ABSTRACT
During the analysis of the La (SS-B) autoantigen for catalytic activities an ATP-dependent double-stranded RNA unwinding activity was detected. Both native and
recombinant La proteins from different species displayed this activity, which
could be inhibited by monospecific anti-La antibodies. La protein was able to melt dsRNA substrates with either
two 3
'
-overhangs or a single 3
'
- and a 5
'
-overhang. Double-stranded RNAs with two 5
'
-overhangs were not unwound, indicating that at least one 3
'
-overhang is required for unwinding. Sequence elements of the La protein
that might be involved in dsRNA unwinding, such as an evolutionarily conserved
putative ATP-binding motif and an element that is homologous to the double-stranded RNA binding protein kinase PKR, are discussed.
Sera from patients with autoimmune diseases such as systemic lupus erythematosus
or primary Sjögren's syndrome frequently develop autoantibodies to RNA- or DNA-binding proteins (
1
). One of the targets of such autoantibodies is the so-called La (SS-B) protein. The La protein can be found associated with all primary
RNA polymerase III transcripts including precursor molecules of ribosomal 5S
RNA, the tRNAs, some 7S RNAs, as well as a portion of the U1 and U6 snRNA (
2
-
4
). Common to all primary RNA polymerase III transcripts is the 3'-terminal oligo(U)-tail, which is transcribed during the transcription
termination step. These oligo(U)-tails were shown to be a binding site for the La protein (
5
). In most cases the binding of the La protein to the primary RNA polymerase III
transcripts is only transient, as their respective oligo(U)-tails are removed after transcription during an unknown processing step.
However, some small cytoplasmic La RNAs retain their oligo(U)-tails even after transport to the cytoplasm (
6
). In addition to the oligo-uridylated RNA polymerase III transcripts an association of the La protein
with some non-oligouridylated RNAs has been reported especially for some viral RNAs
including the leader RNAs of vesicular stomatitis virus and rabies virus (
6
,
7
).
Recently, the La protein was shown to be involved in internal initiation of
translation of poliovirus mRNA and to alleviate translational repression by the
TAR element in HIV-1 mRNA (reviewed in ref.
8
). As a hypothetical mode of action a dsRNA unwinding mechanism has been
suggested (
9
). In a previous report it was described that the La protein is able to melt DNA-RNA hybrids (
10
). This activity could explain how the La protein might release the primary RNA
polymerase III transcripts from their DNA template (
10
-
12
). During these experiments an activation of the La protein-associated ATPase activity was not only observed for DNA-RNA hybrids but also for poly(I)[middot]poly(C), an observation which was confirmed by a recent
report of Xiao and co-workers (
13
) who have shown that La is able to unwind dsRNA to single-stranded forms. In this report we extend these observations and show that
La is an ATP-dependent helicase that unwinds dsRNA substrates with either two 3'-overhangs or a single 3'- and 5'-overhang.
Isopropyl [beta]-d-thiogalactopyranoside (IPTG), anti-human IgG ([gamma]-chain specific) conjugated with alkaline
phosphatase (A3150; 8 U/ml), anti-mouse IgG developed in sheep [(Fab')
2
fragments] adsorbed with human serum proteins (A-0532) were obtained from Sigma (St Louis, MA, USA). The Enhanced
Chemiluminescence (ECL)-Western blotting detection reagents were from Amersham-Buchler (Braunschweig, Germany). 4-Nitro-blue-tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate
(BCIP) from Roth (Karlsruhe, Germany). [
32
P]UTP (specific radioactivity 3000 Ci/mmol) from Dupont (Dreieich, Germany).
Dinatrium 3-(4-methoxyspiro {1,2-dioxetan-3,2'- (5'-chloro) tricyclo[3.3.1.1.
3,7
] decan}-4-yl) phenyl phosphate (CSPD) was obtained from Tropix (Bedford, MA,
USA). pBluescript II SK(-) and KS(-) were obtained from Stratagene (Heidelberg, Germany). pGEM-4Z was from Promega (Heidelberg, Germany). T7-, SP6- and T3 RNA polymerase, pSPT 19 and various
restriction enzymes were obtained from Boehringer Mannheim, (Mannheim,
Germany).
Monospecific anti-La antibodies were isolated by affinity- purification from serum W10 (
14
) using recombinant human La protein covalently linked to Sepharose 4B. Elution
of the bound antibodies was performed as described previously (
10
,
15
-
17
). The anti-La mAb La4B6 was raised against recombinant human La protein and
recognizes the sequence SKGRRFKGKGKGN in the C-terminal part of the La protein (
18
).
For the preparation of total cell extracts, monolayer human XPTA cells and mouse
3T3 fibroblasts (ATCC CCL 92) were grown to confluency in a culture flask. The
cells were washed and 500 [mu]l hot SDS-PAGE sample solution (0.1 M Na
2
HPO
4
, pH 8.3; 20% [w/v] sucrose, 1% SDS, 1 mM PMSF) was added per 200 ml flask. The
lysed cells were heated in an Eppendorf tube for 5 min at 95oC and the resulting protein extract was centrifuged for 15 min at 11 000
g
and frozen at -80oC until use.
The La protein was biochemically purified as described (
16
,
19
) using heparin- and poly(U)-Sepharose chromatography followed by immunoaffinity chromatography
on anti-La Sepharose. The native rat La proteins were isolated from either total
rat liver extracts (female Brown Norway) or from extracts obtained by
solubilisation of isolated rat liver nuclei or nuclear envelopes with Triton X-100 (
20
). The nuclei and nuclear envelopes were prepared by the following procedure
(all steps at 4oC, unless stated otherwise). Rat liver was minced with a pair of scissors
and homogenized in 4 vol 50 mM Tris-HCl, pH 7.4, 5 mM MgSO
4
, 250 mM sucrose, 1 mM PMSF. The homogenate was filtrated through four layers of
cheese cloth. After centrifugation at 800
g
for 10 min, the pellet was washed with the same buffer and resuspended in 50 mM
Tris-HCl, pH 7.4, 5 mM MgSO
4
, 2.1 M sucrose, 1 mM PMSF. After centrifugation for 60 min at 70 000
g
, the pelleted nuclei were resuspended and applied to a 5 ml sucrose cushion.
Nuclei were obtained by centrifugation for 30 min at 70 000
g
. The pelleted nuclei were resuspended in 50 mM Tris-HCl, pH 7.4, 5 mM MgSO
4
, 250 mM sucrose, 1 mM PMSF at a concentration of ~5 * 10
8
nuclei/ml. For preparation of nuclear envelopes the nuclei were incubated with
250 [mu]g/ml DNase I for 60 min at 30oC, after which the nuclei were collected again by centrifugation (800
g
, 10 min). Nuclei were resuspended in 10 mM Tris-HCl, pH 7.4, 0.2 mM MgSO
4
, 1 mM PMSF and subsequently 4 vol 2 M NaCl in 10 mM Tris-HCl, pH 7.4, 0.2 mM MgSO
4
, 1 mM PMSF were added. 2-Mercaptoethanol was added to a final concentration of 1% and after an
incubation for 15 min the nuclear envelopes were collected by centrifugation
(1600
g
, 30 min). Then the NaCl extraction step was repeated without the addition of 2-mercaptoethanol. Finally, the nuclear envelopes were resuspended in 50 mM
Tris-HCl, pH 7.4, 5 mM MgSO
4
, 5 mM 2-mercaptoethanol, 2.1 M sucrose, 1 mM PMSF and stored at -70oC. Nuclei and nuclear envelopes were extracted with Triton X-100 as follows. Pelleted nuclei were resuspended in 25
mM Tris-HCl, pH 7.6, 25 mM KCl, 2.0 mM MgCl
2
, 0.5 mM CaCl
2
, 0.3 mM MnCl
2
, 5.0 mM spermidine-HCl, 250 mM glucose, 5.0 mM 2-mercaptoethanol and Triton X-100 was added to a concentration of 1%. After an incubation
for 10 min the demembranated nuclei were removed by centrifugation (1600
g
, 10 min). Pelleted nuclear envelopes were resuspended in 50 mM Tris-HCl, pH 7.4, 5 mM MgSO
4
, 250 mM sucrose, 1 mM PMSF and were extracted with 2% Triton X-100 for 10 min. The pore complex laminae were removed by centrifugation
(15 000
g
, 20 min).
Two different recombinant human La protein preparations, termed wt1 and wt2,
were used (
21
,
22
). Preparation wt1 was isolated by two ion-exchange chromatographic steps followed by an immunoaffinity
chromatographic step from a bacterial lysate of
Escherichia coli
expressing a La cDNA described previously (
23
,
24
). The recombinant human La preparation wt2 and also the recombinant rat La
protein were isolated via a single immunoaffinity chromatographic step from
bacterial extracts, which were obtained from bacteria expressing either the
human (EMBL accession no. X69804) or rat (EMBL accession no. X67859) La cDNAs (
21
). Bound La protein was eluted as described (
19
). If not stated otherwise La preparation wt2 was used.
SDS-PAGE and immunoblotting were performed as described (
25
). The blocked membrane was incubated with cell culture supernatant of hybridoma
cells secreting the anti-La mAb La4B6 (
18
). Immune complexes were detected with anti-mouse peroxidase conjugate using the ECL-Western blotting detection reagents. After elution of the mouse
immune complexes (
10
) the blot was incubated with monospecific anti-La antibodies isolated from patient serum W10. The immune complexes were
visualized with anti-human antibodies covalently linked to alkaline phosphatase using BCIP/NBT
as substrate.
Plasmid DNAs were obtained from either pSPT 19, pBluescript II SK(-) and KS(-), or pGEM-4Z. Plasmid DNA was prepared according to a Qiagen midiprep
protocol from Diagen (Hilden, Germany) and substrate DNAs were made as follows.
In the case of the substrates S1 and S2, pSPT 19 DNA was cut with either
Pvu
II or
Nar
I, in the case of S3 with either
Hin
dIII or
Nar
I, in the case of S4 with either
Pvu
II or
Eco
RI and in the case of S5 with either
Hin
dIII or
Eco
RI. The substrate S6 was prepared from pBluescript II SK(-) by cutting with either
Xho
I or
Not
I. In the case of the substrate S7 the unlabeled strand was prepared from
pBluescript II KS(-) after cutting with
Sac
I, the labeled strand was prepared from SK(-) after cutting with
Kpn
I. The substrate S8 was transcribed from pGEM-4Z cut with
Pvu
II. The resulting linear DNAs were transcribed
in vitro
by either SP6-, T7- or T3 RNA polymerase.
In vitro
transcription was performed according to the established protocols using 1.5 [mu]g of the respective plasmid DNA in a final volume of 20 [mu]l. In order to label a transcript
in vitro
transcription was performed in the presence of 50 [mu]Ci [[alpha]-
32
P]UTP.
Substrates were prepared by annealing two complementary strands (a 2-3-fold molar excess of the unlabeled strand) in hybridization buffer
[0.4 M NaCl, 1 mM EDTA, 40 mM PIPES, pH 6.4 and 80%(v/v) formamide] as
described (
26
).
The unwinding of the dsRNA substrate was assayed in a final volume of 20 [mu]l containing 0.5-1.2 [mu]l substrate and 0.1-5 [mu]g La protein in a reaction buffer composed of 30 mM
Tris-HCl, pH 7.5, 8 mM MgCl
2
, 1.5 mM DTT, 30 [mu]g/ml BSA, 0.5 U/[mu]l RNasin and 2 mM ATP unless stated otherwise. The reaction mixture was
incubated for 15 min at 37oC. The reaction was stopped by the addition of SDS and EDTA to final
concentrations of 0.3% and 15 mM respectively.
After unwinding RNAs were separated by SDS-PAGE using 10 or 11.5% gels lacking a stacking gel and the reaction
products were visualized by autoradiography.
During the last few years assay systems have been developed to test for
unwinding activity of putative helicases (
26
,
27
). In such an assay we have analyzed the unwinding of dsRNA substrates
consisting of a hybrid of a radioactively labeled strand and an unlabeled
complementary strand, both transcribed
in vitro
from a variety of plasmids (see Materials and Methods). Unwinding of such a
(partially) double-stranded RNA substrate can be detected because the released labeled strand
will usually migrate faster than the dsRNA in an SDS-polyacrylamide gel.
The dsRNA substrate S1 consists of an unlabeled ssRNA molecule of 357
nucleotides (nt) and a labeled ssRNA of 107 nt, which, after hybridization,
form a substrate with 3'-overhangs of 285 and 35 nt respectively, and a dsRNA region of 72
base pairs (bp) (A in Fig.
1
To confirm that the dsRNA unwinding activity was an inherent activity of the La
protein a monospecific polyclonal anti-La antibody preparation was added to the unwinding assay. As shown in
Figure
3
The results described above indicated that also the rat La protein displayed
dsRNA unwinding activity. To substantiate these data and to investigate whether
the unwinding activity indeed represented an endogenous activity of the rat La
protein, the protein was extensively purified from rat liver. Rat liver extract
was fractionated by ammonium sulphate precipitation, Sephadex G-150 gel filtration, depletion by anti-Ro antibodies (to remove Ro RNPs which are known to be stably
associated with a subset of La molecules) and finally heparin chromatography.
The La containing fractions of the heparin column were further fractionated by
poly(U)-Sepharose chromatography followed by anti-La immunoaffinity chromatography. The presence of the La protein in
all fractions was assayed by Western blotting using monoclonal and patient anti-La antibodies. When La containing fractions from the last three columns
were analysed in the dsRNA unwinding assay, all were able to unwind dsRNA (Fig.
4
, lanes b-g). Also La protein isolated by immunoaffinity chromatography from lysed
rat liver nuclei or nuclear envelopes was able to unwind substrate S1 (Fig.
4
, lanes h-k).
As depicted before substrate S1 was composed of a double-stranded region of 72 bp bordered by two 3'-overhangs. When the 357 nt strand was radiolabeled (substrate
S2) instead of the shorter 107 nt strand as in S1, unwinding could not be
assessed due to the fact that the relatively long radiolabeled strand
comigrated with the substrate in the gel (results not shown).
To learn more about the substrate specificity of unwinding by the La protein, a
number of other substrates were prepared. Substrate S3 consisted of a hybrid
between an unlabeled ssRNA of 357 nt and a labeled ssRNA of 61 nt, which was
completely complementary to a region of the longer strand. This resulted in a
substrate containing a double-stranded region of 61 bp and both a 3'- and a 5'-overhang (Fig.
5
In this report we describe an intrinsic enzymatic property of the La protein,
namely an ATP-dependent dsRNA unwinding activity. We further present evidence that only
dsRNA substrates with a 3'-overhang are unwound.
It is obvious that these results are only of interest when it can be shown
convincingly that this unwinding activity is displayed by the La protein and
not due to some minor contamination of the La preparation used. The following
arguments support the notion that dsRNA unwinding is indeed exerted by the La
protein.
(i) The unwinding activity was displayed by recombinant (human, rat) and native
(rat liver) La protein purified from different organisms (
E.coli
, rat). Various purification protocols including ammonium sulphate precipitation
followed by gel filtration, heparin chromatography and various affinity
chromatography steps were unable to separate or remove the unwinding activity
from the La protein. Although SDS-PAGE analyses of the various preparations (not shown) revealed almost
identical protein profiles [full length La protein and its well-known degradation products (
6
,
7
,
18
)] such protein staining patterns can of course not exclude the possibility that
some minor contamination of
E.coli
or rat liver proteins is present.
(ii) Affinity-purified patient anti-La antibodies are able to inhibit the unwinding activity. This
result further corroborates our conclusion that the unwinding activity is an
intrinsic property of the La protein, because it is very unlikely that the
antibodies would also bind to a possible contamination.
(iii) Some mutant La proteins are unable to unwind the dsRNA substrate
(unpublished observations). These mutants were purified from
E.coli
in the same way as the active wt La proteins, and as a consequence would be
expected to contain the same set of contaminating proteins.
(iv) Purified and active La preparations are able to unwind dsRNAs with a 3'-overhang to completion but are unable to unwind similar substrates
with merely 5'-overhangs.
Taken together we feel confident to conclude that the La protein is not only
able to melt DNA-RNA hybrids, as has been reported before (
10
), but is also an ATP-dependent dsRNA unwinding enzyme. While this work was in progress dsRNA
unwinding by La has also been reported in relation with the ability of La to
inhibit activation of the dsRNA dependent protein kinase PKR (
13
).
Presently, the mechanism by which La unwinds dsRNA is unknown. The observation
that a 3'-overhang is required might suggest that unwinding is initiated at
the border of double-stranded and single-stranded RNA in the substrate. In the
in vitro
unwinding assay the concentration of recombinant La was in the micromolar range
and the concentration of substrate RNA in the nanomolar range implying that the
single-stranded RNA binding activity of La may act to change the equilibrium
between dsRNA and its single-stranded components. Although we cannot exclude yet that La acts in a
stoichiometric rather than a catalytic fashion, preliminary unwinding data
obtained with mutants of La suggest that in addition to the RNA binding domain
at the N-terminus of La also elements in the C-terminal part are required for unwinding (unpublished observations).
In addition, it is not known which percentage of the recombinant La protein
molecules is biologically active and thus the effective concentration of
recombinant La in the unwinding assay might be much lower than the total
concentration of La protein. Based upon these considerations and the fact that
ATP is required for unwinding, we favour a mechanism in which La acts in a
catalytic fashion, but additional experiments are required to confirm this.
During the last few years a rapidly growing protein family of putative RNA
helicases with the common amino acid motif DEAD or DEXH have been described (
29
). Sequence comparisons of these putative unwinding enzymes resulted in the
identification of up to eight common elements (
27
,
29
) including the so-called A or Walker motif, representing the ATP binding site, and the B-motif also known as DEAD/DEXH box. For only a few of the DEAD box
proteins, e.g. eIF-4A and the protein p68 (
30
,
31
), an ATP-dependent helicase activity has been demonstrated. In some other cases,
for example the SrmB protein and the prp16 protein, only an RNA-dependent ATPase activity was established (
32
,
33
). An RNA-dependent ATPase activity associated with the capability to melt DNA-RNA hybrids has been described for the procaryotic transcription
termination factor rho (
34
) and for the autoantigen La (SS-B) (
10
). For the latter protein also a dsRNA unwinding activity was proposed and has
now been firmly established (
13
, this paper).
The question arises whether the La protein contains amino acid sequences related
to the A- and the B-motifs or to other conserved regions of DEAD/DEXH box proteins.
Recently Topfer and co-workers (
28
) pointed out that the La protein contains the evolutionarily conserved (
36
) sequence motif A/GXXXXGKG (amino acids 333 to 340 in human La). This sequence
represents the major part of the A-motif with only one difference, the final amino acid T of the A motif
A/GXXXXGKT is replaced by a G. A similar deviation has been found in the ATP-binding site of some other ATP-binding proteins as for example adenylate kinase and the H(+)-ATPase of
E.coli
(
35
). Most recently, a monoclonal anti-La antibody (La4B6) directed to this putative ATP-binding site was generated. It could subsequently be shown that ATP
abolished the binding of La4B6 to the La protein (
18
), indicating that this site indeed interacts with ATP. Although the La protein
does not belong to the DEAD or DEXH families of RNA helicases, a sequence
sharing some similarity to the B-motif can be discerned in the human La protein: DDEHDEHDENG, starting at
position 367. Further experiments are required to investigate whether this
element is required for the unwinding activity.
Finally, Clemens (
37
) indicated that the La protein shares some sequence homologies with the
interferon induced dsRNA dependent protein kinase PKR (also known as DAI),
primarily in regions reminiscent of a dsRNA binding motif (
38
). Interestingly, most of the amino acids suggested to be conserved in these PKR
related motifs appear to be conserved in La from different species. Moreover,
the B-motif related cluster of acidic amino acids flanks the PKR dsRNA binding
motif-like element at the C-terminal side. Experiments are in progress to investigate whether
(some of) these elements are involved in the unwinding activity.
Members of the DEAD/DEXH protein families are known, or suggested, to be
involved in diverse cellular functions including translation initiation,
splicing, ribosome assembly, cell growth and division, embryogenesis and
spermatogenesis (
39
). Also the La protein can be expected to be involved in a number of cellular
processes. Its function in the nucleus as an RNA polymerase III transcription
termination factor has been firmly established (
11
,
12
,
40
), but cytoplasmic functions of La cannot be ruled out. It has been suggested
previously that La might shuttle between nucleus and cytoplasm in dependence on
transcription (
15
,
16
) or infection with various viruses such as herpes-, adeno- and poliovirus (
9
,
41
). Indeed, Peek and co-workers provided further evidence for a partially cytoplasmic localization
of the La protein using cell-enucleation as fractionation method (
14
), which is substantiated by the recent demonstration that a subset of La
molecules is associated with small ribosomal subunits (
23
). In line with these findings, it has been suggested that the La protein could
be involved in the internal initiation of poliovirus mRNA translation (
9
,
42
). In this respect an interesting observation of the present study is that a 3'-overhang in the RNA is required for unwinding. Secondary structures
in 5'-UTRs of mRNAs and in internal ribosomal entry sites will always be
accompanied by `3'-overhangs' and therefore it is tempting to speculate that the dsRNA
unwinding activity of the La protein melts such secondary structures thereby
stimulating initiation of translation. Internal initiation may not only occur
on viral RNAs but could also be used by other cellular mRNAs, such as
protooncogene mRNAs and mRNAs of growth factors or in translation of proteins
involved in the regulation of mitotic events (
9
). It is conceivable that the dsRNA unwinding activity of La is involved in the
internal initiation process.
This investigation was supported by a grant from the Deutsche
Forschungsgemeinschaft (Ba 1145/4-1) and in part by the Netherlands Foundation for Chemical Research (SON)
with financial aid from the Netherlands Organization for Scientific Research
(NWO). M. Bachmann is a recipient of a professorship granted by the `Hermann-and-Lilly-Schilling Stiftung' in the `Stifterverband für die Deutsche Wissenschaft'. The work of Dr G. J. M.
Pruijn has been made possible by a fellowship of the Royal Netherlands Academy
of Arts and Sciences.
REFERENCES
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