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J. Biol. Chem., Vol. 277, Issue 2, 1066-1075, January 11, 2002

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The mRNA of DEAD Box Protein p72 Is Alternatively Translated into an 82-kDa RNA Helicase^*

Heike Uhlmann-Schiffler, Oliver G. Rössler, and Hans Stahl

From the Fachbereich Medizinische Biochemie und Molekularbiologie, Fachrichtung Theoretische Medizin, Universität des Saarlandes, D-66421 Homburg, Germany

Received for publication, August 7, 2001, and in revised form, October 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

p68 and p72 are two highly related DEAD box proteins with similar biochemical activities in the nucleus of vertebrate cells; it is unknown whether they have redundant or differential in vivo functions. We report on a third member of this subfamily that is alternatively expressed from p72 mRNA. A detailed analysis of HeLa p72 mRNA was performed. It has an overall length of more than 5 kb and contains a 0.75-kb 5'-untranslated region and a 3'-untranslated region of 2.5 kb. Its open reading frame extends to nucleotide 243 upstream of the first in-frame AUG (A in the AUG triplet is +1) which serves as the p72 translation initiator codon. We provide evidence that alternative translation at a non-AUG within the extra coding region of this mRNA yields an 82-kDa protein (p82). Immunological studies substantiate that p82 is a naturally existing p72 variant and that both proteins are expressed at similar concentrations. p82 purified from HeLa cells is an ATP-dependent RNA helicase with biochemical properties almost identical to those of p72.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Control of transcription is generally considered the main regulation level of mammalian gene expression, although mRNA maturation and modulation of mRNA stability or translational efficiency also contribute to expression control (1). Generation of protein variants by alternative pre-mRNA splicing or alternative initiation of mRNA translation enlarges the coding capacity and may explain the relatively low number of genes actually found in the human genome. According to recent estimations, on average one gene encodes three proteins (2).

Although the 5'-untranslated leader sequences (UTRs)¹ of most vertebrate mRNAs are less than 100 nucleotides (nt) long (3), those of tightly controlled genes usually are longer and are GC-rich. Such structure-prone 5'-UTRs can modulate translation by the presence of upstream (up) open reading frames (ORFs) in addition to the main ORF, by formation of secondary structures, and/or by RNA-protein interactions (4). Indeed, they appear particularly characteristic of mRNAs encoding growth factors, receptor proteins, transcription factors, signal transduction components, proto-oncogenes, tumor suppressor proteins, and even proteins that mostly are constitutively expressed at a low level ("housekeeping" proteins).

DEAD/DEXH box proteins belonging to superfamily II of RNA helicases play a universal role in RNA metabolism of pro- and eukaryotes. Not surprisingly, they are involved in gene expression control, for instance during germ cell and embryonic development or in cell proliferation and differentiation (5). This in turn requires tight control of at least some DEAD/DEXH box proteins like CsdA of Escherichia coli, which is regulated by an 11-nt "cold box" sequence in its 5'-UTR (6), or the Saccharomyces cerevisiae Dbp2, the gene of which contains an unusually large intron that is part of a post-transcriptional autoregulatory feedback loop (7). A similarly complex transcription regulation has been shown for nuclear p68 (8), a mammalian homologue of Dbp2. In addition, the 5'-UTR of p68 mRNA is long (170 nt) and GC-rich (57.6% as compared with a genome-wide average of 41%; see Ref. 9). This suggests a regulatory mechanism of p68 expression on mRNA level and thus an important cellular function of the protein. In fact, p68 is differentially expressed during embryogenesis, and its expression is induced by serum (10, 11).

p72, another human DEAD box protein (12), is highly homologous to p68. It is, like p68, transcribed in a tissue-specific manner and seems to be involved in neuronal differentiation (13). p68 and p72 share 78.6% homology (identical amino acids plus conservative substitutions). The two proteins (plus possibly not yet identified ones) apparently form a subfamily of DEAD box proteins. p68 and p72 are ATP-dependent RNA helicases with similar biochemical properties, and both proteins show RNA annealing activity (14). In vitro, p68 and p72 are capable of catalyzing RNA secondary structure rearrangements in an ATP-dependent manner via the formation of RNA branch migration structures (14). Both proteins may therefore participate in structural reorganization of ribonucleoprotein assemblies in vivo. In fact, p68 has been found in spliceosomes (15) and in a protein-RNA complex of 5-methylcytosine-DNA glycosylase (16). A recently reported (17) interaction with protein kinase A anchoring protein AKAP95 supports a role of p68 in hormonally responsive transcription complexes, and indeed, p68 and p72 act as transcriptional co-activators of estrogen receptor  in cooperation with an RNA co-activator (18, 19).

Recently, several transcription factors and other regulatory proteins have attracted great interest due to the existence of alternatively transcribed/translated species, and functional importance is attributed to this feature. Intriguingly, two different p72-specific RNAs (5.3 and 9.3 kb) occur in vertebrate cells that are much longer than the p72 coding sequence (cds; 1953 nt, GenBank^TM accession number NM_006386; see Ref. 12). It is unknown as yet which of those RNAs represents the p72 mRNA. In this work, p72 mRNA including its 5'- and 3'-UTRs has been analyzed in detail. We report that in vitro and in vivo translation of the complete ORF of the p72 mRNA leads to an alternative polypeptide, p82, in addition to p72. The specific biochemical activities of p82 isolated from HeLa cells are characterized and compared with those of p72 and p68.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents and Routine Procedures-- Restriction endonucleases were purchased from Roche Molecular Biochemicals or MBI Fermentas, and plasmids pGEM7Zf() and pGEM3 were from Promega. pMOS, chromatography media, and columns were supplied by Amersham Biosciences. All other reagents were of the highest purity grade commercially available. cDNA library screening, PCR, cloning procedures, plasmid preparations, and electrophoreses were carried out according to standard protocols (20). DNA sequencing was done by SEQLAB GmbH, Goettingen, Germany.

Oligonucleotides, Probes, and Antibodies-- Oligonucleotides used are listed in Table I. For HeLa  cDNA library screening, a p72-specific digoxigenin-labeled cDNA probe (dig-p72), which corresponds to positions +93 to +631 of p72 cDNA, was synthesized by PCR of human p72 cDNA using oligonucleotide primers dig5 and dig3. A p72 gene exon 13-specific antisense RNA probe (complementary to nt 1467-1986 of the p72 cDNA) was synthesized for Northern blotting by AvaII digest of clone 2423 (see below), followed by transcription with T7 RNA polymerase to yield the respective antisense RNA. A p72 gene intron 11-specific probe was prepared by RT-PCR of HeLa poly(A)⁺ RNA with primers 5_i11 and 3_i11, followed by XhoI/BamHI recloning into pGEM7Zf() and transcription with SP6 RNA polymerase.

For preparation of p82-specific antibodies (-p82^N), a peptide (NH₂-TVASATGDSASERES-COOH) corresponding to p82-specific N-terminal amino acids (cf. Fig. 6) was chemically synthesized on an Applied Biosystems 433A Peptide Synthesizer, coupled to hemocyanin, and used as an antigen for multiple immunizations of rabbits. Antibodies -p72^C were raised against a p72 deletion mutant (consisting of amino acids 437-650) expressed in E. coli (21). Antibodies -p68-(45-59) (22) were raised against an N-terminal peptide of p68 (amino acids 45-59) and also recognized p72, which contains two conservative substitutions in this sequence. Affinity purification of polyclonal antibodies was carried out on N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (Amersham Biosciences) to which the respective antigen had been coupled. C10 is a p68-specific monoclonal antibody directed against the C-terminal amino acids 600-614 (23). DEAD box-specific monoclonal antibody MaD1 (24) is directed against the amino acid sequence NH₂-LVLDEADRMLDMGFEPQ-COOH. Monoclonal antibody 10C4 was raised against a peptide (NH₂-VGAKP-COOH) of Tms1 protein of Schizosaccharomyces pombe (25).

Genomic Sequence Analysis and Predictions-- DNA and protein sequence homology searches were done using the BLAST and FASTA service of the NCBI (www.ncbi.nlm.nih.gov/) and the ExPASY Molecular Biology server (www.expasy.ch/). Gene sequence analysis was carried out using the Baylor College of Medicine Search Launcher (searchlauncher.bcm.tmc.edu/) and splice site prediction by Neural Network (www.fruit fly.org/se). RNA secondary structure predictions were performed with mfold version 3.1 (26) that gives credible results for RNAs up to 1 kb (27).

RNA Isolation from HeLa Cells, Northern Blotting, Reverse Transcription, and RT-PCR-- Poly(A)⁺ RNA was isolated from 3.6 × 10⁷ HeLa cells using the Oligotex Direct mRNA kit (Qiagen) according to supplier's recommendations except that RNA was eluted with 200 µl of preheated (85 °C) elution buffer. Northern blotting of HeLa poly(A)⁺ RNA was carried out using dig-labeled probes (Digoxigenin System, Roche Molecular Biochemicals) corresponding to p72 gene exon 13-specific antisense RNA and to p72 gene intron 11-specific antisense RNA, respectively, and detected with CSPD (Roche Molecular Biochemicals). For HeLa cDNA synthesis, 1 µg of poly(A)⁺ RNA and 10 pmol of Marathon cDNA synthesis primer (CLONTECH) were heated at 70 °C for 2 min and immediately chilled on ice. Then, first and second strand DNA synthesis was performed exactly as described by the Marathon cDNA amplification protocol (CLONTECH). RT-PCRs with DNase-treated HeLa poly(A)⁺ RNA as the template were performed according to a two-step protocol using M-MLV reverse transcriptase, RNase H minus (Promega), 1 µg of RNA, 1 µg of the respective 3'-oligonucleotide primer (Table I) at 42 °C for 1 h. Taq (Amersham Biosciences) or Pfu DNA polymerase (Promega) were used in the subsequent PCR. In respective negative controls, the poly(A)⁺ RNA was directly used in PCRs and reconfirmed the absence of traces of genomic DNA. Sequences of the RT-PCR products were analyzed by DNA sequencing.

In Vitro Transcription and Translation-- A p72 cDNA (position 297 to position +2125) was obtained by HeLa  cDNA library screening with the dig-p72 probe, cloned into pMOS, and subsequently recloned into pGEM7Zf() via EcoRI to yield clone 2423. The p72 cds was PCR-amplified with BamHI- and HindIII-tagged primers BamN72 and HinC72 (Table I) and cloned into pGEM7Zf() to yield clone 1953. Insert sequences were verified by DNA sequencing. Plasmids were linearized by XhoI digest and transcribed in vitro with SP6 RNA polymerase using the Riboprobe run-off system (Promega) to yield clone 2423 RNA and clone 1953 RNA, respectively. Concentrations of transcripts were determined photometrically, and their quality was checked by agarose gel electrophoresis. In vitro translation in a reticulocyte lysate was performed using [³⁵S]methionine according to the Flexi Rabbit protocol (Promega).

Transient Expression in COS Cells-- Stop codons (TGA) were removed from the cDNA of clones 2423 and 1953, respectively, and an oligonucleotide coding for the 10C4 epitope (5'-GCTCAAAAGTATGTTGGTGCAAAGCCC-3') was introduced by PCR cloning at the 3' ends of their coding sequences with primers i11 (containing an NcoI site) and 10C472 (containing an XhoI site; Table I). The cDNAs obtained were then recloned into pCMV, a vector suitable for transient expression in COS cells, yielding pCMVp82 and pCMVp72, respectively. DNA constructs were verified by nucleotide sequencing. Particular attention was paid to avoid any vector-based ATG in front of the p72 5'-ORF. COS cell transfection was done by the DEAE-dextran method using 10 µg of pCMVp82 and pCMVp72, respectively.

SDS-PAGE, Autoradiography, and Western Blotting-- Standard SDS-PAGE gels were prepared and run according to Ref. 28. For autoradiography, gels containing ³⁵S-labeled protein samples were incubated in enhancer solution (2 M salicylic acid, pH 8) for 30 min, dried, and exposed at 70 °C for 5-16 h. Western blotting was carried out according to Ref. 20 using alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate for detection.

Immunoprecipitation-- 3.6 × 10⁹ HeLa cells were harvested by centrifugation at 300 × g for 5 min, resuspended in 1 ml of lysis buffer (10 mM MES, pH 6.2, 10 mM NaCl, 1.5 mM MgCl₂, 5 mM dithiothreitol, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol, 1% Nonidet P-40, supplemented with Complete Protease Inhibitor Mixture (Roche Molecular Biochemicals) according to manufacturer's recommendations), and recentrifuged at 3000 × g for 5 min. Nuclei were extracted at pH 10.5 as described previously (29) and mixed with protein A-Sepharose-coupled antibodies. After washing with NET buffer (50 mM Tris·Cl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.05% Nonidet P-40), bound proteins were eluted with SDS-PAGE loading buffer and directly analyzed by SDS-PAGE and Western blotting.

Metabolic ³³P Labeling of HeLa Cells-- HeLa cells grown to 80% confluency were incubated in phosphate-free medium supplemented with 250-500 µCi of [³³P]orthophosphate for 16 h. A nuclear extract of the cells was prepared and subjected to immunoprecipitation with the indicated antibodies as described. Immunoprecipitation of p53 was carried out as a positive control with antibody PAb421 (30). Immunoprecipitates were subsequently analyzed by SDS-PAGE and autoradiography.

Purification of p82 from HeLa Cells-- A nuclear extract was subjected to hydroxyapatite chromatography and ammonium sulfate precipitations according to Ref. 29. Briefly, the buffered (pH 8.3) nuclear extract was applied to a hydroxyapatite column (10 × 30 mm) and washed with 50 mM potassium phosphate, pH 8.0, containing 10 mM -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride. Proteins were eluted from the column by a salt gradient (starting from 50 to 350 mM potassium phosphate). p82-containing fractions were subjected to a fractionated ammonium sulfate precipitation (15 and 33% saturation, respectively). Proteins precipitated at 15-33% saturation were washed in 25 mM Tris·Cl, pH 8.5, 2 mM EDTA, 10 mM -mercaptoethanol, 7.5% glycerol, 300 mM NaCl, diluted 2-fold and applied to a DEAE-Sepharose Fast Flow column, followed by a HiTrap Heparin HP column. p82 was eluted by a salt gradient (270 mM to 1 M NaCl), diluted to 100 mM NaCl (final concentration), and applied onto an ssDNA cellulose column, from which it was eluted by a salt gradient (150-700 mM NaCl). p82 fractions were pooled and dialyzed in 25 mM Tris·Cl, pH 7.8, 50 mM NaCl, 1 mM EDTA, 10 mM -mercaptoethanol, 20% glycerol, 0.1% Triton X-100. Sucrose gradient (5-15%) centrifugation was performed with 2 µg of p82 in a Beckman SW55 rotor at 270,000 × g, 0 °C for 24 h. Aldolase (160-kDa tetramer) and bovine serum albumin (68 kDa) were carried along as external 7.4 S and 4.2 S sedimentation markers, respectively.

ATPase Assays-- ATP hydrolysis was assayed according to Ref. 31. Standard 50-µl assays contained varying amounts of p82 in 35 mM HEPES, pH 7.8, 7.5 mM MgCl₂, 2 mM dithiothreitol, 1% glycerol, 0.1 mg/ml bovine serum albumin, 4 µg of poly(C) or poly(A)⁺ RNA, 0.1 mM ATP, and 0.5 µCi of [-³²P]ATP. To check for RNA dependence of the p82 ATPase activity, poly(C) or poly(A)⁺ RNA was omitted from the respective samples. Incubation was carried out at 37 °C for 45 min, stopped by addition of 200 mg/ml activated charcoal (which binds remaining ATP), and centrifuged at 20,000 × g for 5 min. Amounts of released ³²P_i in the supernatants were determined by scintillation counting.

RNA Binding Assays-- Binding of p82 to RNA that had been ³²P-labeled by in vitro transcription of HaeIII-digested pGEM-CAT was assayed by nitrocellulose filter retention of the respective protein-RNA complex as described previously (14).

RNA Helicase Assays-- The 17-bp partial dsRNA substrate, the 3'-substrate (25-bp ds region), and the 5'-substrate (28-bp ds region) were constructed exactly as described (32). One strand of the 44-bp substrate was produced by run-off transcription of NheI-linearized plasmid pGEM3 by T7 RNA polymerase. The other strand was transcribed by SP6 RNA polymerase from EcoRI-linearized pGEM-MO1/2 (32). Hybridization of both transcripts yielded the 44-bp substrate. RNA helicase activity assays of p82 were performed exactly as described (14).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Structure of the p72 mRNA-- The mRNA of p72 was analyzed by RT-PCR and by Northern blotting (Fig. 1 and Table I). When a p72 cDNA was used as a probe in these experiments, a prominent p72-specific RNA species of 5.3 kb was detected. This RNA contains the entire p72 cds region without introns and hence seems to represent functional p72 mRNA. In addition, after overexposure of the Northern blot, much less abundant smaller and one larger (9.3 kb) RNA species became visible. The smaller RNAs varied in size from experiment to experiment, while the 9.3-kb species was also detected with a p72 intron 11-specific probe (Fig. 1A, lane 2), and according to our RT-PCR analysis includes intron 9 (0.25 kb) and intron 11 (4 kb) or intron 11 only in addition to the p72 cds. The occurrence of partially unspliced p72 RNA in the poly(A)⁺ RNA fraction may hint at a post-transcriptional autoregulatory mechanism of p72 similar to that of the highly related DEAD box protein p68 (21) and its yeast homologue Dbp2 (7). Expression of a p72 mRNA containing introns 9 or 11 would lead to polypeptides of 363 and 407 amino acids, respectively, which were not detected in HeLa cells (see below). Two p72 RNA species of about 5 and 9 kb had also been found previously in Northern blot analyses of human tissues (12) and vertebrate cell lines (13) in variable amounts and ratios.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Structure of the p72 mRNA. A, p72-specific RNA in HeLa cells. Northern blot analysis of poly(A)+ RNA performed with an exon 13-specific (lane 1) or an intron 11-specific (lane 2) p72 antisense RNA. The position of RNA size markers is given on the left, and the arrows indicate the p72 RNAs. B, RT-PCR analysis of the p72 5'- (lanes 2-4) and 3'-UTRs (lanes 6-11). Poly(A)+ RNA prepared from HeLa cells was subjected to reverse transcription and PCR with the oligonucleotide primer pairs indicated above the lanes followed by agarose gel electrophoresis and DNA sequencing of the amplified fragments. M1 and M2 (lanes 1, 5, and 12) denote DNA markers, the sizes of which are shown on the left and right, respectively. C, comparison of the p72 mRNA structure predicted from the genomic sequence with that found by RT-PCR analysis. Sizes of amplification products obtained with the indicated primer pairs (cf. B) are shown.

The size of the 5.3-kb p72 mRNA indicates the existence of long untranslated region(s). To rationalize the molecular weight of p72 mRNA, we analyzed the genomic sequence of p72 (encoded by human chromosome 22; GenBank^TM accession number NT_011520.4) with respect to potential promoters and polyadenylation signals. In good agreement with the Northern blotting experiments, these studies predicted a p72 mRNA of 5213 nt (Fig. 1C), provided that no splicing occurs in the UTR(s). However, the use of a potential alternative promoter positioned about 3 kb further upstream could as well result in a similarly sized mRNA, but only if the 5'-UTR was processed by splicing. To distinguish between these two possibilities, the 3'- and 5'-UTRs of p72 mRNA were analyzed by scanning RT-PCR (followed by DNA sequencing) with oligonucleotide primers (Table I) derived from the genomic sequences adjacent to the p72 cds (Fig. 1, B and C). The p72 3'-UTR found by this approach consists of 2.5 kb and represents the uninterrupted gene sequence downstream of the p72 cds. Its 3' end does not match with the poly(A) addition sites predicted in positions +4441 or +4449 (the A of the p72 AUG initiator codon is designated as +1, with positive and negative integers proceeding 3' and 5', respectively) just behind the polyadenylation signal consensus sequence in position 4428, rather it appears to be located at least 50 nt further downstream; an alternative polyadenylation signal (AGTAAA) is found in position +4663 (Fig. 1, B and C).

The p72 5'-UTR (upstream of the first in-frame AUG, the p72 translation initiation site identified previously (12)) verified by RT-PCR and DNA sequencing is 0.75 kb long and GC-rich (60.2%) and also corresponds to the respective continuous gene sequence. The transcription initiation site is predicted in position 772, i.e. 25 nt upstream of the 5' end verified by RT-PCR, and just behind a weak TATA box (TATAGGA, position 798; cf. Fig. 6). Interestingly, the p72 ORF extends up to nt 243 upstream of the p72 translation initiator AUG.

Inspection of the genomic sequence revealed no AUG triplet which could be used as a translation start codon in this additional part of the ORF. Furthermore, a careful analysis of the sequencing data (not shown) obtained from RT-PCR products also excluded an RNA editing event in this part of the RNA which might create an upstream AUG start codon in a relevant portion of the p72 mRNA population.

Initiation of p72 mRNA Translation from a Non-AUG Codon in Vitro and in Vivo-- Few eukaryotic mRNAs are known in which a non-AUG codon serves as translation initiation site in addition to an AUG triplet located further downstream (33). To analyze whether the p72 5'-ORF possesses a similar capability, in vitro translation experiments were performed in a rabbit reticulocyte lysate with RNA obtained by in vitro transcription of clone 2423. This clone contains the p72 sequence from position 297 to +2125, i.e. the complete p72 ORF plus parts of its 5'- and 3'-UTRs, isolated from a HeLa  cDNA expression library. An in vitro transcript of clone 1953 (containing the p72 cds, nt +1 to +1953) served as a control. Both transcripts were of similar quality as checked by agarose gel electrophoresis (Fig. 2A). Clone 1953 RNA was efficiently translated into a 72-kDa product, whereas clone 2423 RNA gave rise to a, albeit weaker, single polypeptide band of 82 kDa (p82; Fig. 2B). This result implied that translation had started upstream of the p72 translation initiator AUG at the beginning of the ORF at a non-AUG start codon, leading to an N-terminal extension of the p72 polypeptide of 80-90 amino acids. The low translational efficiency apparently results from the usage of a non-AUG start codon, whereas the p72 AUG start codon was not at all used for translation initiation of clone 2423 RNA in the reticulocyte lysate. This result and the absence of higher molecular weight products in the clone 1953 translation sample renders p82 generation by post-translational modification in the reticulocyte lysate unlikely. Also, translation efficiency and quality of both p72 RNAs was independent of the presence or absence of a 5'-cap structure (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   In vitro translation of p72 RNA containing part of the 5'-UTR. A, analysis of in vitro transcribed RNA from clone 2423 (nucleotides 297 to +2125 of the p72 cDNA sequence; lane 3), clone 1953 (p72 coding sequence; nucleotides +1 to +1953; lane 4), and luciferase cDNA (positive control; lane 1) by agarose gel electrophoresis. The sizes of RNA marker fragments (M, lane 2) are given on the left. B, in vitro translation products of the transcripts shown in A were analyzed by SDS-PAGE and autoradiography. Translation of clone 2423 RNA yields an 82-kDa product (lanes 2 and 3), whereas p72 cds RNA is translated into a 72-kDa product (lanes 4 and 5) as indicated on the right. Translational efficiency was compared with that of luciferase RNA used as a positive control. The sizes of protein molecular weight markers are shown on the left.

Rabbit reticulocytes are highly specialized cells containing an ill-balanced translation system (34). Therefore, expression of the p72 cDNAs from clones 2423 and 1953 was analyzed by transient expression studies in COS cells (Fig. 3). To provide a specific means of detection of the heterologous p72 cDNA-derived polypeptides, the p72 cDNAs from clones 2423 and 1953 were tagged at their 3' ends with the coding sequence of an unrelated epitope recognized by monoclonal 10C4 antibody and subsequently cloned into pCMV, a eukaryotic expression vector, to yield pCMVp82 and pCMVp72, respectively. In a nuclear extract from COS cells transfected with pCMVp82, the 10C4 antibody clearly recognized two polypeptides of 82 and 72 kDa in a Western blot analysis (Fig. 3A). As both proteins were expressed at a similar concentration, a non-AUG start codon in addition to the previously identified p72 AUG is efficiently used for translation initiation in a p72 mRNA containing part of the 5'-UTR (up to nt 297). As expected, transfection of plasmid pCMVp72 into COS cells led to the expression of only the 72-kDa polypeptide (Fig. 3A).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   p82 and p72 are expressed from one mRNA and are native constituents of HeLa and COS cells. A Western blot analysis of nuclear extracts of HeLa cells and COS cells is shown. The sizes of protein molecular weight markers are shown on the left and the positions of p82, p72, and p68 on the right of each panel, respectively. pCMVp82 contains the p72 cDNA sequence from nucleotide 297 to +2125 under the control of the human cytomegalovirus major immediate early gene promoter 3'-terminally tagged with nucleotides encoding the epitope of monoclonal antibody 10C4. pCMVp72 is identical to pCMVp82 except that it contains the p72 cDNA sequence from nucleotide +1 to +1953. pCMV, used as a negative control, is the expression vector without p72 insert. A-C, lanes 1-3, respectively, were loaded with aliquots of the same samples. A, detection of polypeptides expressed from plasmids transiently transfected in COS cells by 10C4. p72 is expressed from pCMVp72 (lane 1), and p82 and p72 are expressed from pCMVp82 (lane 2). Lane 3, negative control. B, -p82N antibodies specifically identify p82. p82 is detected in COS cells (lanes 1 and 3) and HeLa cells (lane 4) as a native protein and is overexpressed in COS cells after transfection with pCMVp82 (lane 2) but not with pCMVp72 (lane 1) or pCMV (lane 3). Lanes 5 and 6 represent an analysis of an immunoprecipitation obtained from a HeLa nuclear extract with pre-immune serum (lane 5) and -p72C (lane 6). C, -p72C antibodies verify alternative expression of p82 and p72 from pCMVp82. Only p72 is expressed from pCMVp72 (lane 1), and p82 and p72 are expressed from pCMVp82 (lane 2). Endogenous p82 and p72 is detected in COS cells (lane 3) and HeLa cells (lane 4). D, the p68 subfamily of DEAD box proteins recognized by an antibody raised against a p68 N-terminal peptide (-p68-(45-59)). Endogenous p82, p72, and p68 are detected in HeLa nuclear extracts by -p68-(45-59) and MaD1 (lanes 1 and 2), and C10, which binds to the p68 C terminus, recognizes p68 only (lane 3).

p82 Is a Native Constituent of Higher Eukaryotic Cells-- To investigate whether endogenous p72 mRNA is subject to alternative translation initiation yielding p82 and to confirm the presence of the p82-specific N-terminal peptide, an antiserum directed against amino acids in putative positions 23-37 of p82, -p82^N, was produced in rabbits using the respective oligopeptide as an antigen. Indeed, 82-kDa endogenous proteins were specifically detected by -p82^N in the nuclear fraction of HeLa, COS (Fig. 3B), and mouse 3T3 cells indicating that they contain the amino acids encoded by the 5'-extended p72 ORF. Moreover, affinity-purified antibodies, raised against amino acids 437-650 of p72 (-p72^C), recognized both p72 and p82 (Fig. 3C). When HeLa cell nuclear extracts were immunoprecipitated with -p72^C, the 82-kDa protein was recognized by -p82^N in a Western blot of the immunoprecipitate (Fig. 3B). However, when immunoprecipitation was performed with -p82^N, p82 was not detected with -p72^C in a subsequent Western blotting experiment, indicating that the epitope of -p82^N is not exposed in the native protein thus precluding it from precipitation. Furthermore, p82 and p72 were detected in nuclei exclusively (not in the cytoplasm; not shown) and can only be extracted under stringent conditions (pH 10.5; cf. "Experimental Procedures") like it had been demonstrated for p68 which is closely associated with the nuclear matrix (17, 29).

COS and HeLa cells expressed p72 and p82 under normal growth conditions, although the expression level of both proteins seemed to be lower in COS cells (Fig. 3C). p82 and p72 are overexpressed in COS cells after transfection with the respective expression plasmids (Fig. 3, B and C).

Although -p72^C did not detect p68 (Fig. 3C), most probably due to the less conserved C-terminal part of the two proteins (amino acids 433-650 of p72, 50% homology of p72 and p68), -p68-(45-59) antibodies (raised against amino acids 45-59 of p68) bound to p68, p72, and p82 (Fig. 3D) which was expected because p72 (and hence p82) and p68 differ only by two conservative amino acid substitutions in this region. All three proteins were also recognized by monoclonal antibody MaD1, which is directed against the p68 DEAD box motif common also to p72 (and p82). Although the Western blots were certainly not in the linear range allowing quantitation, the p72 signal obtained with MaD1 suggested a very low p72 in vivo level relative to p82 and p68. However, we note that the relative intensity of this signal increased after a hydroxyapatite affinity chromatographic step (see "Purification of p82 from HeLa Cells" under "Experimental Procedures"), indicating a masking effect. Indeed, a Coomassie-stained gel loaded with an immunoprecipitate obtained with -p72^C antibodies showed similar amounts of p72 and p82 in nuclear extracts (Fig. 5, lane 5). Not surprisingly, monoclonal antibody C10 (raised against C-terminal amino acids 600-614 of p68) recognized neither p72 nor p82 (Fig. 3D).

No clue was found for the existence of an individual p82 cDNA in a respective HeLa cDNA by PCR experiments using degenerate oligonucleotide primers (Table I) deduced from amino acid sequences of the -p68-(45-59) and MaD1 antibody-binding sites (not shown). In conclusion, endogenous p82, as well as recombinant p82 expressed from pCMVp82, represents an alternatively translated p72 variant, distinguished from p72 by the amino acids encoded by the 5'-extended ORF of the p72 mRNA.

Biochemical Characterization of p82 RNA Helicase-- Apparently, the additional N-terminal amino acids do not alter the cellular localization of p82 but might affect the biochemical activities of the protein. p72 has been shown to be an RNA-dependent ATPase (14), and we found that p82 purified from HeLa cells (Fig. 4A) is capable of this activity in the presence of poly(C) or poly(A)⁺ RNA as well but not in the absence of nucleic acids (data not shown). RNA binding by p82 was confirmed in a nitrocellulose filter binding assay (not shown).

View larger version (43K): [in this window] [in a new window]   Fig. 4.   RNA helicase activity of p82. The helicase assays were performed with 0.25 nM partially dsRNA substrates (with one strand 32P-labeled) at 37 °C for 30 min and monitored by gel shift analysis. A, co-sedimentation of p82 and RNA helicase activity in a sucrose gradient. The left panel shows a Coomassie-stained SDS-PAGE of p82, purified from HeLa cells, after the ammonium sulfate precipitation and the ssDNA chromatography steps. Sizes of protein molecular weight markers are shown on the left. The upper right panel shows a Western blot analysis of sucrose gradient fractions with -p68-(45-59) antibodies. The position of p82 is indicated on the right. The lower panel represents RNA helicase activity of the respective sucrose gradient fractions with a 17-bp dsRNA substrate. The positions of dsRNA and ssRNA are marked. B, nucleotide specificity of p82 RNA helicase. Unwinding of a 17-bp substrate was monitored using the indicated NTPs (lanes 3-6). p72 RNA helicase activity (lanes 9-12) was carried along as a control. Native (lanes 1 and 7) and denatured substrates (lanes 2 and 8) indicate the positions of the respective dsRNA and released ssRNA. Similar results were obtained with dNTPs used instead of the respective NTPs. C, direction of dsRNA unwinding. RNA helicase assays were performed with substrates containing 3' (lanes 1-5) and 5' single-stranded overhangs (lanes 6-10) as shown schematically. Native (lanes 1 and 6) and denatured substrates (lanes 2 and 7) indicate the positions of the respective dsRNA and ssRNA. The reaction was carried out with 1 ng of p82 (lanes 4 and 9) or 2 ng of p82 (lanes 5 and 10). Lanes 3 and 8, samples contained 2 ng of p82 but ATP was omitted.

To test for p82 RNA helicase activity, we used partial dsRNA substrates formed from appropriate in vitro transcripts. A 17-bp substrate was efficiently unwound by purified p82 in an ATP-dependent manner (Fig. 4A), and the helicase activity co-sedimented with p82 in a sucrose gradient. As checked by Western blotting with -p68-(45-59) antibodies, the gradient fractions did not contain traceable amounts of p68 and/or p72 (Fig. 4A). The strands of RNA substrates with double-stranded regions longer than 40 bp could not be separated by the p82 in the RNA helicase assay, and this result resembles a feature of p68 and p72 (data not shown). As was observed with p68 (29) and p72 (14), ATP hydrolysis is essential for the helicase activity of p82 (Fig. 4, B and C). Also, a 3'-ss overhang of the RNA substrate is crucial for dsRNA unwinding by p82, and no helicase activity was observed when a 5'-ss overhang was present only (Fig. 4C).

The helicase activities of p68 and p72 slightly differ with respect to their optimum reaction conditions as has been reported recently (14). p82 resembles p72 regarding its (d)ATP specificity (Fig. 4B), whereas p68 RNA helicase is less nucleotide-specific (35). Also, the optimum salt concentration of p82 in the helicase assay was similar to that of p72 (0-25 mM NaCl; not shown) and differed from that of p68 which is considerably higher (80-100 mM NaCl) (35).

Phosphorylation modulates the activity of several RNA helicases, like eIF4A8 (36) and human Upf1 RNA helicase (37), and hence could be regulative for p72 and p82 RNA helicase activity as well. Phosphorylation of p72 and p82 has been checked by metabolic labeling of HeLa cells with inorganic ³³P (Fig. 5). Weak signals of ³³P-labeled p72 and p82 were observed after immunoprecipitation with -p72^C and SDS-PAGE followed by autoradiography. Notably, there was no indication for a preferred labeling of p82 as compared with p72 excluding that p82 represents a highly phosphorylated form of p72. p68, on the other hand, was not at all phosphorylated under these conditions (Fig. 5). In addition, no other post-translational modifications like glycosylation or sumosylation were observed with p72 and p82 (data not shown).

View larger version (63K): [in this window] [in a new window]   Fig. 5.   In vivo phosphorylation of p82 and p72. Metabolic labeling of HeLa cells was performed with inorganic 33P followed by immunoprecipitation with pre-immune serum (lane 3) or -p72C (lanes 4 and 5), SDS-PAGE and autoradiography (lanes 3 and 4) or Coomassie staining (lane 5). An immunoprecipitate with the p68-specific monoclonal antibody C10 (lane 2) shows that p68 is not phosphorylated. An immunoprecipitate with the p53-specific monoclonal antibody PAb421 (lane 1) served as a control and shows phosphorylated p53.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The size of full-length p72 mRNA determined by scanning RT-PCR is 5.25 kb. It contains the p72 cds (12) and additional long upstream and downstream sequences, which correspond to the respective continuous gene regions (Fig. 1). A much less abundant 9.3-kb RNA (cf. Fig. 1A and Ref. 12) was found to represent a partially unspliced p72 gene transcript, containing intron 11 or introns 9 and 11 in addition to the aforementioned sequences. Interestingly, partially unspliced p68 RNA species have also been reported, the abundance of which increases upon overexpression of p68 suggesting post-transcriptional expression control (21) similar to that found with Dbp2 of S. cerevisiae (7).

The 2.5-kb p72 3'-UTR ends downstream of the poly(A) addition sites in positions +4441 and +4449. Vertebrate 3'-UTRs are on average 500 bases, some up to 1 kb long. Extended 3'-UTRs have been ascribed a regulative function in protein expression by affecting message stability and/or translational efficiency by long range interactions with the 5'-UTR (38). Whereas most of the 3' elements analyzed so far are gene-specific, AU-rich elements (consensus sequence, 5'-AUUUA-3' in U-rich stretches) have been detected in several mRNAs and seem to affect mRNA stability. The AUUUA motif occurs twice in the p72 3'-UTR in a moderately U-rich region. However, AU-rich element-directed turnover mechanisms differ in extent of decay as well as in decay characteristics (39), and the stability of p72 mRNA needs to be analyzed in detail.

The 5'-UTR of p72 mRNA is 0.75 kb (Fig. 6), and even when compared with those of highly regulated proteins like c-Myc (0.56 kb (40)) and FGF-2 (0.3 kb (41)), this is an unusual size. It resides entirely within a CpG island (GC content 65%) which may explain why it could not be amplified by rapid amplification of cDNA ends experiments.² Secondary structure prediction confirmed that the sequence is structure-prone with an extensively base-paired stem (positions 590 to 436 and 222 to 116; G = 80 kcal/mol) and an overall free energy of 250 kcal/mol. Such a leader sequence is expected to reduce markedly translation of the respective mRNA (33) at least under certain conditions (42) most probably because the 40 S scanning ribosomal subunit is inefficient in resolving hairpin structures with G > 50 kcal/mol. Nevertheless, several cellular, highly structured mRNAs have been reported to be translated, although at reduced efficiency, even under conditions where cap-dependent translation is inhibited (43).

View larger version (50K): [in this window] [in a new window]   Fig. 6.   Structure of the complete 5'-UTR of the p82/p72 mRNA. The TATA box and initiator element (Inr) are indicated in boldface type. The predicted transcription start site in position 772 is double underlined. The 5'-most nucleotide (position 747) verified by RT-PCR is boxed. The upstream ORFs are marked by wavy lines, and their respective start and stop codons are in boldface type. Position 297 denotes the 5' end of clone 2423 cDNA. The p82/p72 ORF (italic lowercase letters) starts in position 243 downstream of the boxed stop codon. The positions of the upstream 237CUG (along with the calculated molecular weight of the polypeptide) and other theoretical non-AUG translation initiators (uppercase letters, underlined) are given. The peptide sequence of the -p82N epitope is indicated below the respective nucleotide sequence (positions 171 to 127), and the first six triplets encoding p72 are depicted in larger type size.

Cap-independent initiation of translation is thought to function via internal ribosome entry sites (IRES; reviewed in Refs. 44-46), yet convincing evidence for IRES-mediated translation initiation of cellular mRNAs is difficult to obtain (47). In our studies, part of the natural p72 mRNA 5'-UTR (in clone 2423 RNA) leads to a complete inhibition of p72 translation in the reticulocyte lysate system (Fig. 2B), and it remains to be analyzed whether the full-length 5'-UTR of p72 mRNA exerts a similar inhibitory effect known for other mRNAs with long 5'-UTRs as well, like that of c-Myc (48, 49). Interestingly, various picornavirus-encoded IRES elements (type I) generally also function poorly in standard cell-free systems (see Ref. 50 and reviewed in Ref. 44), supporting the idea that p72 (and possibly also c-Myc) translation may similarly be started by internal ribosome loading. Moreover, the function of viral IRES elements is independent of any virus-encoded protein(s). Therefore, the host cell translation apparatus must be capable of performing internal initiation, possibly using cellular trans-acting factor(s) that may be expressed in a cell type-specific manner and seem(s) to be absent, e.g. in the reticulocyte lysate.

Another feature not uncommon to tightly regulated genes is the occurrence of upstream ORFs, which according to the ribosome scanning mechanism throttle translation of the main ORF (51). It is beginning to be understood, however, that upstream ORFs participate in translational control by different mechanisms (52), and efficient translation of mammalian NF-B-repressing factor mRNA has been described despite the presence of multiple upstream ORFs (53). Upstream of the main ORF, the p72 5'-UTR contains three short upstream ORFs consisting of 32, 9, and 53 potential codons, respectively (Fig. 6). As deduced from the considerable expression of endogenous p72 and p82 in HeLa cells (Fig. 3, see below), their 5'-UTR seems to have no general inhibitory effect on the expression of both proteins in vivo. In conclusion, our studies on the structure of cellular p72 RNAs show that the 5.3-kb species is the functional mRNA from which p82 and p72 are alternatively translated. Therefore, we propose to actualize the gene nomenclature into "p82/p72 gene."

Sequence analysis (Fig. 6) as well as in vitro translation studies implicated that synthesis of p82 results from the usage of a non-AUG translation initiator codon, most probably from the ²³⁷CUG triplet. Natural non-AUG initiator codons are very rare in eukaryotes but have been found in the expression of some growth factors like c-Myc (54) and FGF-2 (41) where initiation takes place at respective upstream CUG triplets in addition to the first in-frame AUG codons. "Standard" non-AUG codons in eukaryotes (CUG, GUG, and ACG; see Refs. 55 and 56) mostly are poor initiators and depend on an optimal translation initiation context (particularly A³ and/or G⁺⁴) even more than conventional AUG start codons (33). ⁺¹AUG, the translational start site of p72, as well as ²³⁷CUG both have an A in the 3 position, but no G in the +4 position and thus can be regarded as moderate translation initiators whereby ²³⁷CUG should work even less efficiently. Yet, p72 and p82 seem to occur at similar concentrations in HeLa and COS cells (Fig. 5, lane 5).² Initiation in a suboptimal context may be aided by pausing of the scanning ribosome subunit at a moderately stable downstream secondary structure (57), and indeed, a small hairpin (G = 8 kcal/mol) is predicted about 10 nt downstream of ²³⁷CUG. Leaky ribosome scanning beyond this start site might lead to initiation of p72 translation at ⁺¹AUG, alternative to the hypothesis of an internal ribosome entry mechanism discussed above. In any case, the long p72 5'-UTR seems to represent a means to regulate differentially the synthesis of p82 and p72 under specific conditions or in different tissues (13). Translation of c-Myc, for instance, constantly proceeds from an upstream CUG, whereas translation starting from the first in-frame AUG is developmentally controlled (58).

p82 was shown to be a native component of HeLa and COS cells by our immunological studies using an antibody (-p82^N) produced against the N-terminal most hydrophilic 15 amino acid stretch of p82 (Fig. 6). In this context, we detected lower levels of endogenous p72 and p82 in COS as compared with HeLa cells. Reduced affinities of the antibodies used due to possible interspecies amino acid sequence differences seem unlikely on the basis of the high homology of human, rat, and chick p72 (13). Lower p72 and p82 levels in COS cells (which are SV40-transformed) may be due to the presence of SV40 T antigen, which is an RNA helicase and might render cellular helicases unnecessary. Remarkably, the ratio of endogenous p82 to p72 is not changed at the lower expression rate, indicating that alternative translation is not affected.

Alternatively translated proteins, like FGF-2, may differ in their subcellular localization (59), but we found that p82, like p72 and p68, is exclusively located in the nucleus. On the other hand, addition of N-terminal amino acids could affect functional activities of a protein. Biochemical characterization of p82 revealed that it is an ssRNA-dependent ATPase and an RNA helicase, thus the possibility that it might represent an inactive precursor or storage form of p72 can be dismissed. Biochemical activities of p82 closely resemble those of p72 (14) and p68 (35). The p82 RNA helicase proceeds in 3'  5' direction with regard to the binding strand (Fig. 4C), and like p72, it shows a low processivity. Whereas p68, albeit less efficiently, instead of (d)ATP can use (d)CTP, UTP, and dTTP for RNA unwinding (35), p82 and p72 are strictly dependent on (d)ATP (Fig. 4B). In addition, the helicase activity of p82, like that of p72, is salt-sensitive. Remarkably, p82 and p72 appear to be phosphorylated in HeLa cells, whereas p68 is not. It remains to be elucidated, however, whether the phosphorylation state of p82 and p72 modulates their functional activities. Very recently, an ATP-independent RNA annealing activity of p68 and p72 has been described. This novel activity in conjunction with the helicase function enables these proteins to catalyze RNA strand exchange reactions (14). Preliminary studies show that p82 mediates annealing of partially complementary RNA strands as well. One could envision that catalysis of RNA secondary structure rearrangements is a specific feature of all p68 subfamily members, although it is unknown how these activities are connected to their in vivo roles. Given the co-activator function of p68 and p72 (19), it will be of particular interest to uncover possible specific functions of p82 and p72 in transcription.

	ACKNOWLEDGEMENTS

We are grateful to S. Jungbluth for excellent technical assistance. We thank W. Nastainczyk and P. Scholtes for peptide synthesis and immunization of rabbits. 10C4 was kindly provided by M. Montenarh and pCMV by G. Thiel (Universität des Saarlandes, Homburg, Germany). MaD1 was a kind gift of R. Iggo (Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland).

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-6841-16-26019; Fax: 49-6841-16-26521; E-mail: bchsta@med-rz.uni-saarland.de.

Published, JBC Papers in Press, October 23, 2001, DOI 10.1074/jbc.M107535200

² H. Uhlmann-Schiffler, O. G. Rössler, and H. Stahl, unpublished results.

	ABBREVIATIONS

The abbreviations used are: UTR, untranslated region; ORF, open reading frame; nt, nucleotide; RT, reverse transcriptase; MES, 4-morpholineethanesulfonic acid; ds, double-stranded; ss, single-stranded; cds, coding sequence; dig, digoxigenin; IRES, internal ribosome entry sites; CSPD, disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1^3,7]decan}-4-yl)phenyl phosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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