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p72: A Human Nuclear DEAD Box Protein Highly Related to p68

p72: A Human Nuclear DEAD Box Protein Highly Related to p68  1996 Oxford University Press Nucleic Acids Research, 1996, Vol. 24, No. 19 3739–3747 p72: a human nuclear DEAD box protein highly related to p68 1 1 2, Gábor M. Lamm, Samantha M. Nicol , Frances V. Fuller-Pace and Angus I. Lamond * Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria, Department of Molecular and Cellular Pathology, University of Dundee Medical School, Ninewells Hospital, Dundee DD1 9SY, Scotland and University of Dundee, Department of Biochemistry, Dundee DD1 4HN, Scotland Received June 20, 1996; Revised and Accepted August 12, 1996 DDBJ/EMBL/GenBank accession no. U59321 ABSTRACT hrpA gene product of hitherto unknown function (8) and HRH1, the putative human homologue of PRP22 (9). Interestingly, all of these P72, a novel human member of the DEAD box family of proteins are exceptionally large. The Drosophila Maleless (Mle) putative RNA-dependent ATPases and ATP-dependent protein involved in X chromosome dosage compensation and its RNA helicases was isolated from a HeLa cDNA library. human orthologe, RNA helicase A, are most similar to the DEAH The predicted amino acid sequence of p72 is highly box proteins although they have the D-E-I-H motif (10,11). Another homologous to that of the prototypic DEAD box sub-family, containing a D-E-x-H motif, includes RNA helicases of protein p68. In addition to the conserved core domains positive strand RNA viruses (12). characteristic of DEAD box proteins, p72 contains To date, four human members of the DEAD box family have several N-terminal RGG RNA-binding domains and a been reported. Apart from the prototypic member, p68 (13), there serine/glycine rich C-terminus likely involved in me- are currently three other human DEAD box proteins: p54, which diating protein–protein interactions. A p72-specific was cloned from a human lymphoid cell line (14); NP52, isolated probe detects two mRNAs of approximately 5300 and from a HeLa expression library due to a cross reaction with a 9300 bases which, although ubiquitously expressed, monoclonal antibody raised against human aldolase A (15) and show variability in their expression levels in different DDX1, which was found amplified in two retinoblastoma cell tissues. Purified recombinant p72 exhibits ATPase lines (16). P54, NP52 and DDX1 have not been further activity in the presence of a range of RNA moieties. characterised biochemically and their function(s) remains Immunocytochemical studies of p68 and p72 show unknown, although DDX1 has been found amplified in some that these proteins localise to similar locations in the primary neuroblastomas (17). In the case of p68, the purified nucleus of HeLa cells, suggesting their involvement in protein has been shown to exhibit RNA-dependent ATPase a nuclear process. activity and functions as an RNA helicase in vitro (18,19). In this paper we describe the identification and characterisation INTRODUCTION of p72, a novel human nuclear DEAD box protein, which shows a striking homology to p68. We demonstrate that p72 is an The D-E-A-D box protein family (1) of putative RNA helicases ATPase activated by a variety of RNA species but not by dsDNA. includes over 40 proteins from a wide range of organisms, The localisation and possible functional roles of p72 are discussed spanning bacteria to humans, that share a group of conserved and compared with other DEAD box proteins. motifs including the sequence Asp-Glu-Ala-Asp (D-E-A-D) which provides their name [for review see refs (2–5)]. These MATERIALS AND METHODS proteins are implicated in diverse cellular functions including splicing, ribosome assembly, translation initiation, spermatogenesis, cDNA cloning and sequencing mRNA stability, embryogenesis and cell growth and division. The DEAD box family is characterised by a core region The cDNA clone #461 coding for the N-terminal part of p72 was represented by eIF-4A [eukaryotic (translation) initiation factor isolated from a random primed expression library of HeLa 4A] and contains eight conserved amino acid regions, one of poly(A) RNA prepared in pUEX (20) (gift of Dr T. Kreis) due which is the D-E-A-D motif [also called DEAD box (1,4)]. The to a cross reaction with an unrelated monoclonal antibody. Clone conserved core region is flanked by N- and C-terminal extensions #461 was subcloned into the KpnI site of Bluescript KS which share little sequence homology and are probably involved in (Stratagene) and sequenced on both strands using oligonucleotide mediating specialised functions of the individual proteins. The primers either with the dideoxy chain termination method (21) DEAH (Asp-Glu-Ala-His) sub-family includes the Saccharomyces using [α- S]dATP or with fluorescent primers by the EMBL cerevisiae gene products PRP2, PRP16 and PRP22 involved in sequencing service. Radiolabelled clone #461 was then used as a pre-mRNA splicing [reviewed in refs (6,7)], the Escherichia coli probe to screen a λ Zap HeLa cDNA library (Stratagene) to To whom correspondence should be addressed 3740 Nucleic Acids Research, 1996, Vol. 24, No. 19 isolate additional clones spanning the missing 3′ terminus of the 50 mM Tris–HCl (pH 8.0), 1 mM DTT, 1 mM benzamidine], p72 cDNA. A cDNA encoding full-length p72 was assembled loaded onto poly(U)–Sepharose swollen in Buffer F and eluted from the resulting clones and subcloned into the SmaI site of using 100 mM KCl steps from 0.5 to 1 M KCl. The eluate was pBluescript SK(–). This construct is henceforth referred to as again concentrated over an Amicon filter column to 1/5 of its p72-pBS SK. 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/μl. Construction of expression vectors To express the recombinant p72 in the E.coli strain BL21(DE3) Antibody production a BamHI–BamHI fragment of p72 (purified from p72-pBS SK) BL21(DE3) cells expressing a fragment of p72 corresponding to which encoded for the full-length cDNA was cloned in-frame amino acids 1–343 were treated as described above for the with the poly His-tag into the T7 driven pRSET expression vector 2+ purification of full-length recombinant p72. After Ni -NTA- (Invitrogen). For antibody production a fragment of p72 containing Agarose chromatography the pooled fractions were electro- amino acids 1–343, was subcloned into pRSET in-frame with the phoresed through an SDS–PAGE gel, Coomassie stained and the poly His-tag. p72 fragment excised from the gel. The acrylamide slice was For expression in HeLa cells the p72 cDNA was subcloned in macerated and 300 μg of recombinant p72 was mixed with 2 vol frame into the BamHI site of the eukaryotic expression vector Feund’s complete adjuvant (Sigma) and injected into rabbits. pSG5 (22) containing a myc-tag (MEQKLISEEDL) (23). In all Further injections were carried out at three week intervals using cases, correct orientation of the constructs was confirmed by 300 μg protein and Freund’s incomplete adjuvant. restriction digestion analysis and DNA sequencing. ATP hydrolysis assays Growth and induction of bacteria expressing p72 ATP hydrolysis assays were carried out as described in (25) Fresh overnight cultures of BL21(DE3) containing p72 cDNAs containing RNA or DNA species as described in the appropriate in the pRSET plasmid under the IPTG-inducible T7 promoter figure legends. The amount of phosphate hydrolysed from (24) were diluted 30-fold, grown to an OD (650 nm) of 0.3–0.4 [γ- P]ATP was determined by counting the relevant areas of the at 37C and induced by the addition of 0.75 mM IPTG. The TLC plate (as Cerenkov counts) in a liquid scintillation counter. cultures were transferred to 26C and grown for a further 4 h E.coli 16S and 23S rRNA was purchased from Boehringer. before being harvested by centrifugation. The cell pellets were washed in 50 mM Tris–HCl (pH 7.4), harvested by centrifugation In vitro transcription and stored at –70C. Uniformly labelled, capped rabbit β-globin pre-mRNA and wild- type adenovirus pre-mRNA were transcribed as described in (26). Purification of p72 The bacterial pellet was resuspended in ice cold Buffer A (1 ml Northern blotting per 100 mg pellet) containing 6 M guanidine–HCl, 0.1 M NaPi The BamHI–SspI 5′ fragment of p72 was radiolabelled by random (pH 8.0), 10 mM Tris–HCl (pH 8.0), 5 mM imidazole, 1 mM priming (27) and used to probe a commercial multiple tissue phenylmethylsulphonylfluoride (PMSF), 1 mM benzamidine, Northern blot of human poly(A) RNA (Clontech) as according 2 μg/ml leupeptin, 2 μg/ml aprotinin and placed in an ice/salt water to the manufacturer’s recommendations. bath for 30 min with intermittent vortexing. The resuspended bacterial pellet was then sonicated twice for 30 s to shear DNA and SDS–PAGE and Western blotting the insoluble material was pelleted by centrifugation. A denaturing protocol was necessary for the purification of p72 as the protein SDS–PAGE gel analysis was performed according to (28) and was found in bacterial inclusion bodies. The supernate was then transferred onto nitrocellulose membrane (Schleicher and 2+ incubated on a rotating wheel at 4C with Ni -NTA-Agarose Schuell). Membranes were blocked in 2% non-fat milk powder (3 ml packed volume for every 10 ml of supernate) for 3 h, was in phosphate buffered saline (PBS), incubated with the primary washed once with Buffer A and then resuspended again in Buffer antibody for 2 h at room temperature, washed and incubated with A and poured into a disposable BioRad column. The resin was the appropriate secondary antibody (Amersham) coupled to washed with 10 column volumes of Buffer A followed by 2 horseradish peroxidase. Immunoblots were developed with the column volumes of Buffer B (identical to Buffer A but containing ECL detection kit (Amersham) as according to the manufac- 10 mM imidazole). The recombinant protein was eluted with 2.5 turer’s recommendations. column volumes Buffer C (as Buffer A but containing 200 mM imidazole) and eluates were collected as 1 ml fractions. Fractions Cell culture, transfection and immunofluorescent containing recombinant p72 (determined by running an aliquot on microscopy SDS–PAGE and Coomassie staining) were pooled and dialysed at 4C overnight into Buffer D [20% glycerol, 500 mM KCl, HeLa cells were grown on coverslips at 37C with 5% CO in 50 mM Tris–HCl (pH 8.0), 0.5 mM EDTA, 1M guanidine–HCl, Dulbecco’s modified Eagle’s medium (Gibco BRL) supplemented 1 mM DTT, 1 mM benzamidine, 1 mM PMSF] (using with 10% foetal calf serum, 100 U/ml penicillin and streptomycin 0.25 litres/1 ml fraction) and then 3 h into Buffer E (same as (Gibco BRL) and 1% glutamine. The myc-tagged p72 construct in Buffer D but containing 250 mM KCl). The protein was then pSG5 was transfected with LipofectAMINE transfection reagent concentrated to 1/10 of its original volume over an Amicon filter (Gibco BRL) according to the manufacturer’s protocol and the cells column, diluted 1:20 into Buffer F [15% glycerol, 50 mM KCl, were fixed with 3.7% paraformaldehyde in CSK buffer [100 mM 3741 Nucleic Acids Research, 1996, Vol. 24, No. 19 3741 Nucleic Acids Research, 1994, Vol. 22, No. 1 NaCl, 300 mM sucrose, 10 mM PIPES (pH 6.8), 3 mM MgCl , 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 Figure 1. Amino acid sequence of p72. The motifs typical of the DEAD box from Dianova and diluted 1:500. family of helicases are boxed. The N-terminal RGG boxes and the C-terminal glycine hinge region and proline tract are underlined. The p72 DNA sequence has been deposited in GenBank—accession number U59321. Sequence analysis The compilation and analysis of DNA sequences was done using the University of Wisconsin Genetics Computer Group N- and C-terminal extensions. The N-terminus contains four (UWGCG) programmes (31) on a Vax computer cluster at repeats of the RGG box originally identified as an RNA binding EMBL, Heidelberg. The molecular weight and amino acid motif in the hnRNP U protein (35). A run of seven consecutive composition of p72 was determined using the Peptidesort glycines separate the last conserved DEAD box family domain programme (31). The TFasta or BLAST (32) programmes were (HRIGR) and the serine/glycine rich (13.2 and 17.8%, respectively) used to search for homologies between p72 and the GenEMBL C-terminus of p72. The extreme C-terminus of p72 additionally data banks. The CLUSTAL V programme (33) was used to search contains nine consecutive prolines. Serine/glycine rich regions for amino acid homologies in the Swissprot database. The Motifs have been shown to mediate protein–protein interactions in programme (31) was used to search for p72 protein motifs in the cytokeratins (36) and proline rich motifs appear to fulfil a similar ProSite data bank. function in hnRNP, snRNP and poly(A)-binding proteins (37–39) as well as in several transcription factors (40,41). We conclude that RESULTS p72 encodes a novel human DEAD box protein which, in addition to the conserved core motifs, contains domains that may modulate Cloning and structural organisation of p72 p72–RNA and p72–protein interactions. A 1.3 kb cDNA fragment encoding the N-terminal portion of p72 was isolated from a HeLa expression library during expression p72 shows striking homology to p68 screening with an unrelated antibody. The cDNA fragment was used to further screen HeLa cDNA libraries and a 1.1 kb fragment The deduced amino acid sequence of p72 was used to carry out encoding the C-terminal portion of p72 was isolated. The a BLAST (basic local alignment search tool) search of the overlapping cDNAs contain an open reading frame (ORF) of Swiss-Prot database. This search revealed a striking homology 1950 bp capable of encoding a protein with a predicted molecular between p72 and p68, a prototypic member of the DEAD box mass of 71.9 kDa and an isoelectric point of 8.73 (Fig. 1). The family (13). A multiple alignment of the first 481 amino acids of presumed ATG initiation codon is 259 bp from the 5′-end of the p72, encompassing the conserved DEAD box motifs, with the isolated cDNA and the upstream sequence contains stop codons translated, most closely related DEAD box protein entries in the in all three reading frames (data not shown). The 3′ untranslated DDBJ/EMBL/GenBank database is presented in Figure 2. Out of sequence is at least 59 bp in length and contains neither a poly(A) 650 residues in p72, 453 residues (69.7%) are identical in human tail nor a consensus polyadenylation signal (34), suggesting that p68 and 457 residues (70.3%) are identical in mouse p68. An p72 mRNA contains additional 3′ untranslated sequence. In vitro additional 53 residues in human and 52 residues in mouse p68 are translation of the assembled p72 cDNA in a reticulocyte lysate similar amino acid substitutions (77.6 and 78.4% similarity, system yields a labelled translation product that migrates with an respectively). Within the region spanning the conserved motifs apparent molecular weight of 79 kDa on SDS-polyacrylamide characteristic of this family (2) the homology between p72 and gels (data not shown) and an antibody raised against recombinant p68 is ~ 90%, which is considerably higher than that seen between p72 specifically detects a protein migrating at 79 kDa on Western other members of the family. However, C-terminal to the last blots of HeLa nuclear or cytoplasmic extracts (Fig. 4B, lane 7). conserved DEAD box domain (HRIGR) (Fig. 1) the identity This indicates that both recombinant and endogenous p72 between human p68 and p72 drops to 27.5%, suggesting that migrates aberrantly at 79 kDa on SDS–PAGE. these proteins have different functions in the cell. This also The deduced amino acid sequence of p72 demonstrates that it supports the established view that DEAD box proteins have a is a new member of the DEAD box family of proteins containing similar core region encompassing the conserved domains but all the conserved domains which are hallmarks of this family have N- and C- terminal extensions which endow the proteins (Fig. 1). In addition to the conserved core domain, p72 contains with specialised functions [for review see refs (3,4)]. 3742 Nucleic Acids Research, 1996, Vol. 24, No. 19 Figure 2. Amino acid sequence alignment of the first 481 amino acids of p72 with other DEAD box proteins. Multiple alignment was done using the CLUSTAL V programme (33) and generated by the PRETTYPLOT programme. Consensus positions are calculated from at least two most often occurring residues at a particular position taking into account amino acid similarity values. Boxed residues represent plurality values above 4.0. The accession numbers of the aligned sequences are: mmp68 (X65627), hsp68 (X15729), dmRM62 (X52846), spdbp2 (L11574) and scDBP2 (X55993). P72 is also very similar to the Drosophila RM62 protein (60% 5300 bases, the isolated p72 cDNA sequence only spans 2268 identity, 73% similarity) (42). The DEAD box proteins DBP2 and contiguous base pairs which lacks a poly(A) signal and poly(A) dbp2, which are the putative S.cerevisiae and S.pombe homologues tail. It is, therefore, likely that p72 mRNA contains additional of p68 (43), also show strong similarity with p72 (Fig. 2). downstream and perhaps also upstream untranslated regions. The Interestingly, full-length p72, in comparison to p68, appears to be two p72 transcripts may arise by transcription of independent slightly more similar to both dbp2 and DBP2 (p72/dbp2 = 58.2%, genes, differential transcription of a common gene or by p68/dbp2 = 54.4%, p72/DBP2 = 55.3%, p68/DBP2 = 53.2%). A alternative splicing of a common pre-mRNA. These results search of the complete S.cerevisiae genome sequence (Martinsried suggest that the expression of separate p72 transcripts is regulated Protein Sequence database) for p72-like sequences found only in a tissue specific manner. Interestingly, when the same blot was DBP2, suggesting that either (a) there is some redundancy in the probed for p68 mRNA two transcripts were also observed. function of these two proteins, or (b) multicellular organisms However, these p68 transcripts differed from p72 in both their require both proteins. 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 P72 is encoded by two transcripts to tissue specific regulation. A human multiple tissue northern blot of poly(A) RNA was probed with two non-overlapping cDNA fragments encompassing Purification of p72 the 5′ half (Fig. 3) and 3′ portion of p72 (data not shown). Both cDNA fragments gave identical patterns of mRNA distribution in Histidine-tagged p72 was expressed in E.coli and purified to the different tissues and both recognise mRNA transcripts of homogeneity as described in Materials and Methods (Fig. 4A and approximately 5300 and 9300 bases (Fig. 3). The 5300 transcript B). Bacteria transformed with the p72 plasmid and induced with appears to be ubiquitously expressed in all tissues tested with IPTG abundantly express the histidine-tagged protein, as is similar levels of expression in heart, brain, placenta, lung and apparent by the appearance of an extra protein band migrating at liver and apparently higher levels of expression in skeletal 79 kDa on Coomassie stained gels (compare Fig. 4, lanes 1 and 2+ muscle, kidney and pancreas. The 9300 transcript is also 2). Ni -NTA-Agarose chromatography of the bacterial lysate ubiquitously expressed, although extremely low levels are harbouring recombinant p72 yielded a substantial purification of detected in heart and placenta and the transcript is most the protein (Fig. 4, lane 3). A final poly(U)–Sepharose abundantly expressed in kidney and pancreas. The ratio between chromatography step yielded recombinant p72 purified to the two transcripts is also highly variable in the different tissues. homogeneity (Fig. 4A, lane 4). The additional bands detected While in brain, liver, kidney and pancreas the two transcripts are after the poly(U)–Sepharose purification step are degradation expressed at similar levels, in heart, placenta, lung and skeletal products of p72 (see below). muscle predominantly the 5300 base transcript is present. A In order to verify the purification protocol of recombinant p72, cDNA probe encompassing the p68 coding region verified that Western blot analysis of the various purification steps was carried neither transcript represents a cross-reaction with p68 mRNA out using an anti-p72 antibody and the MAD1 monoclonal (data not shown). Although the smallest transcript detected is antibody (Fig. 4B). The MAD1 monoclonal antibody was raised 3743 Nucleic Acids Research, 1996, Vol. 24, No. 19 3743 Nucleic Acids Research, 1994, Vol. 22, No. 1 Figure 3. Multiple tissue Northern analysis. Poly(A) RNA (2 μg/ lane) from human tissues was probed with an AvaI–SspI fragment of p72 cDNA encompassing the 5′ portion of p72. Tissues are marked above the lanes, position of migration of RNA size markers are shown on the right and the arrows indicate the p72 transcripts. 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. 4B, 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. 4B, lane 7). The MAD1 monoclonal antibody detects recombinant p72 in the lysate of E.coli carrying the p72 plasmid (Fig. 4B, lane 2) but not in bacteria transformed with the vector alone (Fig. 4B, lane 1). Although MAD1 detects recombinant p72 Figure 4. Purification of recombinant p72. Coomassie stain (A) or Western blot 2+ as a single protein band of 79 kDa after Ni -NTA-Agarose analysis (B) of proteins recovered during the various purification steps, separated on 10% SDS–PAGE. (A) BL21, total protein lysate of E.coli BL21 chromatography (Fig. 4B, lane 3) it detects several bands after transformed with expression plasmid alone (lane 1). BL21 + p72, as above but poly(U)–Sepharose chromatography (Fig. 4B, lane 4). These with expression plasmid containing p72 (lane 2). Proteins present after additional bands correspond to degradation products of p72 as 2+ Ni -NTA-Agarose chromatography and after poly(U)–Sepharose chromatog- after poly(U)–Sepharose purification an identical pattern is raphy are separated on lanes 3 and 4, respectively. (B) Western blot analysis of purification steps probed with both MAD1 monoclonal and anti-p72 antibodies. observed with an anti-p72 antibody (Fig. 4B, lane 6). This Abbreviations are as in (A). The antibody used to probe the different lanes is purification protocol routinely yielded 1–1.5 mg of homogenous indicated under the brackets. NE, HeLa nuclear extract (lanes 5 and 7). Protein p72 from a one litre bacterial culture. size markers are indicated in kDa on the left (A + B) and recombinant p72 is shown by the arrowhead on the right. ATPase activity of p72 A common feature of DEAD box protein family members is their ability to hydrolyse ATP in the presence of RNA (2). The ATPase (100–1000 μM) (18), DbpA (150 μM) (25) and eIF-4A (50 μM) activity of purified p72 was tested by its ability to release (45). Taken together, the results above clearly demonstrate that radioactive phosphate from [γ- P]ATP. P72 hydrolysed ATP in p72 is an RNA-dependent ATPase. the presence of total HeLa RNA and exhibited no ATPase activity Further ATPase assays were carried out to determine whether in the absence of RNA (Fig. 5). Moreover, the ATPase activity of the ATPase activity of p72 could be preferentially stimulated by p72 was abolished when total HeLa RNA was pre-treated with a specific RNA moiety or by DNA. As shown in Figure 6A, the RNAse A indicating that the ATPase activity of the protein was ATPase activity of p72 was stimulated by a variety of RNAs. dependent on the added RNA. The E.coli DEAD box protein These include total RNA and tRNA from HeLa cells, E.coli and DbpA, which is specifically activated by E.coli ribosomal RNA yeast; rabbit and E.coli rRNA; purified E.coli 16S and 23S rRNA; (25), was used as a positive control in these assays. ATPase and both adenovirus and β-globin pre-mRNA. The amount of reactions containing [γ- P]ATP and a 10-fold excess of cold released phosphate in each reaction was measured as described in nucleoside triphosphates showed competition only by unlabelled Materials and Methods and is depicted in graphic form in Figure ATP, suggesting that only ATP is a substrate for p72 (data not 6B. (Interestingly, ssDNA from phage M13 also stimulated a low shown). The K of p72 for ATP was found to be 170 μM (data level of ATP hydrolysis. The ssDNA preparation was treated with not shown). This value is within the range reported for p68 RNAse A prior to use to preclude an RNA contamination.) No 3744 Nucleic Acids Research, 1996, Vol. 24, No. 19 Figure 5. Assay of ATP hydrolysis by p72. Radioactive phosphate released from [γ- P]ATP was separated (migration from left to right) on a polyethyleneimine thin layer chromatography (PEI TLC) plate. The presence or absence of protein and RNA in the reaction is indicated. HeLa, total HeLa RNA. HeLa + RNAse A, total HeLa RNA pre-incubated with RNAse A. 200 ng of purified p72 or DbpA and 800 ng of either total HeLa RNA or E.coli 16S and 23S rRNA were used per reaction. activity was observed in the presence of total HeLa DNA or poly(U) RNA. The latter observation is particularly relevant since p72 can obviously bind poly(U) RNA as is shown by its purification over a poly(U)–Sepharose column. The ATPase activity of p72 is, therefore, likely to be dependent on RNA secondary structure. We therefore conclude that the ATPase activity of p72 can be stimulated by a variety of RNAs from various species and that this activity appears to require RNA secondary structure. Figure 6. Stimulation of p72 ATPase activity by various RNA substrates. (A) Sub-cellular localisation of p72 32 Radioactive phosphate released from [γ- P]ATP was separated (migration from bottom to top) on a PEI TLC plate. The substrates used to stimulate the We were interested in determining the sub-cellular localisation of ATPase activity of p72 are indicated. ‘Yeast’ indicates S.cerevisiae. Controls p72. Since the anti-p72 antibody did not give a specific signal in include incubation of [γ- P]ATP without p72 (lane 1) or with p72 (lane 2) in the absence of nucleic acids. 200 ng of purified p72 and 1500 ng of nucleic acids immunolocalisation experiments, we constructed a plasmid in were used per reaction except for poly(U) RNA (10 000 ng) and β-globin and which p72 was fused to a myc-epitope tag at its N-terminus and adeno pre-mRNA (5000 ng). (B) Released phosphate and [γ- P]ATP from expressed under the control of the SV40 early promoter. This each lane above were measured by c.p.m.-Cerenkov and plotted as the tagged construct was used in transient expression studies using percentage of c.p.m.-Cerenkov of Pi/c.p.m.-Cerenkov total. Percentage values are indicated at the top of the columns. HeLa cells and detected using a monoclonal anti-myc antibody. Myc-tagged p72 localises to the nucleus of HeLa cells (Fig. 7A, E and I) as determined by co-staining with DAPI (Fig. 7C). Tagged p72 shows a predominantly granular nuclear staining that of tagged p72 (Fig. 7K). In all cases the cells show normal cell pattern (Fig. 7A) with occasional elevated levels of peri-nucleolar morphology as judged by DIC microscopy (Fig. 7D, H and L). staining (Fig. 7E and I, indicated by arrowheads). Consistent with previous studies untransfected cells are not labelled by anti-myc DISCUSSION antibody (data not shown). The high homology between p72 and p68 prompted us to compare the sub-cellular localisation of these In this study we have identified and characterised p72, a novel two proteins. P68 as previously reported (43) shows a diffuse human member of the DEAD box family of proteins. P72 is a granular nuclear distribution in interphase cells (Fig. 7F) and nuclear protein and we have detected p72 mRNA ubiquitously colocalises with tagged-p72 (Fig. 7G). Since several DEAH-box expressed in all human tissues tested. Biochemical studies using proteins from Saccharomyces cerevisiae such as PRP2, PRP16 recombinant p72 protein expressed in E.coli showed that it has and PRP22 have been shown to be involved in pre-mRNA RNA-dependent ATPase activity which is stimulated in vitro by splicing [for review see ref. (6)] we were interested in whether a range of RNAs, including preparations of tRNA, mRNA and p72 localises to splicing snRNP-enriched nuclear organelles rRNA from bacteria, yeast and mammals. called ‘coiled bodies’ [for review see ref. (46)]. HeLa cells P72 is strikingly similar to the human p68 protein, which is one transiently expressing tagged p72 show an average of 2–5 coiled of the prototypic DEAD box proteins, originally isolated due to bodies (Fig. 7J) and their staining pattern does not overlap with a cross-reaction with a monoclonal antibody (DL3C4/PAb204) 3745 Nucleic Acids Research, 1996, Vol. 24, No. 19 3745 Nucleic Acids Research, 1994, Vol. 22, No. 1 Figure 7. Transient expression of p72 in HeLa cells. Localisation pattern of myc-epitope-tagged p72 was determined by indirect immunofluorescence analysis using anti-myc antibodies on HeLa cells fixed 24 h after transfection. Cells were double labelled with anti-myc antibody ( A, E and I) (red) and either DAPI (B) (blue), anti-p68 peptide antibody 2907 (F) (green) or with anti-coilin antibody (J) (green). The respective overlays are shown in (C, G and K). Colocalisation of both antibodies in (G) and (K) produce a yellow colour. Corresponding DIC images are shown in parallel in (D, H and L). The bar represents 10 μm, peri-nucleolar localisation of tagged p72 is indicated by arrowheads. Anti-myc antibody was visualised with anti-mouse secondary antibodies coupled to Texas Red and the anti-p68 peptide antibody 2907 was visualised with an anti-rabbit secondary antibody coupled to fluorescein (FITC). raised against SV40 large T antigen (13). These two proteins are ~ 90% identical in the core domains, their N- and C-termini are more closely related to each other than to any other members of much less conserved. the DEAD box family analysed to date. Interestingly, S.cerevisiae The p68 and p72 proteins also show a functional as well as has only one apparent homologue of p68/p72, called DBP2 (43). structural relationship. P68 has previously been shown to have While DBP2 was previously identified as the yeast homologue of RNA-dependent ATPase and RNA helicase activities in vitro p68, and can be complemented by human p68 (47), it in fact (18,19). As described above, p72 also exhibits RNA-dependent shows a higher degree of sequence homology to p72. We propose ATPase activity. However, we have so far been unable to detect that p68 and p72 represent a specific subfamily of the DEAD box RNA helicase activity for p72 (data not shown). There are several protein family. The fact that mammals appear to have at least two possible explanations for this finding: (a) the recombinant p72 members of this subfamily suggests either that there is some does not unwind RNA under the assay conditions used; (b) since functional redundancy or, perhaps more likely, that these proteins the recombinant p72 was purified from bacterial inclusion bodies, exhibit some specialisation in their substrate specificities. This it may not have the correct conformation for helicase activity, would be consistent with the observation that while they are even though it shows ATPase activity; (c) other factors are 3746 Nucleic Acids Research, 1996, Vol. 24, No. 19 necessary for p72 to unwind RNA [e.g. eIF-4A, which is a much ACKNOWLEDGEMENTS more efficient RNA helicase when part of the eIF-4F complex The authors wish to thank Kerstin Bohmann for the anti-p80 (48,49), and RhlB which exhibits ATP-dependent RNA helicase coilin antibody and Dr G. Evan for monoclonal anti-myc activity when part of the ‘degradosome’ but not as free protein antibodies as well as the EMBL sequencing service for technical (50)]; or (d) unlike p68, p72 is not actually an RNA helicase in assistance. We are also especially grateful to Karsten Weis and Joe vivo. In this regard it is worth noting that relatively few DEAD Lewis for critical reading of the manuscript. Parts of this work box proteins have been shown to exhibit helicase activity (2). were supported by a grant from Boehringer Ingelheim Fonds, an Although the DEAD box proteins are usually referred to as EMBO short-term fellowship to GML and a Medical Research ‘helicases’ it may in fact be the case that their common function Council Senior Fellowship to FFP. 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. 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p72: A Human Nuclear DEAD Box Protein Highly Related to p68

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Oxford University Press
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© 1996 Oxford University Press
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0305-1048
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10.1093/nar/24.19.3739
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Abstract

 1996 Oxford University Press Nucleic Acids Research, 1996, Vol. 24, No. 19 3739–3747 p72: a human nuclear DEAD box protein highly related to p68 1 1 2, Gábor M. Lamm, Samantha M. Nicol , Frances V. Fuller-Pace and Angus I. Lamond * Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria, Department of Molecular and Cellular Pathology, University of Dundee Medical School, Ninewells Hospital, Dundee DD1 9SY, Scotland and University of Dundee, Department of Biochemistry, Dundee DD1 4HN, Scotland Received June 20, 1996; Revised and Accepted August 12, 1996 DDBJ/EMBL/GenBank accession no. U59321 ABSTRACT hrpA gene product of hitherto unknown function (8) and HRH1, the putative human homologue of PRP22 (9). Interestingly, all of these P72, a novel human member of the DEAD box family of proteins are exceptionally large. The Drosophila Maleless (Mle) putative RNA-dependent ATPases and ATP-dependent protein involved in X chromosome dosage compensation and its RNA helicases was isolated from a HeLa cDNA library. human orthologe, RNA helicase A, are most similar to the DEAH The predicted amino acid sequence of p72 is highly box proteins although they have the D-E-I-H motif (10,11). Another homologous to that of the prototypic DEAD box sub-family, containing a D-E-x-H motif, includes RNA helicases of protein p68. In addition to the conserved core domains positive strand RNA viruses (12). characteristic of DEAD box proteins, p72 contains To date, four human members of the DEAD box family have several N-terminal RGG RNA-binding domains and a been reported. Apart from the prototypic member, p68 (13), there serine/glycine rich C-terminus likely involved in me- are currently three other human DEAD box proteins: p54, which diating protein–protein interactions. A p72-specific was cloned from a human lymphoid cell line (14); NP52, isolated probe detects two mRNAs of approximately 5300 and from a HeLa expression library due to a cross reaction with a 9300 bases which, although ubiquitously expressed, monoclonal antibody raised against human aldolase A (15) and show variability in their expression levels in different DDX1, which was found amplified in two retinoblastoma cell tissues. Purified recombinant p72 exhibits ATPase lines (16). P54, NP52 and DDX1 have not been further activity in the presence of a range of RNA moieties. characterised biochemically and their function(s) remains Immunocytochemical studies of p68 and p72 show unknown, although DDX1 has been found amplified in some that these proteins localise to similar locations in the primary neuroblastomas (17). In the case of p68, the purified nucleus of HeLa cells, suggesting their involvement in protein has been shown to exhibit RNA-dependent ATPase a nuclear process. activity and functions as an RNA helicase in vitro (18,19). In this paper we describe the identification and characterisation INTRODUCTION of p72, a novel human nuclear DEAD box protein, which shows a striking homology to p68. We demonstrate that p72 is an The D-E-A-D box protein family (1) of putative RNA helicases ATPase activated by a variety of RNA species but not by dsDNA. includes over 40 proteins from a wide range of organisms, The localisation and possible functional roles of p72 are discussed spanning bacteria to humans, that share a group of conserved and compared with other DEAD box proteins. motifs including the sequence Asp-Glu-Ala-Asp (D-E-A-D) which provides their name [for review see refs (2–5)]. These MATERIALS AND METHODS proteins are implicated in diverse cellular functions including splicing, ribosome assembly, translation initiation, spermatogenesis, cDNA cloning and sequencing mRNA stability, embryogenesis and cell growth and division. The DEAD box family is characterised by a core region The cDNA clone #461 coding for the N-terminal part of p72 was represented by eIF-4A [eukaryotic (translation) initiation factor isolated from a random primed expression library of HeLa 4A] and contains eight conserved amino acid regions, one of poly(A) RNA prepared in pUEX (20) (gift of Dr T. Kreis) due which is the D-E-A-D motif [also called DEAD box (1,4)]. The to a cross reaction with an unrelated monoclonal antibody. Clone conserved core region is flanked by N- and C-terminal extensions #461 was subcloned into the KpnI site of Bluescript KS which share little sequence homology and are probably involved in (Stratagene) and sequenced on both strands using oligonucleotide mediating specialised functions of the individual proteins. The primers either with the dideoxy chain termination method (21) DEAH (Asp-Glu-Ala-His) sub-family includes the Saccharomyces using [α- S]dATP or with fluorescent primers by the EMBL cerevisiae gene products PRP2, PRP16 and PRP22 involved in sequencing service. Radiolabelled clone #461 was then used as a pre-mRNA splicing [reviewed in refs (6,7)], the Escherichia coli probe to screen a λ Zap HeLa cDNA library (Stratagene) to To whom correspondence should be addressed 3740 Nucleic Acids Research, 1996, Vol. 24, No. 19 isolate additional clones spanning the missing 3′ terminus of the 50 mM Tris–HCl (pH 8.0), 1 mM DTT, 1 mM benzamidine], p72 cDNA. A cDNA encoding full-length p72 was assembled loaded onto poly(U)–Sepharose swollen in Buffer F and eluted from the resulting clones and subcloned into the SmaI site of using 100 mM KCl steps from 0.5 to 1 M KCl. The eluate was pBluescript SK(–). This construct is henceforth referred to as again concentrated over an Amicon filter column to 1/5 of its p72-pBS SK. 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/μl. Construction of expression vectors To express the recombinant p72 in the E.coli strain BL21(DE3) Antibody production a BamHI–BamHI fragment of p72 (purified from p72-pBS SK) BL21(DE3) cells expressing a fragment of p72 corresponding to which encoded for the full-length cDNA was cloned in-frame amino acids 1–343 were treated as described above for the with the poly His-tag into the T7 driven pRSET expression vector 2+ purification of full-length recombinant p72. After Ni -NTA- (Invitrogen). For antibody production a fragment of p72 containing Agarose chromatography the pooled fractions were electro- amino acids 1–343, was subcloned into pRSET in-frame with the phoresed through an SDS–PAGE gel, Coomassie stained and the poly His-tag. p72 fragment excised from the gel. The acrylamide slice was For expression in HeLa cells the p72 cDNA was subcloned in macerated and 300 μg of recombinant p72 was mixed with 2 vol frame into the BamHI site of the eukaryotic expression vector Feund’s complete adjuvant (Sigma) and injected into rabbits. pSG5 (22) containing a myc-tag (MEQKLISEEDL) (23). In all Further injections were carried out at three week intervals using cases, correct orientation of the constructs was confirmed by 300 μg protein and Freund’s incomplete adjuvant. restriction digestion analysis and DNA sequencing. ATP hydrolysis assays Growth and induction of bacteria expressing p72 ATP hydrolysis assays were carried out as described in (25) Fresh overnight cultures of BL21(DE3) containing p72 cDNAs containing RNA or DNA species as described in the appropriate in the pRSET plasmid under the IPTG-inducible T7 promoter figure legends. The amount of phosphate hydrolysed from (24) were diluted 30-fold, grown to an OD (650 nm) of 0.3–0.4 [γ- P]ATP was determined by counting the relevant areas of the at 37C and induced by the addition of 0.75 mM IPTG. The TLC plate (as Cerenkov counts) in a liquid scintillation counter. cultures were transferred to 26C and grown for a further 4 h E.coli 16S and 23S rRNA was purchased from Boehringer. before being harvested by centrifugation. The cell pellets were washed in 50 mM Tris–HCl (pH 7.4), harvested by centrifugation In vitro transcription and stored at –70C. Uniformly labelled, capped rabbit β-globin pre-mRNA and wild- type adenovirus pre-mRNA were transcribed as described in (26). Purification of p72 The bacterial pellet was resuspended in ice cold Buffer A (1 ml Northern blotting per 100 mg pellet) containing 6 M guanidine–HCl, 0.1 M NaPi The BamHI–SspI 5′ fragment of p72 was radiolabelled by random (pH 8.0), 10 mM Tris–HCl (pH 8.0), 5 mM imidazole, 1 mM priming (27) and used to probe a commercial multiple tissue phenylmethylsulphonylfluoride (PMSF), 1 mM benzamidine, Northern blot of human poly(A) RNA (Clontech) as according 2 μg/ml leupeptin, 2 μg/ml aprotinin and placed in an ice/salt water to the manufacturer’s recommendations. bath for 30 min with intermittent vortexing. The resuspended bacterial pellet was then sonicated twice for 30 s to shear DNA and SDS–PAGE and Western blotting the insoluble material was pelleted by centrifugation. A denaturing protocol was necessary for the purification of p72 as the protein SDS–PAGE gel analysis was performed according to (28) and was found in bacterial inclusion bodies. The supernate was then transferred onto nitrocellulose membrane (Schleicher and 2+ incubated on a rotating wheel at 4C with Ni -NTA-Agarose Schuell). Membranes were blocked in 2% non-fat milk powder (3 ml packed volume for every 10 ml of supernate) for 3 h, was in phosphate buffered saline (PBS), incubated with the primary washed once with Buffer A and then resuspended again in Buffer antibody for 2 h at room temperature, washed and incubated with A and poured into a disposable BioRad column. The resin was the appropriate secondary antibody (Amersham) coupled to washed with 10 column volumes of Buffer A followed by 2 horseradish peroxidase. Immunoblots were developed with the column volumes of Buffer B (identical to Buffer A but containing ECL detection kit (Amersham) as according to the manufac- 10 mM imidazole). The recombinant protein was eluted with 2.5 turer’s recommendations. column volumes Buffer C (as Buffer A but containing 200 mM imidazole) and eluates were collected as 1 ml fractions. Fractions Cell culture, transfection and immunofluorescent containing recombinant p72 (determined by running an aliquot on microscopy SDS–PAGE and Coomassie staining) were pooled and dialysed at 4C overnight into Buffer D [20% glycerol, 500 mM KCl, HeLa cells were grown on coverslips at 37C with 5% CO in 50 mM Tris–HCl (pH 8.0), 0.5 mM EDTA, 1M guanidine–HCl, Dulbecco’s modified Eagle’s medium (Gibco BRL) supplemented 1 mM DTT, 1 mM benzamidine, 1 mM PMSF] (using with 10% foetal calf serum, 100 U/ml penicillin and streptomycin 0.25 litres/1 ml fraction) and then 3 h into Buffer E (same as (Gibco BRL) and 1% glutamine. The myc-tagged p72 construct in Buffer D but containing 250 mM KCl). The protein was then pSG5 was transfected with LipofectAMINE transfection reagent concentrated to 1/10 of its original volume over an Amicon filter (Gibco BRL) according to the manufacturer’s protocol and the cells column, diluted 1:20 into Buffer F [15% glycerol, 50 mM KCl, were fixed with 3.7% paraformaldehyde in CSK buffer [100 mM 3741 Nucleic Acids Research, 1996, Vol. 24, No. 19 3741 Nucleic Acids Research, 1994, Vol. 22, No. 1 NaCl, 300 mM sucrose, 10 mM PIPES (pH 6.8), 3 mM MgCl , 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 Figure 1. Amino acid sequence of p72. The motifs typical of the DEAD box from Dianova and diluted 1:500. family of helicases are boxed. The N-terminal RGG boxes and the C-terminal glycine hinge region and proline tract are underlined. The p72 DNA sequence has been deposited in GenBank—accession number U59321. Sequence analysis The compilation and analysis of DNA sequences was done using the University of Wisconsin Genetics Computer Group N- and C-terminal extensions. The N-terminus contains four (UWGCG) programmes (31) on a Vax computer cluster at repeats of the RGG box originally identified as an RNA binding EMBL, Heidelberg. The molecular weight and amino acid motif in the hnRNP U protein (35). A run of seven consecutive composition of p72 was determined using the Peptidesort glycines separate the last conserved DEAD box family domain programme (31). The TFasta or BLAST (32) programmes were (HRIGR) and the serine/glycine rich (13.2 and 17.8%, respectively) used to search for homologies between p72 and the GenEMBL C-terminus of p72. The extreme C-terminus of p72 additionally data banks. The CLUSTAL V programme (33) was used to search contains nine consecutive prolines. Serine/glycine rich regions for amino acid homologies in the Swissprot database. The Motifs have been shown to mediate protein–protein interactions in programme (31) was used to search for p72 protein motifs in the cytokeratins (36) and proline rich motifs appear to fulfil a similar ProSite data bank. function in hnRNP, snRNP and poly(A)-binding proteins (37–39) as well as in several transcription factors (40,41). We conclude that RESULTS p72 encodes a novel human DEAD box protein which, in addition to the conserved core motifs, contains domains that may modulate Cloning and structural organisation of p72 p72–RNA and p72–protein interactions. A 1.3 kb cDNA fragment encoding the N-terminal portion of p72 was isolated from a HeLa expression library during expression p72 shows striking homology to p68 screening with an unrelated antibody. The cDNA fragment was used to further screen HeLa cDNA libraries and a 1.1 kb fragment The deduced amino acid sequence of p72 was used to carry out encoding the C-terminal portion of p72 was isolated. The a BLAST (basic local alignment search tool) search of the overlapping cDNAs contain an open reading frame (ORF) of Swiss-Prot database. This search revealed a striking homology 1950 bp capable of encoding a protein with a predicted molecular between p72 and p68, a prototypic member of the DEAD box mass of 71.9 kDa and an isoelectric point of 8.73 (Fig. 1). The family (13). A multiple alignment of the first 481 amino acids of presumed ATG initiation codon is 259 bp from the 5′-end of the p72, encompassing the conserved DEAD box motifs, with the isolated cDNA and the upstream sequence contains stop codons translated, most closely related DEAD box protein entries in the in all three reading frames (data not shown). The 3′ untranslated DDBJ/EMBL/GenBank database is presented in Figure 2. Out of sequence is at least 59 bp in length and contains neither a poly(A) 650 residues in p72, 453 residues (69.7%) are identical in human tail nor a consensus polyadenylation signal (34), suggesting that p68 and 457 residues (70.3%) are identical in mouse p68. An p72 mRNA contains additional 3′ untranslated sequence. In vitro additional 53 residues in human and 52 residues in mouse p68 are translation of the assembled p72 cDNA in a reticulocyte lysate similar amino acid substitutions (77.6 and 78.4% similarity, system yields a labelled translation product that migrates with an respectively). Within the region spanning the conserved motifs apparent molecular weight of 79 kDa on SDS-polyacrylamide characteristic of this family (2) the homology between p72 and gels (data not shown) and an antibody raised against recombinant p68 is ~ 90%, which is considerably higher than that seen between p72 specifically detects a protein migrating at 79 kDa on Western other members of the family. However, C-terminal to the last blots of HeLa nuclear or cytoplasmic extracts (Fig. 4B, lane 7). conserved DEAD box domain (HRIGR) (Fig. 1) the identity This indicates that both recombinant and endogenous p72 between human p68 and p72 drops to 27.5%, suggesting that migrates aberrantly at 79 kDa on SDS–PAGE. these proteins have different functions in the cell. This also The deduced amino acid sequence of p72 demonstrates that it supports the established view that DEAD box proteins have a is a new member of the DEAD box family of proteins containing similar core region encompassing the conserved domains but all the conserved domains which are hallmarks of this family have N- and C- terminal extensions which endow the proteins (Fig. 1). In addition to the conserved core domain, p72 contains with specialised functions [for review see refs (3,4)]. 3742 Nucleic Acids Research, 1996, Vol. 24, No. 19 Figure 2. Amino acid sequence alignment of the first 481 amino acids of p72 with other DEAD box proteins. Multiple alignment was done using the CLUSTAL V programme (33) and generated by the PRETTYPLOT programme. Consensus positions are calculated from at least two most often occurring residues at a particular position taking into account amino acid similarity values. Boxed residues represent plurality values above 4.0. The accession numbers of the aligned sequences are: mmp68 (X65627), hsp68 (X15729), dmRM62 (X52846), spdbp2 (L11574) and scDBP2 (X55993). P72 is also very similar to the Drosophila RM62 protein (60% 5300 bases, the isolated p72 cDNA sequence only spans 2268 identity, 73% similarity) (42). The DEAD box proteins DBP2 and contiguous base pairs which lacks a poly(A) signal and poly(A) dbp2, which are the putative S.cerevisiae and S.pombe homologues tail. It is, therefore, likely that p72 mRNA contains additional of p68 (43), also show strong similarity with p72 (Fig. 2). downstream and perhaps also upstream untranslated regions. The Interestingly, full-length p72, in comparison to p68, appears to be two p72 transcripts may arise by transcription of independent slightly more similar to both dbp2 and DBP2 (p72/dbp2 = 58.2%, genes, differential transcription of a common gene or by p68/dbp2 = 54.4%, p72/DBP2 = 55.3%, p68/DBP2 = 53.2%). A alternative splicing of a common pre-mRNA. These results search of the complete S.cerevisiae genome sequence (Martinsried suggest that the expression of separate p72 transcripts is regulated Protein Sequence database) for p72-like sequences found only in a tissue specific manner. Interestingly, when the same blot was DBP2, suggesting that either (a) there is some redundancy in the probed for p68 mRNA two transcripts were also observed. function of these two proteins, or (b) multicellular organisms However, these p68 transcripts differed from p72 in both their require both proteins. 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 P72 is encoded by two transcripts to tissue specific regulation. A human multiple tissue northern blot of poly(A) RNA was probed with two non-overlapping cDNA fragments encompassing Purification of p72 the 5′ half (Fig. 3) and 3′ portion of p72 (data not shown). Both cDNA fragments gave identical patterns of mRNA distribution in Histidine-tagged p72 was expressed in E.coli and purified to the different tissues and both recognise mRNA transcripts of homogeneity as described in Materials and Methods (Fig. 4A and approximately 5300 and 9300 bases (Fig. 3). The 5300 transcript B). Bacteria transformed with the p72 plasmid and induced with appears to be ubiquitously expressed in all tissues tested with IPTG abundantly express the histidine-tagged protein, as is similar levels of expression in heart, brain, placenta, lung and apparent by the appearance of an extra protein band migrating at liver and apparently higher levels of expression in skeletal 79 kDa on Coomassie stained gels (compare Fig. 4, lanes 1 and 2+ muscle, kidney and pancreas. The 9300 transcript is also 2). Ni -NTA-Agarose chromatography of the bacterial lysate ubiquitously expressed, although extremely low levels are harbouring recombinant p72 yielded a substantial purification of detected in heart and placenta and the transcript is most the protein (Fig. 4, lane 3). A final poly(U)–Sepharose abundantly expressed in kidney and pancreas. The ratio between chromatography step yielded recombinant p72 purified to the two transcripts is also highly variable in the different tissues. homogeneity (Fig. 4A, lane 4). The additional bands detected While in brain, liver, kidney and pancreas the two transcripts are after the poly(U)–Sepharose purification step are degradation expressed at similar levels, in heart, placenta, lung and skeletal products of p72 (see below). muscle predominantly the 5300 base transcript is present. A In order to verify the purification protocol of recombinant p72, cDNA probe encompassing the p68 coding region verified that Western blot analysis of the various purification steps was carried neither transcript represents a cross-reaction with p68 mRNA out using an anti-p72 antibody and the MAD1 monoclonal (data not shown). Although the smallest transcript detected is antibody (Fig. 4B). The MAD1 monoclonal antibody was raised 3743 Nucleic Acids Research, 1996, Vol. 24, No. 19 3743 Nucleic Acids Research, 1994, Vol. 22, No. 1 Figure 3. Multiple tissue Northern analysis. Poly(A) RNA (2 μg/ lane) from human tissues was probed with an AvaI–SspI fragment of p72 cDNA encompassing the 5′ portion of p72. Tissues are marked above the lanes, position of migration of RNA size markers are shown on the right and the arrows indicate the p72 transcripts. 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. 4B, 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. 4B, lane 7). The MAD1 monoclonal antibody detects recombinant p72 in the lysate of E.coli carrying the p72 plasmid (Fig. 4B, lane 2) but not in bacteria transformed with the vector alone (Fig. 4B, lane 1). Although MAD1 detects recombinant p72 Figure 4. Purification of recombinant p72. Coomassie stain (A) or Western blot 2+ as a single protein band of 79 kDa after Ni -NTA-Agarose analysis (B) of proteins recovered during the various purification steps, separated on 10% SDS–PAGE. (A) BL21, total protein lysate of E.coli BL21 chromatography (Fig. 4B, lane 3) it detects several bands after transformed with expression plasmid alone (lane 1). BL21 + p72, as above but poly(U)–Sepharose chromatography (Fig. 4B, lane 4). These with expression plasmid containing p72 (lane 2). Proteins present after additional bands correspond to degradation products of p72 as 2+ Ni -NTA-Agarose chromatography and after poly(U)–Sepharose chromatog- after poly(U)–Sepharose purification an identical pattern is raphy are separated on lanes 3 and 4, respectively. (B) Western blot analysis of purification steps probed with both MAD1 monoclonal and anti-p72 antibodies. observed with an anti-p72 antibody (Fig. 4B, lane 6). This Abbreviations are as in (A). The antibody used to probe the different lanes is purification protocol routinely yielded 1–1.5 mg of homogenous indicated under the brackets. NE, HeLa nuclear extract (lanes 5 and 7). Protein p72 from a one litre bacterial culture. size markers are indicated in kDa on the left (A + B) and recombinant p72 is shown by the arrowhead on the right. ATPase activity of p72 A common feature of DEAD box protein family members is their ability to hydrolyse ATP in the presence of RNA (2). The ATPase (100–1000 μM) (18), DbpA (150 μM) (25) and eIF-4A (50 μM) activity of purified p72 was tested by its ability to release (45). Taken together, the results above clearly demonstrate that radioactive phosphate from [γ- P]ATP. P72 hydrolysed ATP in p72 is an RNA-dependent ATPase. the presence of total HeLa RNA and exhibited no ATPase activity Further ATPase assays were carried out to determine whether in the absence of RNA (Fig. 5). Moreover, the ATPase activity of the ATPase activity of p72 could be preferentially stimulated by p72 was abolished when total HeLa RNA was pre-treated with a specific RNA moiety or by DNA. As shown in Figure 6A, the RNAse A indicating that the ATPase activity of the protein was ATPase activity of p72 was stimulated by a variety of RNAs. dependent on the added RNA. The E.coli DEAD box protein These include total RNA and tRNA from HeLa cells, E.coli and DbpA, which is specifically activated by E.coli ribosomal RNA yeast; rabbit and E.coli rRNA; purified E.coli 16S and 23S rRNA; (25), was used as a positive control in these assays. ATPase and both adenovirus and β-globin pre-mRNA. The amount of reactions containing [γ- P]ATP and a 10-fold excess of cold released phosphate in each reaction was measured as described in nucleoside triphosphates showed competition only by unlabelled Materials and Methods and is depicted in graphic form in Figure ATP, suggesting that only ATP is a substrate for p72 (data not 6B. (Interestingly, ssDNA from phage M13 also stimulated a low shown). The K of p72 for ATP was found to be 170 μM (data level of ATP hydrolysis. The ssDNA preparation was treated with not shown). This value is within the range reported for p68 RNAse A prior to use to preclude an RNA contamination.) No 3744 Nucleic Acids Research, 1996, Vol. 24, No. 19 Figure 5. Assay of ATP hydrolysis by p72. Radioactive phosphate released from [γ- P]ATP was separated (migration from left to right) on a polyethyleneimine thin layer chromatography (PEI TLC) plate. The presence or absence of protein and RNA in the reaction is indicated. HeLa, total HeLa RNA. HeLa + RNAse A, total HeLa RNA pre-incubated with RNAse A. 200 ng of purified p72 or DbpA and 800 ng of either total HeLa RNA or E.coli 16S and 23S rRNA were used per reaction. activity was observed in the presence of total HeLa DNA or poly(U) RNA. The latter observation is particularly relevant since p72 can obviously bind poly(U) RNA as is shown by its purification over a poly(U)–Sepharose column. The ATPase activity of p72 is, therefore, likely to be dependent on RNA secondary structure. We therefore conclude that the ATPase activity of p72 can be stimulated by a variety of RNAs from various species and that this activity appears to require RNA secondary structure. Figure 6. Stimulation of p72 ATPase activity by various RNA substrates. (A) Sub-cellular localisation of p72 32 Radioactive phosphate released from [γ- P]ATP was separated (migration from bottom to top) on a PEI TLC plate. The substrates used to stimulate the We were interested in determining the sub-cellular localisation of ATPase activity of p72 are indicated. ‘Yeast’ indicates S.cerevisiae. Controls p72. Since the anti-p72 antibody did not give a specific signal in include incubation of [γ- P]ATP without p72 (lane 1) or with p72 (lane 2) in the absence of nucleic acids. 200 ng of purified p72 and 1500 ng of nucleic acids immunolocalisation experiments, we constructed a plasmid in were used per reaction except for poly(U) RNA (10 000 ng) and β-globin and which p72 was fused to a myc-epitope tag at its N-terminus and adeno pre-mRNA (5000 ng). (B) Released phosphate and [γ- P]ATP from expressed under the control of the SV40 early promoter. This each lane above were measured by c.p.m.-Cerenkov and plotted as the tagged construct was used in transient expression studies using percentage of c.p.m.-Cerenkov of Pi/c.p.m.-Cerenkov total. Percentage values are indicated at the top of the columns. HeLa cells and detected using a monoclonal anti-myc antibody. Myc-tagged p72 localises to the nucleus of HeLa cells (Fig. 7A, E and I) as determined by co-staining with DAPI (Fig. 7C). Tagged p72 shows a predominantly granular nuclear staining that of tagged p72 (Fig. 7K). In all cases the cells show normal cell pattern (Fig. 7A) with occasional elevated levels of peri-nucleolar morphology as judged by DIC microscopy (Fig. 7D, H and L). staining (Fig. 7E and I, indicated by arrowheads). Consistent with previous studies untransfected cells are not labelled by anti-myc DISCUSSION antibody (data not shown). The high homology between p72 and p68 prompted us to compare the sub-cellular localisation of these In this study we have identified and characterised p72, a novel two proteins. P68 as previously reported (43) shows a diffuse human member of the DEAD box family of proteins. P72 is a granular nuclear distribution in interphase cells (Fig. 7F) and nuclear protein and we have detected p72 mRNA ubiquitously colocalises with tagged-p72 (Fig. 7G). Since several DEAH-box expressed in all human tissues tested. Biochemical studies using proteins from Saccharomyces cerevisiae such as PRP2, PRP16 recombinant p72 protein expressed in E.coli showed that it has and PRP22 have been shown to be involved in pre-mRNA RNA-dependent ATPase activity which is stimulated in vitro by splicing [for review see ref. (6)] we were interested in whether a range of RNAs, including preparations of tRNA, mRNA and p72 localises to splicing snRNP-enriched nuclear organelles rRNA from bacteria, yeast and mammals. called ‘coiled bodies’ [for review see ref. (46)]. HeLa cells P72 is strikingly similar to the human p68 protein, which is one transiently expressing tagged p72 show an average of 2–5 coiled of the prototypic DEAD box proteins, originally isolated due to bodies (Fig. 7J) and their staining pattern does not overlap with a cross-reaction with a monoclonal antibody (DL3C4/PAb204) 3745 Nucleic Acids Research, 1996, Vol. 24, No. 19 3745 Nucleic Acids Research, 1994, Vol. 22, No. 1 Figure 7. Transient expression of p72 in HeLa cells. Localisation pattern of myc-epitope-tagged p72 was determined by indirect immunofluorescence analysis using anti-myc antibodies on HeLa cells fixed 24 h after transfection. Cells were double labelled with anti-myc antibody ( A, E and I) (red) and either DAPI (B) (blue), anti-p68 peptide antibody 2907 (F) (green) or with anti-coilin antibody (J) (green). The respective overlays are shown in (C, G and K). Colocalisation of both antibodies in (G) and (K) produce a yellow colour. Corresponding DIC images are shown in parallel in (D, H and L). The bar represents 10 μm, peri-nucleolar localisation of tagged p72 is indicated by arrowheads. Anti-myc antibody was visualised with anti-mouse secondary antibodies coupled to Texas Red and the anti-p68 peptide antibody 2907 was visualised with an anti-rabbit secondary antibody coupled to fluorescein (FITC). raised against SV40 large T antigen (13). These two proteins are ~ 90% identical in the core domains, their N- and C-termini are more closely related to each other than to any other members of much less conserved. the DEAD box family analysed to date. Interestingly, S.cerevisiae The p68 and p72 proteins also show a functional as well as has only one apparent homologue of p68/p72, called DBP2 (43). structural relationship. P68 has previously been shown to have While DBP2 was previously identified as the yeast homologue of RNA-dependent ATPase and RNA helicase activities in vitro p68, and can be complemented by human p68 (47), it in fact (18,19). As described above, p72 also exhibits RNA-dependent shows a higher degree of sequence homology to p72. We propose ATPase activity. However, we have so far been unable to detect that p68 and p72 represent a specific subfamily of the DEAD box RNA helicase activity for p72 (data not shown). There are several protein family. The fact that mammals appear to have at least two possible explanations for this finding: (a) the recombinant p72 members of this subfamily suggests either that there is some does not unwind RNA under the assay conditions used; (b) since functional redundancy or, perhaps more likely, that these proteins the recombinant p72 was purified from bacterial inclusion bodies, exhibit some specialisation in their substrate specificities. This it may not have the correct conformation for helicase activity, would be consistent with the observation that while they are even though it shows ATPase activity; (c) other factors are 3746 Nucleic Acids Research, 1996, Vol. 24, No. 19 necessary for p72 to unwind RNA [e.g. eIF-4A, which is a much ACKNOWLEDGEMENTS more efficient RNA helicase when part of the eIF-4F complex The authors wish to thank Kerstin Bohmann for the anti-p80 (48,49), and RhlB which exhibits ATP-dependent RNA helicase coilin antibody and Dr G. Evan for monoclonal anti-myc activity when part of the ‘degradosome’ but not as free protein antibodies as well as the EMBL sequencing service for technical (50)]; or (d) unlike p68, p72 is not actually an RNA helicase in assistance. We are also especially grateful to Karsten Weis and Joe vivo. In this regard it is worth noting that relatively few DEAD Lewis for critical reading of the manuscript. Parts of this work box proteins have been shown to exhibit helicase activity (2). were supported by a grant from Boehringer Ingelheim Fonds, an Although the DEAD box proteins are usually referred to as EMBO short-term fellowship to GML and a Medical Research ‘helicases’ it may in fact be the case that their common function Council Senior Fellowship to FFP. 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. 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Nucleic Acids Research – Oxford University Press

Published: Oct 1, 1996

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