ABSTRACT
P72, a novel human member of the DEAD box family of putative RNA-dependent ATPases and ATP-dependent RNA helicases was isolated from a HeLa cDNA library. The
predicted amino acid sequence of p72 is highly homologous to that of the
prototypic DEAD box protein p68. In addition to the conserved core domains
characteristic of DEAD box proteins, p72 contains several N-terminal RGG RNA-binding domains and a serine/glycine rich C-terminus likely involved in mediating protein-protein interactions. A p72-specific probe detects two mRNAs of approximately
5300 and 9300 bases which, although ubiquitously expressed, show variability in
their expression levels in different tissues. Purified recombinant p72 exhibits
ATPase activity in the presence of a range of RNA moieties. Immunocytochemical
studies of p68 and p72 show that these proteins localise to similar locations
in the nucleus of HeLa cells, suggesting their involvement in a nuclear
process.
The D-E-A-D box protein family (
1
) of putative RNA helicases includes over 40 proteins from a wide range of
organisms, spanning bacteria to humans, that share a group of conserved motifs
including the sequence Asp-Glu-Ala-Asp (D-E-A-D) which provides their name [for review see
refs (
2
-
5
)]. These proteins are implicated in diverse cellular functions including splicing,
ribosome assembly, translation initiation, spermatogenesis, mRNA stability, embryogenesis and cell growth and division.
The DEAD box family is characterised by a core region represented by eIF-4A [
e
ukaryotic (translation)
i
nitiation
f
actor 4A] and contains eight conserved amino acid regions, one of which is the D-E-A-D motif [also called DEAD box (
1
,
4
)]. The conserved core region is flanked by N- and C-terminal extensions which share little sequence homology and are
probably involved in mediating specialised functions of the individual
proteins. The DEAH (Asp-Glu-Ala-His) sub-family includes the
Saccharomyces cerevisiae
gene products PRP2, PRP16 and PRP22 involved in pre-mRNA splicing [reviewed in refs (
6
,
7
)], the
Escherichia coli
hrpA gene product of hitherto unknown function (
8
) and HRH1, the putative human homologue of PRP22 (
9
). Interestingly, all of these proteins are exceptionally large. The
Drosophila
Maleless (Mle) protein involved in X chromosome dosage compensation and its
human orthologe, RNA helicase A, are most similar to the DEAH box proteins
although they have the D-E-I-H motif (
10
,
11
). Another sub-family, containing a D-E-x-H motif, includes RNA helicases of positive strand RNA
viruses (
12
).
To date, four human members of the DEAD box family have been reported. Apart
from the prototypic member, p68 (
13
), there are currently three other human DEAD box proteins: p54, which was
cloned from a human lymphoid cell line (
14
); NP52, isolated from a HeLa expression library due to a cross reaction with a
monoclonal antibody raised against human aldolase A (
15
) and DDX1, which was found amplified in two retinoblastoma cell lines (
16
). P54, NP52 and DDX1 have not been further characterised biochemically and
their function(s) remains unknown, although DDX1 has been found amplified in
some primary neuroblastomas (
17
). In the case of p68, the purified protein has been shown to exhibit RNA-dependent ATPase activity and functions as an RNA helicase
in vitro
(
18
,
19
).
In this paper we describe the identification and characterisation of p72, a
novel human nuclear DEAD box protein, which shows a striking homology to p68.
We demonstrate that p72 is an ATPase activated by a variety of RNA species but
not by dsDNA. The localisation and possible functional roles of p72 are
discussed and compared with other DEAD box proteins.
The cDNA clone #461 coding for the N-terminal part of p72 was isolated from a random primed expression library
of HeLa poly(A)
+
RNA prepared in pUEX (
20
) (gift of Dr T. Kreis) due to a cross reaction with an unrelated monoclonal
antibody. Clone #461 was subcloned into the
Kpn
I site of Bluescript KS (Stratagene) and sequenced on both strands using
oligonucleotide primers either with the dideoxy chain termination method (
21
) using [[alpha]-
35
S]dATP or with fluorescent primers by the EMBL sequencing service. Radiolabelled
clone #461 was then used as a probe to screen a [lambda] Zap HeLa cDNA library (Stratagene) to isolate additional clones
spanning the missing 3' terminus of the p72 cDNA. A cDNA encoding full-length p72 was assembled from the resulting clones and subcloned
into the
Sma
I site of pBluescript SK(-). This construct is henceforth referred to as p72-pBS SK.
To express the recombinant p72 in the
E.coli
strain BL21(DE3) a
Bam
HI-
Bam
HI fragment of p72 (purified from p72-pBS SK) which encoded for the full-length cDNA was cloned in-frame with the poly His-tag into the T7 driven pRSET expression vector
(Invitrogen). For antibody production a fragment of p72 containing amino acids 1-343, was subcloned into pRSET in-frame with the poly His-tag.
For expression in HeLa cells the p72 cDNA was subcloned in frame into the
Bam
HI site of the eukaryotic expression vector pSG5 (
22
) containing a myc-tag (MEQKLISEEDL) (
23
). In all cases, correct orientation of the constructs was confirmed by
restriction digestion analysis and DNA sequencing.
Fresh overnight cultures of BL21(DE3) containing p72 cDNAs in the pRSET plasmid
under the IPTG-inducible T7 promoter (
24
) were diluted 30-fold, grown to an OD (650 nm) of 0.3-0.4 at 37oC and induced by the addition of 0.75 mM IPTG. The cultures
were transferred to 26oC and grown for a further 4 h before being harvested by centrifugation. The
cell pellets were washed in 50 mM Tris-HCl (pH 7.4), harvested by centrifugation and stored at -70oC.
The bacterial pellet was resuspended in ice cold Buffer A (1 ml per 100 mg
pellet) containing 6 M guanidine-HCl, 0.1 M NaPi (pH 8.0), 10 mM Tris-HCl (pH 8.0), 5 mM imidazole, 1 mM phenylmethylsulphonylfluoride
(PMSF), 1 mM benzamidine, 2 [mu]g/ml leupeptin, 2 [mu]g/ml aprotinin and placed in an ice/salt water bath for 30 min with
intermittent vortexing. The resuspended bacterial pellet was then sonicated
twice for 30 s to shear DNA and the insoluble material was pelleted by
centrifugation. A denaturing protocol was necessary for the purification of p72
as the protein was found in bacterial inclusion bodies. The supernate was then incubated
on a rotating wheel at 4oC with Ni
2+
-NTA-Agarose (3 ml packed volume for every 10 ml of supernate) for 3 h,
was washed once with Buffer A and then resuspended again in Buffer A and poured
into a disposable BioRad column. The resin was washed with 10 column volumes of
Buffer A followed by 2 column volumes of Buffer B (identical to Buffer A but
containing 10 mM imidazole). The recombinant protein was eluted with 2.5 column
volumes Buffer C (as Buffer A but containing 200 mM imidazole) and eluates were
collected as 1 ml fractions. Fractions containing recombinant p72 (determined
by running an aliquot on SDS-PAGE and Coomassie staining) were pooled and dialysed at 4oC overnight into Buffer D [20% glycerol, 500 mM KCl, 50 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 1M guanidine-HCl, 1 mM DTT, 1 mM benzamidine, 1 mM PMSF] (using
0.25 litres/1 ml fraction) and then 3 h into Buffer E (same as Buffer D but
containing 250 mM KCl). The protein was then concentrated to 1/10 of its
original volume over an Amicon filter column, diluted 1:20 into Buffer F [15%
glycerol, 50 mM KCl, 50 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM benzamidine], loaded onto poly(U)-Sepharose swollen in Buffer F and eluted using 100 mM KCl steps
from 0.5 to 1 M KCl. The eluate was again concentrated over an Amicon filter
column to 1/5 of its original volume and stored in aliquots in liquid nitrogen.
Immediately prior to use in functional assays the purified protein was diluted
in Buffer F to 50 ng/[mu]l.
BL21(DE3) cells expressing a fragment of p72 corresponding to amino acids 1-343 were treated as described above for the purification of full-length recombinant p72. After Ni
2+
-NTA-Agarose chromatography the pooled fractions were electrophoresed
through an SDS-PAGE gel, Coomassie stained and the p72 fragment excised from the gel.
The acrylamide slice was macerated and 300 [mu]g of recombinant p72 was mixed with 2 vol Feund's complete adjuvant (Sigma)
and injected into rabbits. Further injections were carried out at three week
intervals using 300 [mu]g protein and Freund's incomplete adjuvant.
ATP hydrolysis assays were carried out as described in (
25
) containing RNA or DNA species as described in the appropriate figure legends.
The amount of phosphate hydrolysed from [[gamma]-
32
P]ATP was determined by counting the relevant areas of the TLC plate (as
Cerenkov counts) in a liquid scintillation counter.
E.coli
16S and 23S rRNA was purchased from Boehringer.
Uniformly labelled, capped rabbit [beta]-globin pre-mRNA and wild-type adenovirus pre-mRNA were transcribed as described in (
26
).
The
Bam
HI-
Ssp
I 5' fragment of p72 was radiolabelled by random priming (
27
) and used to probe a commercial multiple tissue Northern blot of human poly(A)
+
RNA (Clontech) as according to the manufacturer's recommendations.
SDS-PAGE gel analysis was performed according to (
28
) and transferred onto nitrocellulose membrane (Schleicher and Schuell).
Membranes were blocked in 2% non-fat milk powder in phosphate buffered saline (PBS), incubated with the
primary antibody for 2 h at room temperature, washed and incubated with the
appropriate secondary antibody (Amersham) coupled to horseradish peroxidase.
Immunoblots were developed with the ECL detection kit (Amersham) as according
to the manufacturer's recommendations.
HeLa cells were grown on coverslips at 37oC with 5% CO
2
in Dulbecco's modified Eagle's medium (Gibco BRL) supplemented with 10% foetal
calf serum, 100 U/ml penicillin and streptomycin (Gibco BRL) and 1% glutamine.
The myc-tagged p72 construct in pSG5 was transfected with LipofectAMINE
transfection reagent (Gibco BRL) according to the manufacturer's protocol and
the cells were fixed with 3.7% paraformaldehyde in CSK buffer [100 mM NaCl, 300
mM sucrose, 10 mM PIPES (pH 6.8), 3 mM MgCl
2
, 1 mM EGTA] for 10 min at room temperature. The cells were permeabilised with
0.5% Triton X100 in CSK buffer for 15 min at room temperature. Using
immunofluorescence analysis we observed that routinely 30-40% of cells were transfected.
Immunofluorescent labelling was carried out as described (
29
) and analysed on a Zeiss Axiophot Epifluorescence microscope. Excitation
wavelengths of 476 nm (FITC) and 529 nm (TexasRed) were used. The two channels
were recorded independently and pseudo-coloured images were generated and superimposed. The pictures were printed on a Canon 700 Colour Laser Copier.
The following antibodies were used: rabbit anti-p80 coilin polyclonal serum 204/5 (dilution 1:350) (
30
), rabbit anti-p68 peptide antibody 2907 (dilution 1:300), mAb 9E10 (dilution 1:500) (
23
). TexasRed and fluorescein (FITC) conjugated anti-rabbit or anti-mouse secondary antibodies were purchased from Dianova and diluted
1:500.
The compilation and analysis of DNA sequences was done using the University of
Wisconsin Genetics Computer Group (UWGCG) programmes (
31
) on a Vax computer cluster at EMBL, Heidelberg. The molecular weight and amino
acid composition of p72 was determined using the Peptidesort programme (
31
). The TFasta or BLAST (
32
) programmes were used to search for homologies between p72 and the GenEMBL data
banks. The CLUSTAL V programme (
33
) was used to search for amino acid homologies in the Swissprot database. The
Motifs programme (
31
) was used to search for p72 protein motifs in the ProSite data bank.
A 1.3 kb cDNA fragment encoding the N-terminal portion of p72 was isolated from a HeLa expression library during
expression screening with an unrelated antibody. The cDNA fragment was used to
further screen HeLa cDNA libraries and a 1.1 kb fragment encoding the C-terminal portion of p72 was isolated. The overlapping cDNAs contain an
open reading frame (ORF) of 1950 bp capable of encoding a protein with a
predicted molecular mass of 71.9 kDa and an isoelectric point of 8.73 (Fig.
1
). The presumed ATG initiation codon is 259 bp from the 5'-end of the isolated cDNA and the upstream sequence contains stop
codons in all three reading frames (data not shown). The 3' untranslated sequence is at least 59 bp in length and contains neither a
poly(A) tail nor a consensus polyadenylation signal (
34
), suggesting that p72 mRNA contains additional 3' untranslated sequence.
In vitro
translation of the assembled p72 cDNA in a reticulocyte lysate system yields a
labelled translation product that migrates with an apparent molecular weight of
79 kDa on SDS-polyacrylamide gels (data not shown) and an antibody raised against
recombinant p72 specifically detects a protein migrating at 79 kDa on Western
blots of HeLa nuclear or cytoplasmic extracts (Fig.
4
B, lane 7). This indicates that both recombinant and endogenous p72 migrates
aberrantly at 79 kDa on SDS-PAGE.
The deduced amino acid sequence of p72 was used to carry out a BLAST (basic
local alignment search tool) search of the Swiss-Prot database. This search revealed a striking homology between p72 and
p68, a prototypic member of the DEAD box family (
13
). A multiple alignment of the first 481 amino acids of p72, encompassing the
conserved DEAD box motifs, with the translated, most closely related DEAD box
protein entries in the DDBJ/EMBL/GenBank database is presented in Figure
2
. Out of 650 residues in p72, 453 residues (69.7%) are identical in human p68
and 457 residues (70.3%) are identical in mouse p68. An additional 53 residues
in human and 52 residues in mouse p68 are similar amino acid substitutions
(77.6 and 78.4% similarity, respectively). Within the region spanning the
conserved motifs characteristic of this family (
2
) the homology between p72 and p68 is ~90%, which is considerably higher than that seen between other members of
the family. However, C-terminal to the last conserved DEAD box domain (HRIGR) (Fig.
1
) the identity between human p68 and p72 drops to 27.5%, suggesting that these
proteins have different functions in the cell. This also supports the
established view that DEAD box proteins have a similar core region encompassing
the conserved domains but have N- and C- terminal extensions which endow the proteins with specialised
functions [for review see refs (
3
,
4
)].
A human multiple tissue northern blot of poly(A)
+
RNA was probed with two non-overlapping cDNA fragments encompassing the 5' half (Fig.
3
) and 3' portion of p72 (data not shown). Both cDNA fragments gave identical
patterns of mRNA distribution in the different tissues and both recognise mRNA
transcripts of approximately 5300 and 9300 bases (Fig.
3
). The 5300 transcript appears to be ubiquitously expressed in all tissues
tested with similar levels of expression in heart, brain, placenta, lung and
liver and apparently higher levels of expression in skeletal muscle, kidney and
pancreas. The 9300 transcript is also ubiquitously expressed, although
extremely low levels are detected in heart and placenta and the transcript is
most abundantly expressed in kidney and pancreas. The ratio between the two
transcripts is also highly variable in the different tissues. While in brain,
liver, kidney and pancreas the two transcripts are expressed at similar levels,
in heart, placenta, lung and skeletal muscle predominantly the 5300 base
transcript is present. A cDNA probe encompassing the p68 coding region verified
that neither transcript represents a cross-reaction with p68 mRNA (data not shown). Although the smallest transcript
detected is 5300 bases, the isolated p72 cDNA sequence only spans 2268
contiguous base pairs which lacks a poly(A) signal and poly(A) tail. It is,
therefore, likely that p72 mRNA contains additional downstream and perhaps also
upstream untranslated regions. The two p72 transcripts may arise by
transcription of independent genes, differential transcription of a common gene
or by alternative splicing of a common pre-mRNA. These results suggest that the expression of separate p72
transcripts is regulated in a tissue specific manner. Interestingly, when the
same blot was probed for p68 mRNA two transcripts were also observed. However,
these p68 transcripts differed from p72 in both their size and tissue
distribution (data not shown) and no cross hybridisation was observed between
the p72 and p68 probes. This indicates that the expression of p68 mRNA may also
be subject to tissue specific regulation.
Histidine-tagged p72 was expressed in
E.coli
and purified to homogeneity as described in Materials and Methods (Fig.
4
A and B). Bacteria transformed with the p72 plasmid and induced with IPTG
abundantly express the histidine-tagged protein, as is apparent by the appearance of an extra protein band
migrating at 79 kDa on Coomassie stained gels (compare Fig.
4
, lanes 1 and 2). Ni
2+
-NTA-Agarose chromatography of the bacterial lysate harbouring
recombinant p72 yielded a substantial purification of the protein (Fig.
4
, lane 3). A final poly(U)-Sepharose chromatography step yielded recombinant p72 purified to
homogeneity (Fig.
4
A, lane 4). The additional bands detected after the poly(U)-Sepharose purification step are degradation products of p72 (see below).
In order to verify the purification protocol of recombinant p72, Western blot
analysis of the various purification steps was carried out using an anti-p72 antibody and the MAD1 monoclonal antibody (Fig.
4
B). The MAD1 monoclonal antibody was raised against a peptide encompassing the
DEAD motif of p68 (
44
). This region is 100% conserved in p72 and MAD1 should, therefore, also
recognise the p72 protein. In HeLa nuclear extracts MAD1 predominantly
recognises a 68 and a 79 kDa protein band (Fig.
4
B, lane 5). The former band corresponds to p68 as identified by staining with
p68-specific antibodies (data not shown). The latter band corresponds to p72
since anti-p72 antibodies detect a protein of similar size in HeLa nuclear extracts
(Fig.
4
B, lane 7). The MAD1 monoclonal antibody detects recombinant p72 in the lysate
of
E.coli
carrying the p72 plasmid
(Fig.
4
B, lane 2) but not in bacteria transformed with the vector alone (Fig.
4
B, lane 1). Although MAD1 detects recombinant p72 as a single protein band of 79
kDa after Ni
2+
-NTA-Agarose chromatography (Fig.
4
B, lane 3) it detects several bands after poly(U)-Sepharose chromatography (Fig.
4
B, lane 4). These additional bands correspond to degradation products of p72 as
after poly(U)-Sepharose purification an identical pattern is observed with an anti-p72 antibody (Fig.
4
B, lane 6). This purification protocol routinely yielded 1-1.5 mg of homogenous p72 from a one litre bacterial culture.
A common feature of DEAD box protein family members is their ability to
hydrolyse ATP in the presence of RNA (
2
). The ATPase activity of purified p72 was tested by its ability to release
radioactive phosphate from [[gamma]-
32
P]ATP. P72 hydrolysed ATP in the presence of total HeLa RNA and exhibited no
ATPase activity in the absence of RNA (Fig.
5
). Moreover, the ATPase activity of p72 was abolished when total HeLa RNA was
pre-treated with RNAse A indicating that the ATPase activity of the protein
was dependent on the added RNA. The
E.coli
DEAD box protein DbpA, which is specifically activated by
E.coli
ribosomal RNA (
25
), was used as a positive control in these assays. ATPase reactions containing [[gamma]-
32
P]ATP and a 10-fold excess of cold nucleoside triphosphates showed competition only by
unlabelled ATP, suggesting that only ATP is a substrate for p72 (data not
shown). The
K
m
of p72 for ATP was found to be 170 [mu]M (data not shown). This value is within the range reported for p68 (100-1000 [mu]M) (
18
), DbpA (150 [mu]M) (
25
) and eIF-4A (50 [mu]M) (
45
). Taken together, the results above clearly demonstrate that p72 is an RNA-dependent ATPase.
We were interested in determining the sub-cellular localisation of p72. Since the anti-p72 antibody did not give a specific signal in immunolocalisation
experiments, we constructed a plasmid in which p72 was fused to a myc-epitope tag at its N-terminus and expressed under the control of the SV40 early promoter.
This tagged construct was used in transient expression studies using HeLa cells
and detected using a monoclonal anti-myc antibody. Myc-tagged p72 localises to the nucleus of HeLa cells (Fig.
7
A, E and I) as determined by co-staining with DAPI (Fig.
7
C). Tagged p72 shows a predominantly granular nuclear staining pattern (Fig.
7
A) with occasional elevated levels of peri-nucleolar staining (Fig.
7
E and I, indicated by arrowheads). Consistent with previous studies
untransfected cells are not labelled by anti-myc antibody (data not shown). The high homology between p72 and p68
prompted us to compare the sub-cellular localisation of these two proteins. P68 as previously reported (
43
) shows a diffuse granular nuclear distribution in interphase cells (Fig.
7
F) and colocalises with tagged-p72 (Fig.
7
G). Since several DEAH-box proteins from
Saccharomyces cerevisiae
such as PRP2, PRP16 and PRP22 have been shown to be involved in pre-mRNA splicing [for review see ref. (
6
)] we were interested in whether p72 localises to splicing snRNP-enriched nuclear organelles called `coiled bodies' [for review see ref. (
46
)]. HeLa cells transiently expressing tagged p72 show an average of 2-5 coiled bodies (Fig.
7
J) and their staining pattern does not overlap with that of tagged p72 (Fig.
7
K). In all cases the cells show normal cell morphology as judged by DIC
microscopy (Fig.
7
D, H and L).
In this study we have identified and characterised p72, a novel human member of
the DEAD box family of proteins. P72 is a nuclear protein and we have detected
p72 mRNA ubiquitously expressed in all human tissues tested. Biochemical
studies using recombinant p72 protein expressed in
E.coli
showed that it has RNA-dependent ATPase activity which is stimulated
in vitro
by a range of RNAs, including preparations of tRNA, mRNA and rRNA from
bacteria, yeast and mammals.
P72 is strikingly similar to the human p68 protein, which is one of the
prototypic DEAD box proteins, originally isolated due to a cross-reaction with a monoclonal antibody (DL3C4/PAb204) raised against SV40
large T antigen (
13
). These two proteins are more closely related to each other than to any other
members of the DEAD box family analysed to date. Interestingly,
S.cerevisiae
has only one apparent homologue of p68/p72, called DBP2 (
43
). While DBP2 was previously identified as the yeast homologue of p68, and can
be complemented by human p68 (
47
), it in fact shows a higher degree of sequence homology to p72. We propose that
p68 and p72 represent a specific subfamily of the DEAD box protein family. The
fact that mammals appear to have at least two members of this subfamily
suggests either that there is some functional redundancy or, perhaps more
likely, that these proteins exhibit some specialisation in their substrate
specificities. This would be consistent with the observation that while they
are ~90% identical in the core domains, their N- and C-termini are much less conserved.
The p68 and p72 proteins also show a functional as well as structural
relationship. P68 has previously been shown to have RNA-dependent ATPase and RNA helicase activities
in vitro
(
18
,
19
). As described above, p72 also exhibits RNA-dependent ATPase activity. However, we have so far been unable to detect
RNA helicase activity for p72 (data not shown). There are several possible
explanations for this finding: (a) the recombinant p72 does not unwind RNA
under the assay conditions used; (b) since the recombinant p72 was purified
from bacterial inclusion bodies, it may not have the correct conformation for
helicase activity, even though it shows ATPase activity; (c) other factors are
necessary for p72 to unwind RNA [e.g. eIF-4A, which is a much more efficient RNA helicase when part of the eIF-4F complex (
48
,
49
), and RhlB which exhibits ATP-dependent RNA helicase activity when part of the `degradosome' but not as
free protein (
50
)]; or (d) unlike p68, p72 is not actually an RNA helicase
in vivo
. In this regard it is worth noting that relatively few DEAD box proteins have
been shown to exhibit helicase activity (
2
). Although the DEAD box proteins are usually referred to as `helicases' it may
in fact be the case that their common function is actually an ATPase activity,
with additional helicase activity being restricted to a subset of the family
members.
Both p72 and p68 localise to the nucleus of HeLa cells. Like p68, p72 shows a
predominantly granular nucleoplasmic staining pattern, excluding nucleoli.
However, it also shows some enhanced peri-nucleolar staining that was not seen with p68. Previous studies have shown
that p68 undergoes dramatic changes in nuclear localisation during the cell
cycle (
18
,
43
). During interphase, p68 is found in the nucleoplasm and is excluded from the
nucleoli. However, it transiently enters pre-nucleolar bodies during telophase. We have not observed such a sub-cellular redistribution of p72 during the cell cycle, again
suggesting that p68 and p72, although highly homologous, may have different
biological functions involving interaction with different RNA targets.
The biological functions of p68/p72 in mammals, and of DBP2 in yeast, remains to
be established. The fact that these are nuclear proteins suggests that their
in vivo
substrates are likely to be nuclear RNAs. Analysis of the sequence of p72 shows
that in addition to the conserved DEAD box family core domains, it contains N- and C-terminal extensions with additional protein motifs. These include
four N-terminal RGG boxes (
35
), a glycine hinge, a serine/glycine-rich C-terminus and a C-terminal proline tract. The analysis of the nucleolin RGG
domain suggests that each RGGF tetrapeptide makes a [beta]-turn and several of these repeats form a [beta]-spiral (
51
) which, due to the presence of glycines, are extremely flexible and can adopt
alternative conformational states (
52
). P72 is shown here to bind polyU sepharose and to have RNA-dependent ATPase activity. It is possible that the RGG boxes may be
involved in the interaction of p72 with RNA. A run of glycines is also seen in
the splicing factors U2AF
35
(
53
) and ASF/SF2 (
54
,
55
) and may function to flexibly hinge different protein domains. Proline rich
motifs have been identified in numerous proteins including hnRNP L (
37
) the U1 snRNP specific A and C proteins (
56
,
57
) and the yeast poly(A)-binding protein (
39
). The proline/glutamine rich motif in the U1 snRNP-specific C protein has been proposed to stabilise the U1 snRNP-5' splice site interaction (
38
) and in the transcription factors CTF/NF-1 and Sp-1, this motif has been demonstrated to represent the transcription
activation domain (
40
,
41
). Thus, proline rich motifs may function as binding sites for additional
factors during the assembly of transcription or splicing complexes. The studies
above suggest that the C-terminal proline tract of p72 may be involved in mediating protein-protein interactions while the RGG domain may be necessary for the
protein to bind to its putative target RNA species.
An important goal for future studies will be to identify the authentic
in vivo
RNA substrates for p68 and p72 and to determine whether they act alone or as
part of larger nuclear complexes. It will also be important to characterise in
more detail the functional properties of the p68/p72 subfamily of DEAD box
proteins and to determine whether there are additional members of this group.
The authors wish to thank Kerstin Bohmann for the anti-p80 coilin antibody and Dr G. Evan for monoclonal anti-myc antibodies as well as the EMBL sequencing service for technical
assistance. We are also especially grateful to Karsten Weis and Joe Lewis for
critical reading of the manuscript. Parts of this work were supported by a
grant from Boehringer Ingelheim Fonds, an EMBO short-term fellowship to GML and a Medical Research Council Senior Fellowship to
FFP.
REFERENCES
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