From the Department of Biochemistry, University of
Nijmegen, P. O. Box 9101, NL-6500HB Nijmegen, The Netherlands and
the § Institute of Cell and Molecular Biology, Swann
Building, King's Buildings, University of Edinburgh, Mayfield
Road, EH9 3JR Edinburgh, Scotland, United Kingdom
Received for publication, August 21, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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The yeast exosome is a complex of 3' In both bacteria and eukaryotes, the processing and degradation of
many RNA species involves multiprotein complexes (reviewed in Refs.
1-4). The Escherichia coli degradosome includes the endoribonuclease E (RNase E), the 3' The 3' processing of many RNAs is affected by the absence or mutation
of exosome components. The nuclear exosome is implicated in the
processing of ribosomal RNA (rRNA), spliceosomal small nuclear RNAs,
and small nucleolar RNAs, as well as the degradation of pre-rRNA
spacers and unspliced pre-mRNAs (12-22). The cytoplasmic exosome
complex is involved in the 3' Human cells also contain a multiprotein complex that is related to the
yeast exosome (11). This complex, initially designated as the
polymyositis/scleroderma
(PM/Scl)1 overlap syndrome
particle and herein referred to as the human exosome, was reported to
contain 11 (25) to 16 (26) subunits ranging from 20 to 110 kDa. Two
proteins of this complex were identified as autoantigens, which are
targeted by autoantibodies present in the serum of patients suffering
from myositis, scleroderma, or PM/Scl overlap syndrome (27). All tested
anti-PM/Scl-positive sera recognize a nuclear protein, known as
PM/Scl-100, while some also recognize a protein migrating at about 75 kDa (PM/Scl-75) in SDS-polyacrylamide gel electrophoresis (28-30).
PM/Scl-100 and -75 are the human homologues of yeast Rrp6p and Rrp45p,
respectively (13, 31). The cloning of five additional human homologues of yeast exosome components has been reported, while ESTs were identified for three further homologues (11, 12, 32-34). However, detailed characterization of human exosome components is limited. The
functional conservation has been reported for hRrp4p, hRrp44p/hDis3p, and hCsl4p, and direct evidence for complex formation has been described for hRrp4p and the PM/Scl autoantigens (11).
Here we report the cloning of the human homologues of yeast Rrp40p,
Rrp41p (also designated Ski6p), and Rrp46p. Subcellular distribution,
coimmunoprecipitation, and in vivo as well as in vitro activity assays show that these three proteins are
components of the human exosome.
Isolation of hRrp40p, hRrp41p, and hRrp46p cDNAs--
Data
base homology searches revealed human ESTs, which could be assembled
into contigs with apparent homology to yeast exosome components (11).
ESTs homologous to yeast Rrp40p (accession numbers AA747303, AA282142,
H25417, and AA057832), Rrp41p/Ski6p (accession numbers U46288 and
AA129848) and Rrp46p (accession numbers AA428915, AA461395, and
AA426493) were selected. Nucleotide sequences of five independent
clones of each EST were determined by the dideoxynucleotide termination method. Complete (hRRP41) or partial
(hRRP40 and hRRP46) open reading frames (ORFs)
were identified. To isolate additional cDNAs corresponding to
sequences required to complete the ORFs, human teratocarcinoma and
placenta cDNA libraries were screened by PCR using gene-specific
primers for hRRP40 and hRRP46 (hRrp40-a or hRrp46-a, respectively; see Table I) in combination with Complementation Experiments--
To test for complementation of
the yeast conditional alleles GAL::rrp40,
GAL::rrp41, and GAL::rrp46,
strains P147, P118, and YCA21 were transformed with plasmids phRRP40,
phRRP41, and phRRP46, respectively (Table
II). Complementation of the conditional
alleles was assayed by growth on repressive YPD medium at 30 °C and
compared with the growth of the isogenic wild-type strain (YDL401).
Plasmids were constructed by inserting the cDNA of hRRP40,
hRRP41, and hRRP46 into plasmid pNOPPATA1L (CEN, LEU2)
(generously provided by E. Hurt) under the control of a NOP1 promotor,
at restriction sites NdeI-BamHI for hRRP40
and hRRP41, and NdeI-SalI for hRRP46.
Expression of Recombinant Proteins--
For prokaryotic
expression, the hRRP40, hRRP41, and hRRP46 cDNAs
were cloned into pET16b and/or pGEX2T'G resulting in N-terminally His-tagged or glutathione S-transferase (GST)-tagged
recombinant proteins, respectively. His-tagged proteins were purified
by Ni2+-NTA-agarose beads (Qiagen) essentially according to
the manufacturer's protocol. GST-tagged proteins were purified as
described previously (35). In addition, recombinant N-terminally
His-tagged hRrp40p and hRrp46p were expressed in and purified from SF-9
cells using the Bac-to-Bac baculovirus expression system according to
the manufacturer's protocol (Life Technologies, Inc.).
Generation of Rabbit Antisera--
Polyclonal antisera were
raised in rabbits by immunization with 100-200 µg of purified
recombinant protein according to standard procedures (36). Serum H70
(anti-hRrp40p), H71 (anti-hRrp41p), and H73 (anti-hRrp46p) were
generated using His-tagged hRrp40p expressed in E. coli,
His-, and GST-tagged hRrp41p expressed in E. coli and
His-tagged hRrp46p expressed in the baculovirus system, respectively.
Western Blot Analysis--
For Western blot analysis, autoimmune
patient and rabbit antisera were diluted 5000- and 500-fold,
respectively, in blocking buffer (3% nonfat milk, phosphate-buffered
saline (PBS), 0.1% Tween 20). As secondary antibody, horseradish
peroxidase-conjugated rabbit anti-human IgG or swine anti-rabbit IgG
(Dako Immunoglobulins) were used at a 2500-fold dilution in blocking
buffer. Visualization was performed by chemiluminescence.
Transient Transfection of HEp-2 Cells and Indirect
Immunofluorescence--
For transfection, hRRP40, hRRP41,
and hRRP46 cDNAs were cloned into the pCI-Neo vector
(Promega), which contained a sequence element encoding the vesicular
stomatitis virus G epitope (VSV) to allow expression of either
N-terminally or C-terminally VSV-tagged proteins. HEp-2 cells were
grown to 80% confluence by standard tissue culture techniques in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
containing 10% fetal calf serum. Approximately 2 × 106 cells were transfected with 10-20 µg of DNA in 800 µl of Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum by electroporation, which was performed at 270 V and
950 microfarads using a Gene Pulser II (Bio-Rad). After transfection,
cells were seeded onto coverslips and cultured for 24 h. Cells
were washed twice with PBS, fixed with methanol for 5 min at
For indirect immunofluorescence, fixed cells were subsequently
incubated for 1 h at room temperature with an affinity-purified mouse anti-VSV tag monoclonal antibody (Roche Molecular Biochemicals) and subsequently with fluorescein isothiocyanate-conjugated rabbit anti-mouse Ig (Dako Immunoglobulins) and fluorescein
isothiocyanate-conjugated swine anti-rabbit Ig (Dako Immunoglobulins).
All antibodies were diluted 50-fold in PBS. Cells were mounted with
PBS/glycerol containing Mowiol, and bound antibodies were visualized by
fluorescence microscopy.
Preparation of HeLa Cell Extracts--
Cytoplasmic and nuclear
HeLa cell extracts were prepared according to a modification of the
Dignam procedure essentially as described by Wahle et al.
(37).
Immunoprecipitation--
Immunoprecipitations were essentially
performed as described previously (38). Per immunoprecipitation, 10 µl of patient serum or 20 µl of rabbit serum was coupled to 10 µl
of protein A-agarose (Biozym) and extract of 2.5 × 106 HeLa cells was used. For Western blotting, the
immunoprecipitates were heated for 5 min in SDS sample buffer and
fractionated by SDS-polyacrylamide gel electrophoresis (39).
Size Exclusion Chromatography--
Nuclear and cytoplasmic
extract of 75 × 106 HeLa cells was fractionated by
automated liquid chromatography (BioLogic, Bio-Rad) using the Superdex
200 HR 10/30 column (Amersham Pharmacia Biotech) in a buffer containing
10 mM Hepes-KOH, pH7.9, and 100 mM KCl at 0.5 ml/min. Thirty samples of ~330 µl were collected, and proteins were
immediately precipitated by the addition of four volumes of acetone.
After overnight incubation at In Vitro Exonuclease Assay--
Immunoprecipitations were
performed as described above. After removal of nonbinding material,
immune complexes bound to the protein A-agarose beads were washed twice
with buffer A (10 mM Tris-HCl, pH 7.5, 50 mM
KCl, 5 mM MgCl2, 1 mM
Na2HPO4). Substrate solution
(32P-labeled substrate in buffer A) was added to the
protein A beads, and the suspension was incubated at 37 °C with
gentle agitation. Formamide loading buffer was added to samples taken
at regular intervals and immediately frozen until analysis. Samples
were analyzed by denaturing polyacrylamide gel electrophoresis followed by autoradiography. Substrates, transcribed in vitro by T3
RNA polymerase from XbaI-linearized pBS( Cloning of Human Homologues of Yeast Rrp40p, Rrp41p/Ski6p, and
Rrp46p--
Full length cDNAs encoding human homologues of the
S. cerevisiae Rrp40p, Rrp41p/Ski6p, and Rrp46p (open reading
frames YOL142w, YGR195w, and YGR095c, respectively) were generated
based upon EST clones, cDNA-specific PCR fragments isolated from a
teratocarcinoma cDNA library, and 5' RACE products obtained from
human placental RNA (Fig. 1). These
sequence data have been submitted to the DDBJ/EMBL/GenBankTM data
bases under accession numbers AF281132, AF281133, and AF281134. From
the cDNAs, hRrp40p, hRrp41p, and hRrp46p have predicted molecular
masses of 31 kDa (275 amino acids), 27 kDa (245 amino acids), and 26 kDa (235 amino acids), respectively. The data in Table
III show that the predicted protein
sequences of hRrp40p, hRrp41p, and hRrp46p are relatively well
conserved to the S. cerevisiae (Sc) homologues, and clear
homologues are also present in M. musculus (Mm), C. elegans (Ce), and S. pombe (Sp). hRrp41p and hRrp46p
are both homologous to E. coli RNase PH (11, 12, 31) and
therefore show homology to each other, whereas hRrp40p is not
significantly homologous to hRrp41p or hRrp46p.
Two genomic sequences for hRRP46 are present in the data base; a
complete sequence on chromosome 19 and a partial sequence (accession
no. L08634) corresponding to nucleotides
Two EST clones encoding a possible splicing variant of hRrp40p
(accession nos. AA282142 and H25417) were identified, and the same
sequence is listed as apoptosis-related protein PNAS-3 mRNA
(accession no. AF229833). These lack an internal region of the ORF
leading to frameshift at Gln-158 and truncation of the polypeptide.
hRrp41p Encodes a Functional Homologue of Yeast
Rrp41p/Ski6p--
To test whether hRrp40p, hRrp41p, or hRrp46p can
complement mutations in the corresponding yeast genes, the full-length
cDNAs were cloned into yeast expression vectors under the control
of the strong constitutive NOP1 promoter (see
"Experimental Procedures"). The constructs were transformed into
yeast strains in which the endogenous genes were subject to
GAL regulation; strains GAL::rrp40, GAL::rrp41, and GAL::rrp46.
Western blotting (data not shown) confirmed that each of the human
proteins was well expressed. In plate assays, the growth inhibition of
the GAL::rrp41 strain on glucose medium was
efficiently suppressed by expression of hRrp41p (Fig.
2), showing it to be the functional
homologue of Rrp41p/Ski6p. The expression of hRrp40p or hRrp46p did not
support the growth of GAL::rrp40 or
GAL::rrp46 strains, respectively, indicating that
the human proteins are unable to perform all of the essential functions
of their yeast homologues.
Detection of hRrp40p, hRrp41p, and hRrp46p in Human Cells--
The
cloned cDNAs were expressed as His-tagged and/or GST-tagged
polypeptides using the bacterial and baculovirus expression systems. In
each case this resulted in the synthesis of proteins with gel
mobilities close to those expected for the predicted molecular weights.
The recombinant proteins were purified by Ni2+ or
glutathione affinity chromatography and used to raise rabbit antisera,
designated H70 (anti-hRrp40p), H71 (anti-hRrp41p), and H73
(anti-hRrp46p). All sera recognized the corresponding recombinant His-tagged protein on Western blots (Fig.
3, A-C, lanes Ag).
The reactivity of all antisera with all three recombinant proteins was
analyzed by Western blotting. Purification of the baculovirus-expressed hRrp46p might, in principle, have led to the copurification of the
endogenous insect exosome complex; moreover, hRrp46p and hRrp41p are
homologous (see Table III). However, none of the anti-sera showed
detectable cross-reactivity.
To demonstrate that hRrp40p, hRrp41p, and hRrp46p are expressed in HeLa
cells, Western blots containing total HeLa cell extract were probed
with the rabbit antisera (Fig. 3, A-C, lanes
T). Although the sera recognized more than one protein in
the total cell extract, prominent species (indicated by
arrowheads) were decorated in each case that migrated
somewhat faster than the corresponding recombinant His-tagged proteins
(Fig. 3, A-C, compare lanes Ag and
T). Immunoprecipitation experiments (see below) confirmed that these correspond to the hRrp40p, hRrp41p, and hRrp46p.
We conclude that hRrp40p, hRrp41p, and hRrp46p are present in HeLa cell
lysates and show gel mobilities close to those expected for their
predicted molecular weights.
Subcellular Localization of hRrp40p, hRrp41p, and hRrp46p--
In
yeast, 10 out of 11 known components are associated with both the
cytoplasmic and the nuclear forms of the exosome, while Rrp6p is
detected only in the nucleus (11). Western blotting experiments showed
that hRrp4p and PM/Scl-75 are components of both nuclear and
cytoplasmic complexes in human cells, while PM/Scl-100 (the homologue
of Rrp6p) appears to be solely nuclear (11).
To determine the subcellular distribution of hRrp40p, hRrp41p, and
hRrp46p, HeLa cells were separated into three fractions: cytoplasmic
extract (C), nuclear extract (N), and the nuclear material retained
after salt extraction (R). The material of an equal number of cells
(5 × 105 cells) from each fraction was used for
Western blotting. As controls for the fractionation, we analyzed the
largely cytoplasmic histidyl-tRNA synthetase (Jo-1, Fig.
3F), the nuclear DNA topoisomerase I (DNA Topo I, Fig.
3G), and the nuclear matrix-associated protein lamin B (Fig.
3H). The positions of migration are indicated by
arrowheads. These results confirmed that the cells were
fractionated as expected.
Western blots were probed with the rabbit sera H70 (Fig.
3A), H71 (Fig. 3B), and H73 (Fig. 3C).
hRrp40p, hRrp41p, and hRrp46p were each detected in both the nuclear
(N) and cytoplasmic (C) fraction, with lower yields in the cytoplasm
(C). Weaker signals were seen in residual nuclear material after salt
extraction (R). A similar distribution was found for hRrp4p (Fig.
3D). In contrast, PM/Scl-100 was recovered almost entirely
in the nuclear fraction (Fig. 3E).
To confirm the subcellular localization of hRrp40p, hRrp41p, and
hRrp46p, polypeptides carrying N-terminal and C-terminal VSV tags were
expressed in HEp-2 cells. Indirect immunofluorescence of cells
transfected with these constructs showed that all of these VSV-tagged
proteins were strongly enriched in the nucleoli (Fig.
4). In addition, a weaker diffuse
staining was widely distributed in the nucleoplasm and cytoplasm.
Indirect immunofluorescence using the rabbit antisera H70 and H73 also
resulted in prominent staining of the nucleoli of HEp-2 cells. Rabbit
antiserum H71 did not give signals in indirect immunofluorescence (data
not shown).
We conclude that hRrp40p, hRrp41p, and hRrp46p are present in both the
cytoplasm and nucleus, with the highest concentration in the nucleolus.
hRrp40p, hRrp41p, and hRrp46p Are Part of the PM/Scl
Complex--
The previously identified components of the human exosome
(hRrp4p, PM/Scl-75, and PM/Scl-100) cosedimented in a large complex on
glycerol gradient centrifugation of HeLa cell lysates (11). To
determine whether hRrp40p, hRrp41p, and hRrp46p are associated with
complexes of similar size, HeLa cell extracts were fractionated by size
exclusion chromatography.
Cytoplasmic and nuclear extracts were prepared from 75 × 106 cells and separately fractionated by chromatography on
a Superdex 200 column. For each extract, 30 fractions were collected
and analyzed by Western blotting with rabbit antisera H70, H71, H73, anti-hRrp4p, and anti-PM/Scl-100 (Fig.
5). These analyses revealed that hRrp40p,
hRrp41p, and hRrp46p are associated with relatively large complexes in
the cytoplasmic (Fig. 5A) and nuclear (Fig. 5B)
extracts. All three proteins cofractionated with PM/Scl-100 and hRrp4p
in both the cytoplasmic and nuclear extracts.
To estimate the size of the complexes, gel filtration standards were
fractionated using the same conditions. In the cytoplasmic extract, the
exosome components peaked in fractions three and four corresponding to
a molecular mass of ~700 kDa. In the nuclear extract, all exosome
proteins analyzed except PM/Scl-100 showed a broader distribution over
fractions 3-11, corresponding to estimated molecular masses of
~250-700 kDa. The peak of PM/Scl-100 was limited to fractions 3 and
4, suggesting association with only the higher molecular weight
complexes. Similar results were obtained following fractionation of
nuclear extracts by glycerol gradient centrifugation, which showed
cosedimentation of hRrp40p and hRrp41p with hRrp4p and the PM/Scl
autoantigens (data not shown).
Coimmunoprecipitation experiments were performed to confirm the
physical association of hRrp40p, hRrp41p, and hRrp46p with the known
human exosome components. Five anti-PM/Scl-positive patient sera were
used to immunoprecipitate the exosome complex from a HeLa cell nuclear
extract; three anti-PM/Scl-negative sera served as controls (Fig.
6A). The immunoprecipitates
were analyzed by Western blotting using the rabbit antisera H70, H71,
and H73. hRrp40, hRrp41, and hRrp46 were each immunoprecipitated by the anti-PM/Scl-positive sera, but not by the control sera. Although the
rabbit antisera showed reactivity with several proteins present in the
total nuclear extract (Fig. 6A, lanes
i), each rabbit antiserum consistently stained only one
protein in the immunoprecipitates. In each case the gel mobility
corresponded with the predicted molecular weight, confirming that these
represent the cognate proteins.
In the converse experiments, sera H70, H71, and H73 were used for
immunoprecipitation and the coprecipitation of PM/Scl autoantigens was
detected with two anti-PM/Scl patient sera, Lun7 and Lun36. Sera H70
and H73 each coimmunoprecipitated PM/Scl-100 from a nuclear HeLa cell
extract (Fig. 6B, right panel). In
addition, both patient sera stained several smaller proteins that were
immunoprecipitated by H70 and H73 from the nuclear extract, which may
represent other components of the PM/Scl particle. Neither pre-immune
sera (PI lanes) precipitated the PM/Scl-100
autoantigen or any of the smaller proteins. PM/Scl-100 was also
coimmunoprecipitated at low levels from a cytoplasmic extract by sera
H70 and H73 (Fig. 6B, left panels).
Serum H71 failed to precipitate PM/Scl-100 (Fig. 6B) and
also failed to decorate the nucleoli of HEp-2 cells in
immunofluorescence microscopy (data not shown). This may be due either
to inaccessibility of hRrp41p in the complex or to the inability of the
antibodies to recognize native hRrp41p.
We conclude that hRrp40p, hRrp41p, and hRrp46p are present in a complex
containing the known human exosome components, PM/Scl-100 and hRrp4p.
Exoribonuclease Activity of the Complexes Containing hRrp40p,
hRrp41p, and hRrp46p--
The human exosome complex was
immunoprecipitated from a HeLa cell extract using rabbit antisera H70,
H71, and H73 or anti-PM/Scl-positive patient serum R212. The pre-immune
rabbit sera and a pool of 10 normal human sera served as negative
controls. Associated exoribonuclease activity was assayed in
vitro with an internally labeled 37-nucleotide single-stranded RNA
substrate. The immunoprecipitates obtained with rabbit antisera H70 and
H73 and patient serum R212 exhibited ribonuclease activity, with
progressive disappearance of the RNA substrate and the accumulation of
the labeled end-product (Fig. 7,
left panel, lanes 2-4 and
8-13). Analysis of the reaction products by thin layer
chromatography revealed that the accumulating end products are
nucleoside monophosphates consistent with exonucleolytic degradation of
the substrate (data not shown). Omission of phosphate from the buffer
used in this assay, which has been shown to affect the activity of
yeast Rrp41p/Ski6p (12), did not significantly inhibit the reaction
(data not shown). Immunoprecipitates obtained with rabbit antiserum H71
(Fig. 7, left panel, lanes
5-7) and the control sera (Fig. 7, right
panel) did not show nucleolytic activities. The similar
patterns of reaction products seen with H70 and H73 indicate that the
complexes immunoprecipitated by each serum are related.
When the substrate was 3'-labeled with 32P, incubation with
the immunoprecipitates obtained with sera H70, H73, or R212 resulted in
the disappearance of the RNA substrate without detectable intermediate products. We conclude that the immunoprecipitated complexes exhibit 3'
These results show that hRrp40 and hRrp46 are associated with a complex
displaying 3' We have characterized three novel human polypeptides, hRrp40p,
hRrp41p, and hRrp46p, encoded by cDNAs that were isolated on the
basis of homology to the yeast exosome components Rrp40p, Rrp41p/Ski6p,
and Rrp46p, respectively (11). Western blotting experiments using
rabbit antisera raised against each of the recombinant proteins showed
that HeLa cells express proteins of the predicted molecular weights.
Consistent with data previously obtained for human exosome components,
all three proteins are present in both the cytoplasm and nucleus, with
nucleolar enrichment. The novel proteins were shown to be part of a
large complex cofractionating with hRrp4p and PM/Scl-100. The physical
association with the PM/Scl autoantigens was confirmed by
coimmunoprecipitation. Functional assays demonstrated that the
complexes containing hRrp40p and hRrp46p display 3' Subcellular Localization of hRrp40p, hRrp41p and
hRrp46p--
Consistent with previous data using anti-PM/Scl positive
patient sera, hRrp40p, hRrp41p, and hRrp46p were shown by indirect immunofluorescence to be enriched in the nucleolus. However,
subcellular fractionation showed that hRrp40p, hRrp41p, and hRrp46p are
present in both nuclear and cytoplasmic fractions, as is hRrp4p (11). Salt extraction released most of the nuclear hRrp40p, hRrp41p, and
hRrp46p, but a substantial amount of each protein was retained. The
release of DNA topoisomerase I indicated that the high salt extraction
was efficient. We speculate that the extracted and retained fractions
represent nucleoplasmic and nucleolar pools of the exosome,
respectively. Diffusely distributed nucleoplasmic and cytoplasmic
populations are presumably less visible in immunofluorescence than is
the nucleolar population.
The distribution of the human exosome components is similar to that
previously seen for yeast Rrp4p and Rrp43p (11, 40). The yeast exosome
is implicated in RNA processing reactions in the nucleolus (pre-rRNA
processing and spacer degradation), nucleoplasm (pre-small nucleolar
RNA and pre-small nuclear RNA processing and pre-mRNA degradation)
and cytoplasm (mRNA degradation) (12-23), and the distribution
observed for exosome components presumably reflects these functions.
The similarities in the distribution patterns, and the complementation
of yeast mutants by the human proteins, make it likely that the human
exosome will carry out many or all of the same functions.
The yeast exosome is implicated in the degradation of unspliced
pre-mRNAs (18, 22) and might therefore influence the outcome of
alternative splicing events. The identification of cDNAs that apparently result from alternative splicing of hRRP40 and
hRRP46 therefore raises interesting possibilities for
autogenous regulation.
Characterization of the Complex Containing hRrp40p, hRrp41p, and
hRrp46p--
Immunoprecipitation with anti-PM/Scl-positive patient
sera, using metabolically labeled human cell extracts, indicated that the PM/Scl complex consists of at least 11 proteins (25, 26). The
estimated molecular masses of the proteins found in these studies were
110, 90, 80, 39, 37, 33, 30, 27, 26, 22, and 20 kDa. Comparison of
these molecular masses with those of the proteins characterized in the
present study suggested that hRrp40, hRrp41 and hRrp46 correspond to
the 30-, 27-, and 26-kDa proteins, respectively.
Size exclusion chromatography of the exosome complex in a HeLa cell
extract, indicated that the cytoplasmic complex has a molecular mass of
~700 kDa. The nuclear complex gave a broad distribution between 250 and 700 kDa. These complex sizes were estimated based upon the
separation of gel filtration standards. However, this fractionation may
not be dependent solely on the mass of the complex, but may also
reflect its structure relative to the protein size markers. It is also
unclear whether the size distribution of the nuclear exosome reflects
the existence of multiple, heterogeneous complexes or whether it is due
to instability of the complex. The size of the human exosome complex
estimated by size exclusion chromatography was larger than previously
estimated from glycerol gradient centrifugation (12), presumably
reflecting differences in the physical basis of the separation techniques.
The complexes immunoprecipitated with anti-hRrp40p (H70) or
anti-hRrp46p (H73) showed very similar in vitro activities.
In each case, both processive and distributive activities are suggested by the data. A distributive exonuclease activity removes one (or a few)
nucleotide(s) before dissociating from the substrate. In consequence,
its activity is "distributed" over the entire substrate population,
which is therefore progressively shortened in approximate synchrony.
Such an activity would be consistent with the shortening of the RNA
population near the top of the gel in the H70 and, particularly, H73
lanes in Fig. 7 (lanes 2-4 and 8-10,
respectively). In contrast, binding of a processive exonuclease to the
a molecule of substrate results in its rapid degradation to a short
residual fragment, at which point the RNA is too short for the enzyme
to bind. In consequence, a fraction of the substrate is rapidly
shortened, while most of the substrate in untouched. Such an activity
may be seen in the early time points of using the H70 and H73
immunoprecipitates shown in Fig. 7.
5'
exoribonucleases. Sequence analysis identified putative human
homologues for exosome components, although several were found only as
expressed sequence tags. Here we report the cloning of full-length
cDNAs, which encode putative human homologues of the Rrp40p,
Rrp41p, and Rrp46p components of the exosome. Recombinant proteins were
expressed and used to raise rabbit antisera. In Western blotting
experiments, these decorated HeLa cell proteins of the predicted sizes.
All three human proteins were enriched in the HeLa cells nucleus and
nucleolus, but were also clearly detected in the cytoplasm. Size
exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p
were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demon- strating their physical association. The immunoprecipitated complex exhibited 3'
5' exoribonuclease activity in vitro. hRrp41p was expressed in yeast and shown
to suppress the lethality of genetic depletion of yeast Rrp41p. We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel
components of the human exosome complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' exonuclease polynucleotide phosphorylase, the DEAD box RNA helicase RhlB, and several additional proteins whose role is unclear (5-7). Related complexes are implicated in RNA processing in chloroplasts and mitochondria (8-10). The yeast
exosome contains at least 11 components, which are known or predicted
to be 3'
5' exoribonucleases (11, 12). Ten of these (Rrp4p,
Rrp40-46p, Mtr3p, and Csl4p) have been demonstrated to be encoded by
essential genes. These 10 components were found in both cytoplasmic and
nuclear complexes, whereas the nonessential RRP6 gene
product was detected only in the nucleus (11, 13).
5' pathway of mRNA degradation (22). The activity of the exosome complex may be regulated by cofactors
including, for example, the putative ATP-dependent DEVH box
RNA helicases Dob1p and Ski2p (23, 24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
LacZ library primers. The largest PCR products were identified by Southern blotting and cloned into the pCRII-TOPO vector (Invitrogen). Sequence analysis revealed putative start codons for both hRRP40 and
hRRP46. The cDNAs isolated from the teratocarcinoma
cDNA library were ligated to the coding sequence present in the EST
clones. To further investigate the 5' ends of the corresponding
mRNAs, a 5' RACE analysis was performed using total human placenta
RNA (Smart RACE cDNA amplification kit,
CLONTECH). Gene specific primers used for the 5'
RACE analysis were hRrp40-b, hRrp40-c, hRrp41-b, hRrp41-c, hRrp46-b,
and hRrp46-c. Finally, primers (
5S and
3AS) were designed to
introduce restriction sites at the termini of the isolated cDNAs to
allow cloning into appropriate expression vectors (see below). The
sequences of the oligonucleotides used in the cloning of
hRRP40, hRRP41, and hRRP46 are listed
in Table I.
Sequences of gene-specific oligonucleotides used in the cloning of
hRrp40p, hRrp41p, and hRrp46p
Yeast strains used in this study
20 °C, briefly rinsed in acetone, air-dried, and stored at
20 °C until use.
70 °C, pellets were collected by
centrifugation at 13,000 × g for 30 min. Air-dried pellets were solubilized in SDS sample buffer, and proteins were analyzed by Western blotting (20% of each fraction). To estimate complex sizes, gel filtration standards (Bio-Rad), including
thyroglobin (670 kDa), bovine IgG (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B12 (1.35 kDa), were analyzed as described above.
) template
(Stratagene), were labeled randomly using [32P]UTP
(Amersham Pharmacia Biotech) or at the 3' end using
[32P]pCp (Amersham Pharmacia Biotech), respectively
(38).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the cloning
strategy used to generate the cDNAs encoding hRrp40p, hRrp41p, and
hRrp46p. Sequencing of human EST clones, which were selected based
upon homology with yeast Rrp40p, Rrp41p/Ski6p, and Rrp46p proteins,
resulted in sequence information represented by the cDNA clones.
Additional 5' RACE analyses and PCR analyses on cDNA libraries
complemented the cDNA clones with additional 5' end
sequences.
23 to +150 of the isolated
hRRP46 cDNA clone. A T/C polymorphism is evident in these
sequences, resulting in an amino acid substitution (methionine/threonine) at position 5 of hRrp46p. Both types of cDNA
were obtained from the 5' RACE analyses, and ESTs exist with each
nucleotide. In the studies described below, the cDNA clone encoding
threonine at position 5 was used. Polymorphism was also evident in the
region 5' to the ORF, with an in frame upstream stop codon present in
L08634, but not in the chromosome 19 sequence. In the latter, the ORF
could potentially extend 33 amino acids further 5'. However, no
cDNA that extends beyond 23 nucleotides of the 5'-untranslated
region was isolated and the extended ORF sequence would be in poorer
agreement with the molecular weight predicted from the observed gel
migration of the protein from HeLa cell lysates, compared with that
synthesized in E. coli. Therefore, we believe that the
cDNA used encodes the full-length protein. An EST with an internal
truncation in the hRrp46p ORF was also found, presumably as a
consequence of alternative splicing, that leads to a frameshift at
Gly-85 and truncation of the polypeptide.
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Fig. 2.
Complementation of growth defects in yeast
strain depleted of Rrp40p, Rrp41p/Ski6p, or Rrp46p by the human
homologues. Figure shows plate assay for growth of
GAL::Rrp40, GAL::Rrp41, and GAL::Rrp46
transformed with phRrp40p, phRrp41p, and phRrp46p, respectively, on
repressive YPD medium. WT, wild-type.
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Fig. 3.
Western blot analyses of fractionated HeLa
cells to determine the subcellular distribution of exosome
components. HeLa cells were lysed by hypotonic Dounce homogenizer,
nuclei were extracted with 0.3 M NaCl, and the retained
nuclear material was recovered after sonification of the salt-extracted
nuclei. Western blots containing total (T), cytoplasmic
(C), nuclear (N), and retained nuclear
(R) HeLa cell fractions, each obtained from an equal number
of cells, were probed with rabbit antisera reactive with hRrp40p
(panel A), hRrp41p (panel
B), hRrp46p (panel C), hRrp4p
(panel D), or PM/Scl-100 (panel
E). Western blots were also decorated with autoimmune
patient sera reactive with histidyl-tRNA synthetase (Jo-1,
panel F), with DNA topoisomerase I
(panel G), or with monoclonal antibodies reactive
with lamin B (panel H). The relevant proteins are
indicated by arrowheads. Western blots in panels
A-C also contain the corresponding recombinant His-tagged
proteins (Ag lanes).
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Fig. 4.
Nucleolar accumulation of the VSV-tagged
hRrp40p, hRrp41p, and hRrp46p in HEp-2 cells. HEp-2 cells were
transfected with VSV-tagged constructs by electroporation and cultured
for 24 h at 37 °C. The localization of C-terminally VSV-tagged
hRrp40p (A), hRrp41p (B), and hRrp46p
(C) was determined by indirect immunofluorescence using a
monoclonal anti-VSV tag antibody (left panels).
The corresponding phase-contrast images are shown on the
right.
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Fig. 5.
hRrp40p, hRrp41p, and hRrp46p are associated
with high molecular weight complexes in HeLa cells. A,
cytoplasmic extract. B, salt-extractable nuclear extract.
Lysates (from an equal number of cells) were fractionated by size
exclusion chromatography and fractions 2-27 were analyzed by Western
blotting using rabbit antisera specific for hRrp40p (I),
hRrp41p (II), hRrp46p (III), hRrp4p
(IV), and PM/Scl-100 (V). In the left
lanes (i), total cytoplasmic or nuclear extract
was loaded. Note that serum H73 is also reactive with a cytoplasmic
polypeptide that migrates slightly faster than hRrp46p (see also Fig.
3C), but fractionated only in the lower molecular weight
fractions (panel A, III, indicated by
*).
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Fig. 6.
hRrp40p, hRrp41p, and hRrp46p are physically
associated with the human exosome. A, coimmunoprecipitation
experiments were performed using five anti-PM/Scl-positive patient sera
(lanes 1-5) and three anti-PM/Scl-negative
patient sera (lanes 6-8). Immunoprecipitated
material was analyzed by Western blotting using the rabbit antisera
raised against hRrp40p (H70), hRrp41p (H71), or hRrp46p (H73) to detect
coimmunoprecipitation of these proteins. In lanes
i, total nuclear extract (30%) used for immunoprecipitation
was loaded. B, Western blot analyses of the converse
experiment in which the preimmune (PI, lanes
2, 4, 6, 10, 12,
and 14) and immune (I, lanes
3, 5, 7, 11, 13,
and 15) rabbit antisera were used for the
immunoprecipitations and two anti-PM/Scl-positive patient sera, Lun7
(lower panels) and Lun36 (upper
panels), were used for detection. Cytoplasmic
(lanes 1-8) and salt-extractable nuclear
(lanes 9-16) HeLa cell extracts were used for
immunoprecipitations. Total cytoplasmic and nuclear extract was loaded
in lanes i (lanes 1 and
8 (cytoplasmic) and lanes 9 and
16 (nuclear)). Arrowheads indicate the relevant
proteins.
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Fig. 7.
hRrp40p- and hRrp46p-containing complexes
display ribonuclease activity in vitro.
Immunoprecipitated hRrp40p, hRrp41p, hRrp46p, and PM/Scl antigens
containing complexes from HeLa cells were assayed for exoribonuclease
activity in vitro. A uniformly 32P-labeled RNA
substrate was incubated with the immunoprecipitates of anti-hRrp40p
(H70), anti-hRrp41p (H71), and anti-hRrp46p (H73) rabbit sera or an
anti-PM/Scl-positive patient serum (R212). Samples taken after 10, 20, and 30 min of incubation at 37 °C were analyzed by 10% denaturing
polyacrylamide gel electrophoresis followed by autoradiography
(left panel). Control experiments
(right panel) were performed using the
immunoprecipitates of the corresponding preimmune rabbit antisera
(PI-H70, PI-H71, and PI-H73) or a pool of ten normal human sera
(NHS). In the left lanes
(i), input RNA substrate was loaded.
5' exonuclease activity. Removal of the 3' phosphate group, inherently introduced by the [32P]pCp labeling procedure,
resulted in a slightly enhanced reaction (data not shown) indicating a
preference for a 3'-hydroxyl residue, as seen for the yeast exosome
(12).
5' exoribonuclease activity in vitro. We
conclude that these proteins are indeed novel human exosome components.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5'
exoribonuclease activity in vitro. Moreover, expression of
hRrp41p in yeast is able to support the growth of cells depleted of
yeast Rrp41p/Ski6p. We conclude that hRrp40p, hRrp41p, and hRrp46p are
novel components of the human exosome complex.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. I. Lundberg for providing patient sera and Dr. E. Hurt for providing pNOPPATA1L. We also thank J. Koenderink and J. Vogelzangs for their contribution to the baculovirus expression.
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FOOTNOTES |
---|
* This work was supported in part by the Netherlands Foundation for Chemical Research with financial aid from the Netherlands Organization for Scientific Research, the Netherlands Technology Foundation, and the Wellcome Trust.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF281132-AF281134.
¶ To whom correspondence should be addressed. Tel.: 31-24-3616847; Fax: 31-24-3540525; E-mail: g.pruijn@bioch.kun.nl.
Published, JBC Papers in Press, November 10, 2000, DOI 10.1074/jbc.M007603200
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ABBREVIATIONS |
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The abbreviations used are: PM/Scl, polymyositis/scleroderma; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; VSV, vesicular stomatitis virus G epitope; EST, expressed sequence tag; GST, glutathione S-transferase; ORF, open reading frame; RACE, rapid amplification of cDNA ends; contig, group of overlapping clones.
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REFERENCES |
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