From the Institute of Medical Biochemistry,
University of Vienna, the Vienna Biocenter, Dr. Bohr-Gasse 9, A-1030
Vienna, Austria, the § Division of Rheumatology, University
Hospital of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, and
the
Department of Biochemistry, Nijmegen Center for Molecular
Life Sciences, University of Nijmegen, NL-6500
HB Nijmegen, The Netherlands
Received for publication, February 13, 2001
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ABSTRACT |
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The hY RNAs are a group of four small
cytoplasmic RNAs of unknown function that are stably associated with at
least two proteins, Ro60 and La, to form Ro ribonucleoprotein
complexes. Here we show that the heterogeneous nuclear
ribonucleoproteins (hnRNP) I and K are able to associate with a subset
of hY RNAs in vitro and demonstrate these interactions to
occur also in vivo in a yeast three-hybrid system.
Experiments performed in vitro and in vivo with
deletion mutants of hY1 RNA revealed its pyrimidine-rich central loop
to be involved in interactions with both hnRNP I and K and clearly
showed their binding sites to be different from the Ro60 binding site.
Both hY1 and hY3 RNAs coprecipitated with hnRNP I in
immunoprecipitation experiments performed with HeLa S100 extracts and
cell extracts from COS-1 cells transiently transfected with
VSV-G-tagged hnRNP-I, respectively. Furthermore, both anti-Ro60 and anti-La antibodies coprecipitated hnRNP I, whereas
coprecipitation of hnRNP K was not observed. Taken together, these data
strongly suggest that hnRNP I is a stable component of a subpopulation of Ro RNPs, whereas hnRNP K may be transiently bound or interact only
with (rare) Y RNAs that are devoid of Ro60 and La. Given that functions
related to translation regulation have been assigned to both proteins
and also to La, our findings may provide novel clues toward
understanding the role of Y RNAs and their respective RNP complexes.
Small ribonucleoprotein
(RNP)1 complexes are usually
composed of one molecule of a small RNA and several proteins that bind either directly to the RNA or indirectly via protein-protein
interactions (1, 2). Many of these complexes exert essential functions that are often indispensable for survival, such as the small nuclear RNPs, which are major components of the spliceosomal machinery, or the
signal recognition particle, which plays a key role in protein export.
In contrast to these well defined complexes, the structure of the
cytoplasmic Ro RNPs is still not fully resolved, and their function has
remained enigmatic (3, 4). They are composed of one molecule of a small
Y RNA (transcribed by RNA polymerase III) and at least two proteins,
the 60-kDa protein Ro60 and the 48-kDa phosphoprotein La. However,
although Ro60 and Y RNAs are present in comparable stochiometric
amounts, La is ~50-fold more abundant, and therefore the vast
majority of La molecules is not bound to Y RNAs, in contrast to Ro60
(5).
Y RNAs are highly conserved in evolution (6) and have been found in all
multicellular eukaryotic organisms and may also be present in some
bacteria (7) but, remarkably, have so far not been detected in yeast.
Interestingly, the genome of the nematode Caenorhabditis
elegans contains only one functional Y RNA gene, whereas in humans
and other vertebrates four closely related Y RNA species exist. Unlike
other RNA polymerase III transcripts, Y RNAs retain the oligo(U)
stretch at their 3' end that forms the binding site for La; therefore
these RNAs remain permanently associated with La (5, 8). On the other
hand, nuclear Y RNAs do not seem to be associated with Ro60, and it was
suggested that Ro RNPs assemble upon export to the cytoplasm (3,
9).
Several functions have been proposed for La, including regulation of
RNA polymerase III transcription (10-12), involvement in internal
ribosome entry site-dependent viral and cellular
translation (13, 14), and a role in the assembly of small nuclear RNPs (15), but it is still not entirely clear which role this abundant nuclear and cytoplasmic protein plays in vivo. In
particular, it is completely unknown whether a unique function can be
attributed to Y RNA-associated La. Much less is known about biological
activities of Ro60 (4). In the cytoplasm it may be involved in the
regulation of translation as recently demonstrated for ribosomal
protein L4 in Xenopus laevis oocytes (16) and in the nucleus
Ro60 may be implicated in a discard pathway of 5 S rRNA by recognizing incorrectly processed and misfolded molecules (17, 18). However, these
(proposed) functions do not seem to be dependent on the presence of Y
RNAs in X. laevis nor in C. elegans.
Interestingly, a prokaryotic homologue of Ro60 was recently discovered
in the bacterium Deinococcus radiodurans that seemed to
contribute to the remarkable resistance of this organism to ultraviolet
radiation (7). This may lead to speculation about a RNA chaperoning
function of Ro60 as has been recently proposed for La (19).
Although the stable association of Ro60 and La with Y RNAs is beyond
experimental doubt, the presence of other components in addition to
Ro60 and La is still a matter of debate. Thus, the suggested
associations of a 52-kDa protein (Ro52) and the Ca2+-binding protein calreticulin have remained
controversial (20-25), and recently reported interactions of two novel
proteins with Ro60 observed in yeast two- and three-hybrid systems need
to be confirmed (26, 27). In a previous study, we demonstrated in vitro binding of several proteins contained in a HeLa S100 extract to hY RNAs (28). These proteins (with molecular masses between 53 and 80 kDa) bound specifically to hY1 and hY3 RNA but only weakly or
not at all to hY4 and hY5 RNA. Interestingly, autoantibodies to these
proteins were found in sera from patients with the rheumatic autoimmune
disease systemic lupus erythematosus (SLE) who commonly develop
autoantibodies to Ro and La (29). Using deletion mutants of hY1
RNA we were able to show that the binding sites for these proteins were
distinct from the Ro60 binding site. In this report we demonstrate that
two of these proteins are identical to the heterogeneous nuclear RNP
proteins I (hnRNP I) and K (hnRNP K) and present evidence that they are
able to associate with a subset of hY RNAs in vivo.
Sera and Antibodies--
For immunodetection of proteins binding
to hY1 and hY3 RNA in vitro, an anti-Ro positive serum from
a patient (BM) with SLE containing antibodies to these proteins
was employed (28). Sera from healthy persons and from patients with
rheumatoid arthritis (which do not contain anti-Ro or anti-La
autoantibodies) were used as negative controls. Monoclonal antibodies
used were anti-La SW5 (30), anti-Ro60 2G10 (31), anti-hnRNP K 2G14
(Ref. 32; kind gift of Gideon Dreyfuss, Howard Hughes Medical
Institute, University of Pennsylvania, Philadelphia, PA), anti-PTB 3 (Ref. 33; kind gift of D. M. Helfman, Cold Spring Harbor
Laboratory), and an anti-human interleukin-6 antibody (Janssen
Biochimica, Denmark) as control.
Cellular Extracts--
HeLa S100 extracts for use in
reconstitution assays were prepared essentially as described (28).
Briefly, 1 × 1010 HeLa cells (Computer Cell Culture
Center, Mons, Belgium) were washed twice in isotonic buffer (10 mM Tris-HCl, pH 7.9, 140 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 25%
glycerol), resuspended in 2 pellet volumes of buffer A (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 25% glycerol)
and disrupted by Dounce homogenization. Nuclei were separated by brief
centrifugation (3 min at 3,000 × g), and the
supernatant was first centrifuged for 20 min at 20,000 × g and then for 1 h at 100,000 × g.
After measuring the protein concentration the S100 extract was stored at
For immunoprecipitation experiments with transfected COS-1 cells, total
cell extracts were prepared after trypsinization, harvesting (800 rpm,
5 min), and washing of the cells with ice-cold phosphate-buffered
saline (10 mM sodium phosphate, pH 7.4, 150 mM
NaCl). Cells were sonified with a Branson microtip (three times for
10 s at 4 °C) in lysis buffer (25 mM Tris-HCl, pH
7.5, 100 mM KCl, 1 mM dithioerythritol,
2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40). After 15 min cell debris was removed by
centrifugation (12,000 rpm, 15 min), and the extract was stored at
Western Blot Analyses--
Proteins were separated by SDS-PAGE
and subsequently blotted onto nitrocellulose as described (28, 34). The
nitrocellulose sheets were blocked for 1 h with 3% nonfat dried
milk in incubation buffer (10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 0.1% Triton X-100) and subsequently incubated for
1 h either with human autoimmune serum diluted 1:50 or with
monoclonal antibodies, respectively. After washing the nitrocellulose
filters three times with incubation buffer, bound antibodies were
detected by alkaline phosphatase-conjugated goat anti-human or
anti-mouse IgG (Chemicon, Temecula, CA).
Extracts from transfected cells were fractionated by SDS-PAGE and
blotted onto a nitrocellulose filter. After blocking the filters in
washing buffer (phosphate-buffered saline containing 5% nonfat dried
milk, 0.1% Nonidet P-40) for 1 h at room temperature, filters
were incubated with monoclonal anti-VSV-G tag antibody (Roche
Diagnostics, Almere, Netherlands) diluted 1:500 in washing buffer for
1 h at room temperature. After washing, bound antibodies were
detected by chemiluminescence using horseradish peroxidase-conjugated goat anti-mouse IgG as secondary antibody (Dako, Glostrup, Denmark).
Immunoprecipitation--
Immunoprecipitations with monoclonal
antibodies to Ro60, La, and hnRNP I and K were performed as described
with antibodies coupled to protein A-Sepharose beads (Amersham
Pharmacia Biotech) using dimethylpimelimidate (Sigma) as a
cross-linking agent (28). Twenty µl of a HeLa cell extract was
diluted in 0.5 ml of immunoprecipitation buffer (IPP)-150 (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05%
Nonidet P-40) and incubated for 1 h at 4 °C with immobilized
antibodies (20 µl of packed bead volume). Subsequently, beads were
washed three times with IPP-150 and resuspended in 400 µl of IPP-150 containing 0.5% SDS. RNA was extracted with phenol/chloroform, precipitated with ethanol using 10 µg of glycogen as carrier, and
analyzed by Northern blot hybridization. For isolation of proteins 200 µl of a HeLa cell extract was applied to 1 ml of anti-Ro or anti-La
immunoaffinity columns using a peristaltic pump at a low flow rate.
After washing with at least 10 bed volumes of IPP-150, proteins were
eluted with IPP-1000 (10 mM Tris-HCl, pH 7.5, 1000 mM NaCl, 0.05% Nonidet P-40).
For immunoprecipitations carried out with extracts from transfected
cells, monoclonal antibodies (anti-VSV-G-tag, anti-Ro60, or anti-La)
were coupled to protein A-agarose beads by incubating overnight at
4 °C in IPP-500 buffer (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.05% Nonidet P-40). Subsequently, the
antibody-coated beads were equilibrated with IPP-150 and incubated with
cell extracts for 2 h at 4 °C. Then the beads were extensively
washed with IPP-150, and coprecipitating RNAs were isolated by
phenol/chloroform extraction and ethanol precipitation.
In Vitro Transcription of Y RNAs and Northern Blot
Analyses--
In vitro transcription of human sense and
antisense hY RNAs and of hY1 RNA deletion mutants was performed as
described (28). To generate biotinylated RNAs biotin 16-UTP (Roche
Molecular Biochemicals) was used at 75 µM concentration,
for preparation of radiolabeled RNAs 0.5 mM UTP and 40 µCi of [ Reconstitution and Purification of hY RNPs--
These procedures
were performed essentially as described (28). To dissociate existing
complexes, the salt concentration of the HeLa extract was first
increased to 1 M KCl followed by 30 min of incubation on
ice. Then 10 µg of biotinylated hY RNA or biotinylated control RNA (5 S rRNA) and 200 µg yeast tRNA (Roche) was added and the KCl
concentration was readjusted to 150 mM by diluting with
reconstitution buffer (10 mM Tris-HCl, pH 7.9, 2 mM MgCl2, 1 mM DTT, 5% glycerol).
After incubating for 20 min at 30 °C reconstituted hY RNP complexes
were isolated by adding 20 µl of NeutrAvidin beads (Pierce)
and rotating for 1 h at 4 °C. Beads were then washed five times
with 1 ml of IPP-150, and bound proteins were isolated employing
elution buffer (20 mM Tris-HCl, pH 7.9, 20 mM
DTT, 2% SDS). Eluted proteins were heated for 5 min at 65 °C and
precipitated by adding 1 µl of glycogen (20 mg/ml) and 4 volumes of
acetone. Samples were left at Two-dimensional Gel Electrophoresis--
Proteins binding to
biotinylated hY RNAs in vitro were dissolved in 20-40 µl
of lysis buffer containing 9 M urea, 2% CHAPS (Sigma),
0.8% ampholyte pH 3-10 (40% solution, Fluka, Switzerland), 1% DTT.
High resolution two-dimensional gel electrophoresis on immobilized pH
gradients was carried out on ready-cut IPG Immobiline strips, pH
3-10 nonlinear, 18 cm long (Amersham Pharmacia Biotech). After
overnight incubation in rehydration buffer (8 M urea, 0.5% CHAPS, 15 mM DTT, 0.2% ampholyte pH 3-10), the strips
were focused on a horizontal electrophoresis apparatus (Amersham
Pharmacia Biotech) in a stepwise fashion: 0.5 h at 300 V, 1 h
at 500 V, 1 h at 1500 V, and 15 h at 2500 V (total 40 kVh) at
room temperature. The IPG strips were subsequently incubated for 15 min
in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, traces of bromphenol blue)
containing 10 mg/ml DTT and for 15 min in equilibration buffer
containing 48 mg/ml iodoacetamide (Sigma). Equilibrated IPG strips were
immersed in SDS running buffer for a few seconds and placed on top of a
1-mm vertical SDS 10% polyacrylamide gel. SDS-PAGE was run for 4-5 h
at 20 mA, and the gels were then stained with Coomassie Blue R-250 or
electroblotted onto nitrocellulose membranes for immunodetection.
Coomassie-stained spots corresponding to proteins binding to hY1 RNA
(and not to 5 S rRNA) were excised, completely destained, and subjected
to sequence analysis by tandem mass spectrometry performed after in gel
trypsin digestion (Harvard Microchemistry Facility, Cambridge, MA).
Transfection Experiments--
VSV-G-tagged cDNAs were
constructed as follows. For the human La protein the N-terminal VSV-G
tag (MEIYTDIEMNRLGK) was introduced via PCR using the following
primers: La-1
(5'-GAATTCGCCACCATGGAGATTTATACAGACATAGAGATGAACCGACTTGGAAAGCGCGGCCGCATGGCTGAAAATGGTGATAATG-3') and La-2 (5'-CTCGAGCTACTGGTCTCCAGCACCATT-3') using a
full-length La cDNA (35) as template. Indicated in bold type are
the introduced EcoRI, NotI, and XhoI
sites. The VSV-G tag encoding sequence is underlined. The PCR product
was digested with EcoRI/XhoI and cloned into the
corresponding sites of the pcDNA3 vector (Invitrogen). The
cDNAs encoding hnRNP K and hnRNP I were kindly provided by Gideon
Dreyfuss (Howard Hughes Medical Institute, University of Pennsylvania,
Philadelphia, PA). The human hnRNP K cDNA was modified by PCR to
introduce flanking NotI and XhoI sites, allowing
replacement of the La cDNA with the hnRNP K cDNA, which
resulted in an N-terminally VSV-G-tagged hnRNP K construct in
pcDNA3. An N-terminally Myc-tagged version of a human hnRNP I
cDNA in pcDNA3 was modified to replace the Myc tag with a VSV-G
tag. The integrity of the constructs was confirmed by DNA sequencing.
As a control, the empty pcDNA3 vector was used in transfection experiments.
African green monkey cells (COS-1) were transiently transfected with
the expression constructs. Briefly, COS-1 cells were grown to 80%
confluency in Dulbecco's modified Eagle's medium supplemented with
10% heat-inactivated fetal calf serum and penicillin/streptomycin in
5% CO2 at 37 °C. Cells were trypsinized and resuspended
in phosphate-buffered saline. Approximately 5 × 106
cells were transfected with 20 µg of plasmid DNA in a total volume of
400 µl of phosphate-buffered saline. Electroporation was performed at
300 V and a capacity of 125 microfarads with a Gene Pulser II
(Bio-Rad). Subsequently, the cells were seeded in 75-cm2
culture flasks and cultured overnight.
Yeast Three-hybrid System--
To investigate the interaction of
hnRNP I and hnRNP K with hY RNAs in vivo, a three-hybrid
system (36) was used (RNA-protein hybrid hunter kit; Invitrogen,
Groningen, The Netherlands). The DNAs encoding hY RNAs and hY1 RNA
deletion mutants were introduced into the SmaI and
AvrII sites of the pRH5' hybrid RNA vector downstream of two
copies of the MS2 RNA sequence. hnRNP I and hnRNP K hybrids were
constructed by ligating the PCR amplified fragments into the
EcoRI and XhoI sites of the pYEASTrp2 hybrid
protein vector. The sequence and orientation of all recombinant DNAs
was verified by sequencing. The yeast strain L40uraMS2, which stably
expresses the LexA DBD-MS2 coat protein was double transformed with
plasmids containing the hybrid RNAs and the hybrid proteins,
respectively. Transformants were selected on synthetic medium plates
lacking uracil and tryptophan. Expression of the bait proteins was
checked by Western blotting using appropriate monoclonal antibodies.
Double transformants were then assayed for Identification of Novel hY RNA-associated Proteins--
Recently,
we have described a set of five proteins with molecular masses between
53 and 80 kDa that in addition to Ro60 and La bound to human hY RNAs
in vitro, particularly to hY1 and hY3 RNA (28).
Interestingly, autoantibodies to these proteins were detected in
several sera of patients with SLE who commonly develop antibodies to Ro
and La proteins. To further characterize these hY RNA-binding proteins
Ro RNP complexes were reconstituted in vitro by incubating
HeLa S100 extracts with biotinylated hY1 RNA. Reconstitution reactions
employing biotinylated 5 S rRNA were performed in parallel to control
for nonspecifically binding proteins. All reactions were supplemented
with a 20-fold excess of yeast tRNA as a nonspecific competitor.
Reconstituted complexes were purified by NeutrAvidin affinity
chromatography followed by two-dimensional gel electrophoresis and
subsequently analyzed by immunoblotting using SLE serum BM known to
recognize all five novel hY RNA-binding proteins as well as Ro60 and La
(Ref. 28; see also Fig. 2A). As can be seen in Fig.
1, several protein spots on the membrane were stained by serum BM. The spots corresponding to proteins binding
specifically to hY1 RNA are indicated by arrows. The
positions of Ro60 and La with its typical isoelectric isoforms (37-39)
were confirmed in a separate experiment using monospecific sera and monoclonal antibodies to these proteins (not shown); the two spots visible below the La spots presumably represent La degradation products. Apart from the spots corresponding to Ro60 and La proteins, four major protein spots can be detected migrating above Ro60. The most
acidic of these proteins, which reproducibly migrated at ~68 kDa, was
excised from a Coomassie-stained two-dimensional gel run in parallel,
digested with trypsin, and microsequenced by tandem electrospray mass
spectrometry. This analysis provided a 19-amino acid tryptic peptide
sequence (GSYGDLGGPIITTQVTIPK), which completely matched a sequence
contained in the human hnRNP K protein (residues 378-396). This result
was compatible with the migration of the proteins in our
two-dimensional gels, which largely corresponded to the molecular mass
(66 kDa) and isoelectric point values (6.1-6.4) reported for hnRNP K
(40).
The second protein that we could identify was a basic protein visible
as a 62/60-kDa doublet. Previous experiments had already suggested the
presence of a 60-kDa protein in reconstituted hY1 RNPs that comigrated
with Ro60 in SDS-PAGE and appeared to have similar RNA binding
properties as the 62-kDa protein (28). Because of its migration
behavior and characteristic doublet appearance, we hypothesized that
this protein was hnRNP I, the PTB, which is known to migrate as a
double band of ~60 kDa in SDS-PAGE and has a reported pI of 8.5 (40,
41).
To confirm the presence of hnRNP I and hnRNP K in reconstituted
complexes hY1 RNA-binding proteins were separated by SDS-PAGE and
stained with either serum BM or monoclonal antibodies to hnRNP K or
hnRNP I, respectively (Fig. 2,
lanes 1). Compatible with our previously published data
serum BM recognized proteins of 80, 68, 65, 62, and 53 kDa in addition
to Ro60 and La (28). In contrast to serum BM, the anti-hnRNP K antibody
stained solely the 68-kDa band, whereas the anti-hnRNP I antibody
recognized two bands of 62 and 60 kDa. Neither the serum nor the
monoclonal antibodies recognized a protein in the control lanes
containing proteins binding to 5 S rRNA (Fig. 2, lanes
2).
Of the three other hY1 RNA-binding proteins recognized by the serum,
the 53-kDa protein was horizontally streaked and only weakly reactive
with the serum on two-dimensional immunoblots and could therefore not
be sequenced. For the 65-kDa protein migrating as a double spot at
neutral pI and for the basic protein(s) migrating at ~70-72 kDa
(presumably comigrating with hnRNP K in SDS-PAGE) no unambiguous data
could be obtained, and the 80-kDa protein was not visible on
Coomassie-stained two-dimensional gels.
Interaction of hnRNP I and hnRNP K with hY RNAs in Vitro--
To
study the interaction of hnRNP I and hnRNP K with hY RNAs in more
detail, reconstitution reactions were performed with all four hY RNAs
as well as with several deletion mutants of hY1 RNA. Proteins binding
to hY RNAs were separated by SDS-PAGE, blotted onto nitrocellulose
membranes, and subsequently probed with monoclonal antibodies to hnRNP
I and K as described above (Fig. 3); both proteins showed pronounced binding to hY1 RNA, but only hnRNP I
efficiently bound to hY3 RNA, whereas the interaction of hnRNP K with
hY3 RNA was relatively weak. In this assay, neither of the two proteins
interacted detectably with hY4 or hY5 RNA, confirming previous data
(28).
To map the regions of hY1 RNA involved in binding of the two proteins,
deletion mutants of (biotinylated) hY1 RNA were used. Truncation of the
Ro60 binding site did not have any effect on binding of either hnRNP I
or hnRNP K (Fig. 3B, lane 5), whereas mutation of the La binding site (3'-terminal UU to AG) significantly decreased binding of both proteins (Fig. 3B, lane
6). The strongest effect was observed with a mutant lacking the
pyrimidine-rich central loop 2b, which bound hnRNP I very weakly and
hnRNP K not at all (Fig. 3B, lane 7). Deletion of
stem2-loop1 or of stem3-loop3 had no or only little effect on binding
of hnRNP I, whereas binding of hnRNP K appeared to be reduced by ~50
and 80%, respectively (Fig. 3B, lanes 8 and
9). Finally, the binding of both proteins appeared to be
somewhat increased with a mutant lacking the stem4-loop4 region (Fig.
3B, lane 10). A comparable result was obtained
with 35S-labeled hnRNP I and K proteins translated in
vitro in a wheat germ system (data not shown).
Taken together, these results (i) clearly confirmed the in
vitro binding of hnRNP I and K to hY1 RNA; (ii) showed the
interaction of the two proteins with hY3 RNA to be slightly (hnRNP I)
or considerably (hnRNP K) weaker than with hY1 RNA; (iii) demonstrated
the central loop 2b to be indispensable for efficient binding of both
proteins to hY1 RNA; (iv) suggested that La but not Ro60 is required
for efficient binding; and (v) indicated that the binding sites for the
two hnRNP proteins are closely spaced but not necessarily identical.
Interaction of hnRNP I and K with Native hY RNAs--
To
investigate whether and which hY RNAs are associated with the hnRNP I
and K proteins in vivo, a HeLa cell extract was subjected to
immunoprecipitation using specific monoclonal antibodies to hnRNP I,
hnRNP K, Ro60, and La. RNAs were isolated from the immunoprecipitates by phenol-chloroform extraction and probed with radiolabeled antisense hY RNAs by Northern blot hybridization. As shown in Fig.
4A, coprecipitation of hY1 and
hY3 RNA by the anti-hnRNP I monoclonal antibody was clearly observed
although at a lower level as compared with the precipitates obtained
with the anti-La and anti-Ro60 antibodies, which efficiently
precipitated all four hY RNAs. RNA bands visible just below hY1 RNA and
hY3 RNA (lanes 1 and 3) presumably corresponded to previously reported degradation products of these two hY RNAs known
as hY2 and hY3* RNAs (22, 42), which was consistent with the lack of
coprecipitation of these molecules by the anti-La antibodies. These
results not only confirmed the specific binding of hnRNP I to hY1 and
hY3 RNA observed in vitro but also strongly suggested that
hnRNP I is associated with hY1 and hY3 RNA in vivo. In
contrast, coprecipitation of hY RNAs by the anti-hnRNP K antibody was
not detectable (lane 4).
Although these data suggested that the association of hnRNP I with hY1
and hY3 RNA occurs also in vivo, they did not allow us to
conclude that this protein was present also in Ro RNPs (i.e. hY RNA complexes containing both Ro60 and La). To address this question, Ro and La RNPs were isolated from a HeLa cell extract using
anti-Ro60 and anti-La micropreparative immunoaffinity columns. Proteins
were eluted with 1 M NaCl, separated by SDS-PAGE, and identified by immunoblotting using monoclonal antibodies against hnRNP
I and hnRNP K; proteins isolated from in vitro reconstituted hY1 RNPs served as controls (Fig. 4B, lane 1). In
these experiments the presence of hnRNP I in both anti-La and anti-Ro60
eluates was reproducibly observed (Fig. 4B, lanes
2 and 3), demonstrating that this protein was contained
in Ro RNP complexes. On the other hand, and in agreement with the RNA
precipitation data, hnRNP K could not be detected in these eluates (not shown).
To confirm these data and to account for the possibility that the
epitope recognized by the anti-hnRNP K monoclonal antibody might be
inaccessible when the hnRNP K is complexed with hY RNA, we expressed
both hnRNP proteins and, as a control, La as VSV-G-tagged fusion
proteins in transiently transfected COS-1 cells. Western blot analysis
with a monoclonal antibody directed to the VSV-G tag demonstrated that
the three tagged proteins are expressed to similar amounts (Fig.
5A). This antibody was then
used to immunoprecipitate lysates of transfected cells that were
subsequently analyzed for the presence of (coprecipitated) Y RNAs by
Northern blot hybridization. Also by this approach hnRNP I was found to
associate with Y1 and Y3 RNA (Fig. 5B, lane 5),
and again no association of hnRNP K with Y RNAs could be detected (Fig.
5B, lane 8). As expected, VSV-G-tagged La was
associated with all four Y RNAs (lane 2), and no Y RNAs were
detectable when cells were transfected with an "empty vector"
(lane 11). All four Y RNAs were also coprecipitated by the
anti-La (lane 3) and anti-Ro60 (lanes 6,
9, and 12) antibodies.
Interaction of hnRNP I and K with hY1 and hY3 RNA in a Yeast
Three-hybrid System--
To investigate the interactions of hnRNP I
and hnRNP K with hY RNAs in a living cell, we made use of the yeast
three-hybrid system (36). In analogy to the widely used two-hybrid
system, this system is based on transcriptional activation of the
reporter genes his3 and lacZ upon
interaction of RNA with the protein of interest. In our case the first
(protein) hybrid consisted of the DNA-binding domain of the
transcriptional activator LexA fused to the RNA-binding viral MS2 coat
protein (LexA DBD-MS2), the second (RNA) hybrid consisted of hY RNA (or
hY1 RNA deletion mutants) cloned downstream of the MS2 RNA sequence
(MS2 RNA-hY RNA), and the third (protein) hybrid was composed of the
transcription activation domain of Gal4 fused to either hnRNP I or
hnRNP K (B42AD-hnRNP protein) (Fig.
6A).
To examine the interaction of hY1 and hY3 RNA with hnRNP I or hnRNP K,
a yeast strain expressing the LexA DBD-MS2 coat protein hybrid was
cotransformed with the hybrid plasmids encoding these RNAs and
proteins. Transformants lacking any of the (RNA or protein) hybrid
components were not able to grow on a medium deficient of histidine
(not shown) and showed no or only little
When we examined binding of hnRNP I and K to hY1 RNA deletion mutants
(same as shown in Fig. 3), mutant Y1 Although data from several laboratories suggest that Ro RNP
complexes may contain a number of other proteins in addition to Ro60
and La (20-28), unambiguous evidence for this has not been presented
yet (3, 4). Here we have identified two of the proteins previously
described by us to associate with hY RNAs in vitro (28) as
hnRNP I and hnRNP K. The two proteins interacted strongly with hY1 RNA
and to a lesser extent with hY3 RNA but not with hY4 or hY5 RNA, which
is in agreement with our previous findings. Immunoprecipitation
experiments performed with extracts from HeLa cells and transiently
transfected COS-1 cells provided substantial evidence for in
vivo association of hnRNP I with hY1 and hY3 RNA and indicated the
existence of a subpopulation of Ro RNPs containing hnRNP I in addition
to Ro60 and La. The observation that the anti-hnRNP I antibody
precipitated significantly smaller amounts of hY RNAs than the
anti-Ro60 antibody (although comparable amounts of Ro60 and hnRNP I
appeared to be bound in the reconstitution assays) indicates that, in
contrast to Ro60, hnRNP I is associated with a minor portion (10-20%)
of hY1 and hY3 RNAs. Although these assays did not provide any evidence
for hY RNA-hnRNP K interactions in these cellular extracts, a clearly
positive result was obtained in a yeast three-hybrid system that was
comparable with that obtained with hnRNP I. Thus, hnRNP K may bind only
to a rather minor subset of hY RNAs that are devoid of Ro60 (there is
no Ro60 homologue in yeast) and therefore difficult to detect, or,
alternatively, the interaction with hY RNAs may have been disturbed
during preparation of the cellular extracts or upon antibody binding in
immunoprecipitation assays.
The importance of an intact La binding site for efficient binding of
hnRNP I and hnRNP K was remarkable and leads us to speculate that the
function of La in hY RNP assembly may be that of an RNA chaperone being
required for correct folding of hY RNAs, thus enabling binding of other
proteins (with the notable exception of Ro60). This would be consistent
with recent reports on the chaperoning role of La in small nuclear RNP
assembly and pre-tRNA processing (12, 15, 19).
The results obtained with hY1 RNA deletion mutants showed that the
internal pyrimidine loop (71-86 nucleotides) was indispensable for
efficient association of the two hnRNP proteins with hY1 RNA and
demonstrated their binding sites to be clearly different from the Ro60
binding site. Both hnRNP I and K are known to bind to pyrimidine-rich
sequences (43, 44). Thus, hnRNP I, which is commonly known as PTB,
binds to the polypyrimidine stretch present near the 3' splice site of
many introns and also to pyrimidine-rich sequences of mature mRNAs
(45). For hnRNP K, which shows increased affinity for poly(rC)
sequences but does not bind to the polypyrimidine tract, several
cytosine-rich recognition motives have been described, such as the CT
element (CCCTCCCCA) of the c-myc gene (46) and CU repeats
(CCCCACCCUCUUCCCC) present in the 3'-untranslated region of erythroid
15-lipoxygenase mRNA (47). These sequences as well as those
recognized by hnRNP I (PTB) show significant similarities with the
central loop 2b region of hY1 RNA (UACUCUUUCCCCCCUU), supporting the
assumption that this region may directly interact with both proteins as
suggested by the results obtained with the deletion mutants.
Remarkably, an internal pyrimidine-rich loop is present also in hY3
RNA, whereas it is lacking in hY4 and hY5 RNA, which would be
consistent with their failure to bind the two hnRNP proteins.
The heterogeneous nature of Ro RNPs has been postulated by several
researchers based on biochemical fractionation and in vitro reconstitution data (22, 23, 28). The number of Y RNAs has increased
throughout the evolution from C. elegans, which expresses only one Y RNA, to vertebrates with four Y RNAs (6, 48). Interestingly,
the Ro60 binding site and the pyrimidine-rich loop sequence represent
the most conserved regions, being already present in C. elegans RNA, which shows the greatest homology to hY3 RNA (49).
This strongly suggests that the pyrimidine-rich element is important
for Y RNA functioning, including interactions with other proteins
and/or RNAs. Furthermore, the internal loop of hY1 RNA was shown to be
resistant to enzymatic and chemical cleavage (50), an observation also
made with pyrimidine-rich loops of other small RNAs transcribed by RNA
polymerase III (51-53). Because the four vertebrate Y RNAs show a high
diversity in the central region, this part of the molecule might
determine the fate (i.e. localization and function) of Y
RNPs or Ro RNPs, respectively, because of differential association with
proteins of (more or less) diverse function (such as hnRNP I and hnRNP K).
Several RNA polymerase III transcripts including RNase P RNA, RNase MRP
RNA, and hY RNAs (except hY4 RNA) have been localized to the
perinucleolar compartments together with two hnRNP proteins, hnRNP I
(41, 54), and CUG-binding protein/hNab50 (55, 56). Importantly, the
presence of hnRNP I in the perinucleolar compartments was found to be
sensitive to RNase A treatment (57), which may be considered a further
(though rather indirect) indication for in vivo interactions
of this protein with hY1 and hY3 RNAs. Remarkably, the presence of Ro60
and La could not be detected within the perinucleolar compartments (54), which may suggest the existence of hY RNA complexes
containing hnRNP I (as well as other proteins yet to be identified) but
devoid of both Ro60 and La.
hnRNP I and K are both shuttling proteins that belong to a group of
multifunctional RNA-binding proteins exerting regulatory roles at the
post-transcriptional level, including RNA processing and export as well
as regulation of mRNA stability and translation (44, 58-61). Given
that Ro RNPs are predominantly, if not exclusively, localized in the
cytoplasm, it is important to mention reported functions of hnRNP I and
K that are related to translational events. Thus, hnRNP I has been
shown to bind specifically to several viral internal ribosome entry
sites, thereby promoting ribosome binding in a cap-independent manner
(62-64). Remarkably, a similar activity was also found for La (13),
and cooperation of these two proteins in the regulation of internal
ribosome entry site-dependent translation has been recently
suggested (65). Furthermore, these proteins may be also required for
efficient and correct initiation of cap-dependent translation by inhibiting translation of uncapped mRNAs (66). Apart
from its role as transcription factor (44, 67, 68), hnRNP K protein has
been demonstrated to act as a differentiation regulator by virtue of
its binding to the control elements at the 3' end of the erythroid
15-lipoxygenase mRNA, thereby inhibiting translation (47). In a
similar manner translation of papilloma virus late mRNAs is
inhibited by hnRNP K and the poly(rC)-binding protein (69).
Furthermore, two translation-related functions have been assigned to
the Ro60 protein, namely participation in a degradation pathway for
misfolded 5 S rRNA (17) and a role in the regulation of translation of
ribosomal protein L4 in concert with La and cellular nucleic
acid-binding protein by binding to a polypyrimidine stretch in the
5'-untranslated region of L4 mRNA (16, 70).
Combining our experimental data and those of others one may speculate
about the cellular role of Ro RNPs or hY RNPs, respectively: newly
transcribed hY RNAs associate with La and move through the nucleoplasm
toward the nuclear membrane where Ro60 associates just prior to export
into the cytoplasm, being indispensable for nuclear export of hY RNAs
(3, 9). During their intranuclear migration subsets of hY RNPs are
formed by virtue of association with various proteins including hnRNP I
and K, Ro52, and presumably other proteins including those reported to
interact with hY RNA in vitro or with Ro60 in yeast two- and
three-hybrid systems (26-28). All these proteins may be
multifunctional and upon binding to hY RNAs become destined to exert
specific functions either in the nucleus (particularly in the
perinucleolar compartment) or in the cytoplasm. It is intriguing that
for all four hY RNA-binding proteins identified to date (La, Ro60, and
hnRNP I and K) regulatory roles in translation of certain cellular and
viral mRNAs have been described, although these proteins were
originally identified as nuclear proteins with (nuclear) functions
mainly related to RNA processing and transcription. Thus, hY RNAs may
serve as carriers for (nuclear) proteins involved in regulation of
translation. Taken together, we think that our findings not only allow
better characterization of the structure of Ro RNP (or hY RNP)
complexes but also may provide some clues regarding their function(s),
which must be further investigated in future experiments.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
70 °C.
-32P]UTP (PerkinElmer Life Sciences) were
employed. The plasmid encoding 5 S rRNA was a kind gift of Dr. K. Nierhaus (Max Planck Institute for Molecular Genetics, Berlin,
Germany). For detection of Y RNAs by Northern blot hybridization
RNAs were separated on 10% polyacrylamide, 7 M urea gels,
electroblotted onto nylon membranes (Zeta-probe; Bio-Rad), fixed
by UV cross-linking, and hybridized with 32P-labeled
antisense hY RNA transcripts by incubating overnight at 65 °C as
described (28, 34).
70 °C for at least 30 min and
subsequently centrifuged for 15 min at 15,000 × g. Recovered proteins were dissolved either in SDS sample buffer for
SDS-PAGE or in two-dimensional lysis buffer for application in
two-dimensional electrophoresis.
-galactosidase (
-gal)
expression on a filter using X-gal as a substrate and for growth on
selective medium without histidine. For quantitative determination of
-gal activity cells were disrupted with glass beads, and, after
determination of protein concentration, enzyme activity was measured
photometrically at 420 nm using
2-nitrophenyl-
-D-galactopyranoside as a substrate. As a
positive control the established IRE-IRP interaction was used (36).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (39K):
[in a new window]
Fig. 1.
Characterization of hY1 RNA-binding proteins
by two-dimensional gel electrophoresis and immunoblotting.
Proteins from HeLa cell extracts binding to biotinylated hY1 RNA in
reconstitution assays were separated by two-dimensional
electrophoresis, transferred to a nitrocellulose membrane, and stained
with serum BM from a patient with SLE recognizing several hY1
RNA-binding proteins in addition to Ro60 and La (marked by
arrows). The two stained spots migrating below La presumably
represent La degradation products. Positions of Ro60 and La were
determined in a separate experiment using monoclonal antibodies to
these proteins. The hnRNP K protein was identified by tandem mass
spectrometry, the identity of hnRNP I (migrating as a doublet) was
deduced from its characteristic position on the two-dimensional gel and
immunologically confirmed using a monoclonal antibody against hnRNP I
(see Fig. 2C).
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[in a new window]
Fig. 2.
Detection of hnRNP K and hnRNP I in
reconstituted hY1 RNP complexes. Proteins binding in
reconstitution assays to biotinylated hY1 RNA (lanes 1) or 5 S rRNA (lanes 2) were isolated by streptavidine affinity
chromatography, separated by SDS-PAGE, and probed by Western
blotting with patient serum BM (A), a monoclonal anti-hnRNP
K antibody (B), and a monoclonal anti-hnRNP I antibody
(C).
View larger version (23K):
[in a new window]
Fig. 3.
Interaction of hnRNP K and hnRNP I with hY
RNAs and hY1 RNA deletion mutants. Reconstitution reactions were
performed with biotinylated hY RNAs and deletion mutants of hY1 RNA,
and binding of hnRNP I and K was detected by immunoblotting employing
monoclonal antibodies. Note that mutant S1L1 lacks the Ro binding
site and mutant
S1L1sty lacks the La binding site. A,
proposed secondary structure of human hY1 RNA and hY1 RNA deletion
mutants. wt, wild type; S, stem; L,
loop. B, Western blot analysis of hY RNA association with
hnRNP K and hnRNP I.
View larger version (19K):
[in a new window]
Fig. 4.
Association of hnRNP I with native hY RNAs
and Ro RNPs in HeLa cell extracts. A, Northern blot
analysis of hY RNAs immunoprecipitated from a HeLa cell extract by
monoclonal antibodies to Ro60 (lane 1), La (lane
2), hnRNP I (lane 3), and hnRNP K (lane 4).
A monoclonal antibody to human interleukin-6 was used as a negative
control (lane 5), and 5% of the total input RNA served as a
positive control (lane 6). B, Western blot
analysis of hnRNP I immunoprecipitated by the anti-La (lane
2) and anti-Ro60 (lane 3) monoclonal antibodies; a
monoclonal anti-interleukin-6 antibody served again as a negative
control (lane 4). For comparison hY1 RNA-associated proteins
isolated from in vitro reconstituted complexes are shown
(lane 1).
View larger version (45K):
[in a new window]
Fig. 5.
Association of VSV-tagged hnRNP I with Y RNAs
in transiently transfected cells. COS-1 cells were transfected
with constructs encoding VSV-G-tagged La, hnRNP I, and hnRNP K, and an
empty vector (mock), and 24 h after transfection cell lysates were
prepared and analyzed by Western blotting (A) or Northern
blotting (B). A, expression of the VSV-G-tagged
proteins La, hnRNP I, and hnRNP K analyzed by Western blotting using a
monoclonal anti-VSV-G tag antibody. The positions of the molecular mass
markers are indicated on the right. B, Northern
blot analysis of Y RNAs coprecipitating with VSV-G-tagged proteins. The
lysates were subjected to immunoprecipitation with monoclonal
anti-VSV-G tag, anti-La, or anti-Ro60 antibodies, respectively. RNA
isolated from the total lysates (input) and from the
immunoprecipitates was analyzed by Northern blot hybridization using
antisense hY RNAs as probes. The positions of Y1, Y3, Y4, and Y5 are
indicated. The band indicated with ** probably represents a
degradation product of Y3, previously designated Y3** (42).
View larger version (40K):
[in a new window]
Fig. 6.
Interaction of hnRNP I and hnRNP K with hY1
and hY3 RNA in a yeast three-hybrid system. The yeast three-hybrid
system was used to study interaction of the two hnRNP proteins with hY
RNAs in vivo by measuring -gal activity of transformed
cells. A, schematic diagram showing gene activation by
specific interaction between hY-RNA and hnRNP I or K proteins in the
yeast three-hybrid system. Expression of reporter genes lacZ
and his3 is activated upon positive interaction between
hnRNP I or K and hY1 RNA dragging the following two domains of the
transcriptional activator to a close vicinity: the DNA-binding domain
of LexA (DBD) and the activation domain of Gal 4 (AD). B, analysis of
-gal activity.
Transformants were grown on synthetic medium lacking uracil and
tryptophan, and
-gal activity was tested in the blue color X-gal
filter assay (right) and quantitatively determined by direct
measurement of the enzymatic activity in units/mg yeast mass
(left).
-gal activity when grown on
a synthetic medium lacking uracil and tryptophan (Fig. 6B).
In contrast, elevated
-gal activity was clearly seen in
transformants expressing hY1 RNA and hnRNP I or hnRNP K hybrids, respectively, whereas
-gal activity exceeded background levels only
by approximately 2-fold in hY3 RNA transformants (Fig. 6B). The observed interactions of hY1 RNA with the two hnRNP proteins in the
yeast system were largely consistent with the in vitro reconstitution data described above. Interactions of hnRNP I with hY3
RNA, on the other hand, were considerably weaker than in the in
vitro binding experiments.
L2b lacking the pyrimidine-rich
domain showed only low levels of
-gal activity when cotransformed
with either hnRNP K or hnRNP I, again compatible with the in
vitro results. In contrast,
-gal activity of the Y1
S3L3
mutant strain was even somewhat greater than that of wild-type Y1 RNA
for both hnRNP I and K, whereas deletion of stem4-loop4 (Y1
S4L4) led
to a slightly decreased activation of the lacZ gene with
both hnRNP proteins. The other two hY1 RNA mutants showed similar
-gal activities as wild-type hY1 RNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank G. Dreyfuss for providing cDNAs for hnRNP I and K and monoclonal antibodies, D. M. Helfman for the anti-PTB3 antibody, E. Höfler for expert technical assistance, D. Rendic for computational help, A. Barta and W. van Venrooij for critical comments and invaluable suggestions, Josef Smolen for support, and The Federation of European Biochemical Societies (FEBS) for enabling Gustav Fabini to attend the course on advanced methods for protein analysis held in Hatfield, UK.
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FOOTNOTES |
---|
* This work was supported by a grant from the Austrian Science Fund (project part no. 5 of the Special Research Project "Modulators of RNA Fate and Function") and in part by the Netherlands Foundation for Chemical Research with financial aid from the Netherlands Organization for Scientific Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Inst. of Chemistry, Div. of Glycobiology, Agricultural University, Muthgasse 18, A-1190 Vienna, Austria.
** To whom correspondence should be addressed: Div. of Rheumatology, Dept. of Internal Medicine III, University Hospital of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. Tel.: 431-40400-2121; Fax: 431-40400-4306; E-mail: Guenter.Steiner@akh-wien.ac.at.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M101360200
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ABBREVIATIONS |
---|
The abbreviations used are:
RNP, ribonucleoprotein;
hnRNP, heterogeneous nuclear ribonucleoprotein;
IPP, immunoprecipitation buffer;
PTB, polypyrimidine tract-binding protein;
SLE, systemic lupus erythematosus;
VSV, vesicular stomatitis virus;
-gal,
-galactosidase;
PAGE, polyacrylamide gel electrophoresis;
DTT, dithiothreitol;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PCR, polymerase chain reaction;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside..
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