°C for 45 minutes, 1 hour and 1.5
hours, respectively, just prior to fixation. (e,f) Cells incubated at
37°C for 3 hours or 6 hours, respectively, following a 1 hour heat
shock at 42°C. (g) Cells incubated for 6 hours at 4°C. (H) Cells
incubated at 4°C for 6 hours following a 1 hour heat shock at 42°C.
(i,j) Cells treated for 3 hours and 6 hours, respectively, with 20 µg/ml
cycloheximide (Chx) following a 1 hour heat shock at 42°C.
Fig. 7. Effect of transcription inhibition on HAP subnuclear
distribution. (a) Cells treated with 40 µg/ml DRB for 3 hours at
37°C. (b) Cells treated with 40 µg/ml DRB for 3 hours at 37°C
following a 1 hour heat shock at 42°C. After the indicated
treatments, HeLa cells were fixed with methanol and stained with
affinity purified anti-HAP antibodies; protein localization was
revealed by indirect immunofluorescence with FITC-conjugated anti-
rabbit IgG swine antibodies.
1473 hnRNPs and heat shock
discrete nuclear sites could vary during the cell cycle. To verify
that this was indeed the case, HeLa cells were synchronized at
the G1/S border by two successive thymidine blocks. Following
release from the block, cells were harvested at different time
intervals and costained with anti-HAP antibodies and with anti-
DNA ligase I antibodies (not shown), the latter used to follow
the progression through the cell cycle. In parallel, at the same
time intervals, samples of cells were heat shocked at 42°C for
1 hour and immediately stained with anti-HAP antibodies. As
shown in Fig. 8, the fraction of cells in which HAP
relocalization occurred was maximal in mid and late S-phase
(almost 100% of the cells), while it was drastically reduced in
G2, further reduced in G1phase and was minimal in early S.
Sequential recruitment of HSF1 and HAP to the
same sites in heat shocked cells
The experiments reported in the previous section identify a
number of parameters (transcription and cell cycle) that govern
the heat shock-induced recruitment of HAP to a few nuclear
granules. However, the nature and the protein composition of
these granules remain undefined. In an initial attempt to
investigate these aspects, we asked whether these granules
could be sites of accumulation of other proteins involved in the
heat-shock response and known to relocate in the nucleus
following this treatment.
In effect, we noticed that the heat shock-induced
relocalization of HAP closely resembles that of Heat Shock
Factor 1 (HSF1) (Cotto et al., 1997; Jolly et al., 1997),
suggesting that the two proteins could be recruited to the same
sites. To test this hypothesis, HeLa cells were costained with
anti-HAP and anti-HSF1 antibodies at increasing heat shock
times. As shown in Fig. 9, after a 1 hour heat shock, in cells
where HAP granules were detectable they exactly coincided
with HSF1 granules, as judged by confocal laser microscopy
analysis. However, the two proteins appeared to be recruited to
and released from these sites with different kinetics. As can be
seen, after 15 minutes heat shock, HAP (red) and HSF1 (green)
occupied mutually exclusive nuclear regions; in addition, HAP
concentrated near the nuclear envelope, leaving large regions of
the nucleoplasm virtually devoid of protein. It should be noted
that HAP distribution under these conditions closely resembled
that observed in non-heat-shocked cells after a 3 hour treatment
with DRB (see Fig. 7). At later times (30 minutes), HSF1 was
concentrated in a few sites while HAP left the nuclear envelope
to gather around these sites. At 45 and 60 minutes, sites of HSF1
and HAP accumulation merged (yellow dots; Fig. 9). Following
a 3 hour recovery at 37°C, HSF1 returned to a diffused nuclear
distribution while HAP was still concentrated in the same
discrete granules. Three conclusions can be drawn from these
experiments. First, the identified granules contain, beside HSF1,
one or more hnRNP proteins (HAP and probably hnRNP M and
2H9) (Gattoni et al., 1996; Mahe et al., 1997). Second, the
presence of HAP in granules does not require the concomitant
presence of HSF1 and vice versa, suggesting the existence of an
underlying structure that independently recruits the different
proteins. Third, the appearance of HAP in the granules is
preceded by a migration of the protein toward the nuclear
envelope. This phenomenon can be interpreted by hypothesizing
that HAP accompanies mature mRNAs to the nuclear pore
where, in heat-shocked cells, because of inhibition of nuclear
export (Saavedra et al., 1996; Stutz and Rosbash, 1998), it
temporally stalls before being recruited to nuclear granules. It is
not clear whether such recruitment concerns the unbound protein
or the hnRNP complexes.
Fig. 8. Cell cycle-dependent, heat shock-induced, relocalization of
HAP. Exponentially growing HeLa cells were synchronized at the
G1/S border by two successive thymidine blocks (see Materials and
methods), harvested at different times after release from the second
block and fixed either immediately (left panels) or after a 1 hour
incubation at 42°C (right panels). Non-heat-shocked cells were
costained with anti-HAP antibodies and with anti-DNA ligase I
mouse polyclonal antibodies (Montecucco et al., 1995); the latter
staining (not shown) was used to follow the cell cycle-dependent
localization of the replication foci. Heat-shocked cells were
exclusively stained with anti-HAP antibodies. Protein localization
was revealed with FITC-conjugated anti-rabbit IgG swine antibodies.
In this paper we report the characterization of HAP, a new
member of the hnRNP protein family, isolated with a two-
hybrid search for proteins interacting with hnRNP A1. In vitro,
the interaction occurs with the gly-rich domain of hnRNP A1
and is mediated by the S/K-R/E-rich region of HAP. The
presence of alternating positive and negative residues in this
region is reminiscent of similar dipeptide motifs found in
phosphorylated RS domains of SR proteins that, as we have
previously shown, can interact with hnRNP A1 (Cartegni et al.,
HAP turned out to be identical to a protein previously
described as both a Scaffold Attachment Factor (SAF-B) (Renz
and Fackelmayer, 1996) and a factor (HET) involved in the
transcription regulation of Hsp27 gene (Oesterreich et al.,
1997). Sequence analysis revealed that HAP contains an RNA
binding domain (RBD) (see Fig. 1B), which is a previously
undescribed feature that is diagnostic of RNA binding proteins.
Here we provide experimental evidence that HAP is indeed a
novel member of the hnRNP protein family. In fact,
immunoprecipitation of HAP from HeLa cell nucleoplasm,
under conditions that prevent hnRNP complexes disassembly,
results in the coprecipitation of the whole set of hnRNP
proteins (see Fig. 5A,B). HAP is a minor component of the
complexes, being 100-fold less abundant than hnRNP A1
F. Weighardt and others
Fig. 9. Kinetics of HAP and
HSF1 nuclear relocalization
after heat shock. Exponentially
growing HeLa cells were heat
shocked for the indicated times
immediately before fixation
and costained with affinity-
purified anti-HAP rabbit
antibody and with anti-HSF1
rat mAb 10H8. Protein
localization was revealed by
anti-rabbit IgG sheep
antibodies and FITC-
conjugated anti-rat IgG goat
antibodies. Confocal laser
microscopy images of the same
field were taken and merged;
HAP, red; HSF1, green.
results in yellow staining. Top
row, untreated cells.
1475 hnRNPs and heat shock
(Michael et al., 1995; Renz and Fackelmayer, 1996), which
could explain why it was not previously recognized.
Concerning the identification of HAP with SAF-B, or with
HET, it should be reminded that this is not the first case of an
hnRNP protein for which different roles were proposed. For
example, hnRNP U was described as Scaffold Attachment Factor
A (SAF-A) (Eggert et al., 1997; Fackelmayer et al., 1994;
Gohring and Fackelmayer, 1997), other hnRNPs such as F and
H were reported to be associated to the nuclear matrix (Holzmann
et al., 1997) and, according to some authors, most hnRNP
proteins are found in nuclear matrix preparations (Mattern et al.,
1996). Another example is hnRNP K, which is both an RNA
binding protein and a transcription factor (Michelotti et al., 1996).
On the other hand, it was recently reported that SAF-B (HAP),
initially isolated as a protein that binds to S/MAR sequences,
interacts with RNA polymerase II, with a subset of SR factors
and can influence splicing (Nayler et al., 1998).
As observed with anti-hnRNP antibodies, anti-HAP antibodies
immunoprecipitate other hnRNP proteins from nucleoplasm only
under conditions that preserve the integrity of the hnRNP
complexes (Fig. 4). On the other hand HAP is able to interact
with hnRNP A1 in the absence of nucleic acids, as proved by our
in vitro pull-down experiments (see Figs 2, 3). This point,
therefore, deserves a further comment. In the absence of nucleic
acid, the hnRNP A1-HAP interaction is probably as weak as the
hnRNP A1-hnRNP A1 or the hnRNP A1-SR proteins
interactions for which we estimated a Kdof 10−7M (Cartegni et
al., 1996). Therefore, under nucleic acid-free conditions, its
detection in vitro is only possible at the high protein
concentrations attainable in pull down experiments but not, for
example, at the concentrations used to immunoprecipitate
nucleoplasm.The heat shock-induced relocalization to a small
number of nuclear granules (Figs 6, 8) is a feature that
distinguishes HAP (and hnRNPs M and 2H9) from most other
hnRNP proteins. We show here, that HAP is recruited to the same
nuclear granules as Heat Shock Factor 1 (HSF1). Such granules
are distinct from speckles and coiled bodies (Cotto et al., 1997;
Jolly et al., 1997). The kinetics of recruitment to and release from
these granules speak against a mechanism based on a direct
interaction between these two factors, as also suggested by the
failure to coimmunoprecipitate HAP and HSF1 (data not shown).
Rather, our results are more compatible with the existence of an
underlying nuclear structure that serves as a recruiter for both
proteins. Notably, HAP relocalization is triggered by heat shock,
but does not require continuous exposure to high temperature in
order to occur. In fact, granules appear in most cells only during
the 3 hour recovery period (Fig. 6). This indicates that the heat
shock response entails changes in the nuclear architecture that are
unexpectedly delayed and are probably functional to the ensuing
The significance of the HAP granules is still an open
question. There are two alternative hypotheses. One possibility
is that granules are sites of synthesis and processing of heat
shock-induced transcripts. However, several observations argue
against this interpretation. First, HSF1 granules do not coincide
with sites of transcription of heat-shock genes (Jolly et al.,
1997). Secondly, we have observed (data not shown) that poly
(A)+RNAs do not accumulate in HAP granules. Moreover, the
size of these structures is far larger than that of transcription
factories (Jackson et al., 1993), which supports the alternative
hypothesis that granules are sites of accumulation and storage.
Since the HAP recruitment/release cycle occurs even in the
presence of inhibitors of protein synthesis (Fig. 6), it seems
unlikely that granules are simply precipitates of irreversibly
denatured proteins. In fact, our results are compatible with a
model whereby HAP in untreated cells accompanies RNA from
the transcription sites to the nuclear envelope and is released at
the nuclear pore to be recycled. In heat-shocked cells, HAP
would temporally stall at the nuclear envelope instead, because
of the inhibition of export, to be then directed to the storage
granules either as a free protein or as part of hnRNP complexes.
The hypothesis that granules are sites of accumulation and
storage is supported by the recent observation that in
Drosophila two hnRNP proteins, HRB57A (hnRNP K-like) and
HRB87F (hnRNP A1-like), are sequestered at puff 93D after
heat shock (Buchenau et al., 1997; Zu et al., 1998). In this case
it was proposed that the two proteins are recruited through a
specific binding to the hsr-ω RNA, which appears to contain
reiterated high affinity binding sites for these proteins (Zu et al.,
1998). Given the fact that the 93D locus is required for viability,
it was suggested that protein sequestration at that locus may be
an important survival strategy. It is tempting to speculate that a
similar mechanism could be at the basis of our observation.
Whatever the mechanism of granule formation, an important
consequence of the massive recruitment of HAP (and of hnRNP
M and 2H9) to granules is the drastic drop of their concentration
in the nucleoplasm. If, as some recent reports seem to indicate
(Gattoni et al., 1996; Mahe et al., 1997; Nayler et al., 1998),
these proteins have a role in RNA processing, a change of their
concentration relative to other hnRNPs could in principle affect
the processing of some RNAs. In this regard, our observation
that HAP-containing complexes have a different protein
composition in heat-shocked cells (see Fig. 5) could indicate
that they are assembled on a specific class of RNAs.
This work was supported by a grant from the Associazione Italiana
per la Ricerca sul Cancro (AIRC) and by the ‘MURST-CNR
Biotechnology Program L. 95/95’. F. Weighardt was the recipient of
a post-doctoral fellowship from the University of Pavia. L. Cartegni
and I. Chiodi were supported by fellowships of the Italian PhD
program. The authors thank F. Peverali for helpful discussion.
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