Overexpression of a DEAD Box Protein (DDX1) in Neuroblastoma and Retinoblastoma Cell Lines
1998; Elsevier BV; Volume: 273; Issue: 33 Linguagem: Inglês
10.1074/jbc.273.33.21161
ISSN1083-351X
AutoresRoseline Godbout, Mary Packer, Wenjun Bie,
Tópico(s)Cell death mechanisms and regulation
ResumoThe DEAD box gene, DDX1, is a putative RNA helicase that is co-amplified with MYCN in a subset of retinoblastoma (RB) and neuroblastoma (NB) tumors and cell lines. Although gene amplification usually involves hundreds to thousands of kilobase pairs of DNA, a number of studies suggest that co-amplified genes are only overexpressed if they provide a selective advantage to the cells in which they are amplified. Here, we further characterize DDX1 by identifying its putative transcription and translation initiation sites. We analyze DDX1 protein levels inMYCN/DDX1-amplified NB and RB cell lines using polyclonal antibodies specific to DDX1 and show that there is a good correlation with DDX1 gene copy number, DDX1 transcript levels, and DDX1 protein levels in all cell lines studied. DDX1 protein is found in both the nucleus and cytoplasm ofDDX1-amplified lines but is localized primarily to the nucleus of nonamplified cells. Our results indicate that DDX1 may be involved in either the formation or progression of a subset of NB and RB tumors and suggest that DDX1 normally plays a role in the metabolism of RNAs located in the nucleus of the cell. The DEAD box gene, DDX1, is a putative RNA helicase that is co-amplified with MYCN in a subset of retinoblastoma (RB) and neuroblastoma (NB) tumors and cell lines. Although gene amplification usually involves hundreds to thousands of kilobase pairs of DNA, a number of studies suggest that co-amplified genes are only overexpressed if they provide a selective advantage to the cells in which they are amplified. Here, we further characterize DDX1 by identifying its putative transcription and translation initiation sites. We analyze DDX1 protein levels inMYCN/DDX1-amplified NB and RB cell lines using polyclonal antibodies specific to DDX1 and show that there is a good correlation with DDX1 gene copy number, DDX1 transcript levels, and DDX1 protein levels in all cell lines studied. DDX1 protein is found in both the nucleus and cytoplasm ofDDX1-amplified lines but is localized primarily to the nucleus of nonamplified cells. Our results indicate that DDX1 may be involved in either the formation or progression of a subset of NB and RB tumors and suggest that DDX1 normally plays a role in the metabolism of RNAs located in the nucleus of the cell. DEAD box proteins are a family of putative RNA helicases that are characterized by eight conserved amino acid motifs, one of which is the ATP hydrolysis motif containing the core amino acid sequence DEAD (Asp-Glu-Ala-Asp) (1Linder P. Lasko P.F. Ashburner M. Leroy P. Nielsen P.J. Nishi K. Schnier J. Slonimski P.P. Nature. 1989; 337: 121-122Crossref PubMed Scopus (629) Google Scholar, 2Wassarman D.A. Steitz J.A. Nature. 1991; 349: 463-464Crossref PubMed Scopus (180) Google Scholar, 3Schmid S.R. Linder P. Mol. Microbiol. 1992; 6: 283-292Crossref PubMed Scopus (449) Google Scholar). Over 40 members of the DEAD box family have been isolated from a variety of organisms including bacteria, yeast, insects, amphibians, mammals, and plants. The prototypic DEAD box protein is the translation initiation factor, eukaryotic initiation factor 4A, which, when combined with eukaryotic initiation factor 4B, unwinds double-stranded RNA (4Rozen F. Edery I. Meerovitch K. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1990; 10: 1134-1144Crossref PubMed Scopus (498) Google Scholar). Other DEAD box proteins, such as p68, Vasa, and An3, can effectively and independently destabilize/unwind short RNA duplexes in vitro (5Hirling H. Scheffner M. Restle T. Stahl H. 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Acad. Sci. U. S. A. 1991; 88: 2113-2117Crossref PubMed Scopus (62) Google Scholar). p68, found in dividing cells (15Ford M.J. Anton I.A. Lane D.P. Nature. 1988; 332: 736-738Crossref PubMed Scopus (160) Google Scholar), is believed to be required for the formation of nucleoli and may also have a function in the regulation of cell growth and division (16Iggo R.D. Lane D.P. EMBO J. 1989; 8: 1827-1831Crossref PubMed Scopus (125) Google Scholar,17Buelt M.K. Glidden B.J. Storm D.R. J. Biol. Chem. 1994; 269: 29367-29370Abstract Full Text PDF PubMed Google Scholar). Other DEAD box proteins are implicated in RNA degradation, mRNA stability, and RNA editing (18Iost I. Dreyfus M. Nature. 1994; 372: 193-196Crossref PubMed Scopus (113) Google Scholar, 19Py B. Higgins C.F. Krisch H.M. Carpousis A.J. Nature. 1996; 381: 169-172Crossref PubMed Scopus (481) Google Scholar, 20Missel A. Souza A.E. Nörskau G. Göringer H.U. Mol. Cell. Biol. 1997; 17: 4895-4903Crossref PubMed Scopus (96) Google Scholar). The human DEAD box protein geneDDX1 1The abbreviations used are: DDX1DEAD box 1NBneuroblastomaRBretinoblastomaRACErapid amplification of cDNA endsPAGEpolyacrylamide gel electrophoresisntnucleotide(s)MOPS4-morpholinepropanesulfonic acidbpbase pair(s)kbkilobase(s) or kilobase pair(s). was identified by differential screening of a cDNA library enriched in transcripts present in the two RB cell lines Y79 and RB522A (21Godbout R. Squire J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7578-7582Crossref PubMed Scopus (99) Google Scholar). The longest DDX1 cDNA insert isolated from this library was 2.4 kb with an open reading frame from position 1 to 2201. All eight conserved motifs characteristic of DEAD box proteins are found in the predicted amino acid sequence of DDX1 as well as a region with homology to the heterogeneous nuclear ribonucleoprotein U, a protein believed to participate in the processing of heterogeneous nuclear RNA to mRNA (22Kiledjian M. Dreyfuss G. EMBO J. 1992; 11: 2655-2664Crossref PubMed Scopus (517) Google Scholar, 23Godbout R. Hale M. Bisgrove D. Gene ( Amst. ). 1994; 138: 243-245Crossref PubMed Scopus (20) Google Scholar). The region of homology to heterogeneous nuclear ribonucleoprotein U spans 128 amino acids and is located between the first two conserved DEAD box protein motifs, 1a and 1b. DEAD box 1 neuroblastoma retinoblastoma rapid amplification of cDNA ends polyacrylamide gel electrophoresis nucleotide(s) 4-morpholinepropanesulfonic acid base pair(s) kilobase(s) or kilobase pair(s). The proto-oncogene MYCN encodes a member of the MYC family of transcription factors that bind to an E box element (CACGTG) when dimerized with the MAX protein (24Blackwood E.M. Eisenman R.N. Science. 1991; 251: 1211-1217Crossref PubMed Scopus (1480) Google Scholar, 25Amati B. Dalton S. Brooks M.W. Littlewood T.D. Evan G.I. Land H. Nature. 1992; 359: 423-426Crossref PubMed Scopus (379) Google Scholar). The MYCN gene is amplified and overexpressed in approximately one-third of all NB tumors (26Brodeur G.M. Seeger R.C. Schwab M. Varmus H.E. Bishop J.M. Science. 1984; 224: 1121-1124Crossref PubMed Scopus (1842) Google Scholar, 27Seeger R.C. Brodeur G.M. Sather H. Dalton A. Siegel S.E. Wong K.Y. Hammond D. N. Engl. J. Med. 1985; 313: 1111-1116Crossref PubMed Scopus (1724) Google Scholar). Amplification of MYCN is associated with rapid tumor progression and a poor clinical prognosis (26Brodeur G.M. Seeger R.C. Schwab M. Varmus H.E. Bishop J.M. Science. 1984; 224: 1121-1124Crossref PubMed Scopus (1842) Google Scholar, 27Seeger R.C. Brodeur G.M. Sather H. Dalton A. Siegel S.E. Wong K.Y. Hammond D. N. Engl. J. Med. 1985; 313: 1111-1116Crossref PubMed Scopus (1724) Google Scholar). MYCN overexpression is usually achieved by increasing gene copy number rather than by up-regulating basal expression of MYCN (27Seeger R.C. Brodeur G.M. Sather H. Dalton A. Siegel S.E. Wong K.Y. Hammond D. N. Engl. J. Med. 1985; 313: 1111-1116Crossref PubMed Scopus (1724) Google Scholar, 28Cohn S.L. Salwen H. Quasney M.W. Ikegaki N. Cowan J.M. Herst C.V. Sharon B. Kennett R.H. Rosen S.T. Prog. Clin. Biol. Res. 1991; 366: 21-27PubMed Google Scholar). Because gene amplification involves hundreds to thousands of kilobase pairs of contiguous DNA (29Cowell J.K. Annu. Rev. Genet. 1982; 16: 21-59Crossref PubMed Scopus (270) Google Scholar, 30Zehnbauer B.A. Small D. Brodeur G.M. Seeger R. Vogelstein B. Mol. Cell. Biol. 1988; 8: 522-530Crossref PubMed Scopus (40) Google Scholar, 31Akiyama K. Nishi Y. Nucleic Acids Res. 1991; 19: 6887-6894Crossref PubMed Scopus (12) Google Scholar, 32Schneider S.S. Hiemstra J.L. Zehnbauer B.A. Taillon-Miller P. Le Paslier D. Vogelstein B. Brodeur G.M. Mol. Cell. Biol. 1992; 12: 5563-5570Crossref PubMed Scopus (50) Google Scholar), it is possible that co-amplification of a gene located in proximity to MYCN may contribute to the poor clinical prognosis of MYCN-amplified tumors. TheDDX1 gene maps to the same chromosomal band asMYCN, 2p24, and is located ∼400 kb telomeric to theMYCN gene (33Amler L.C. Schürmann J. Schwab M. Genes Chromosomes Cancer. 1996; 15: 134-137Crossref PubMed Google Scholar, 34Kuroda H. White P.S. Sulman E.P. Manohar C. Reiter J.L. Cohn S.L. Brodeur G.M. Oncogene. 1996; 13: 1561-1565PubMed Google Scholar, 35Noguchi Y. Akiyama K. Yokoyama M. Kanda N. Matsunaga T. Nishi Y. Genes Chromosomes Cancer. 1996; 15: 129-133Crossref PubMed Google Scholar, 36Pandita A. Godbout R. Zielenska M. Thorner P. Bayani J. Squire J.A. Genes Chromosomes Cancer. 1997; 20: 243-252Crossref PubMed Scopus (22) Google Scholar). All four MYCN-amplified RB tumor cell lines tested to date are amplified for DDX1 (21Godbout R. Squire J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7578-7582Crossref PubMed Scopus (99) Google Scholar), 2R. Godbout, unpublished results. while approximately two-thirds of NB cell lines and 38–68% of NB tumors are co-amplified for both genes (37Manohar C.F. Salwen H.R. Brodeur G.M. Cohn S.L. Genes Chromosomes Cancer. 1995; 14: 196-203Crossref PubMed Scopus (55) Google Scholar, 38Squire J.A. Thorner P.S. Weitzman S. Maggi J.D. Dirks P. Doyle J. Hale M. Godbout R. Oncogene. 1995; 10: 1417-1422PubMed Google Scholar, 39George R.E. Kenyon R.M. McGuckin A.G. Malcolm A.J. Pearson A.D.J. Lunec J. Oncogene. 1996; 12: 1583-1587PubMed Google Scholar). George et al. (39George R.E. Kenyon R.M. McGuckin A.G. Malcolm A.J. Pearson A.D.J. Lunec J. Oncogene. 1996; 12: 1583-1587PubMed Google Scholar) found a significant decrease in the mean disease-free survival of patients withDDX1/MYCN-amplified NB tumors compared withMYCN-amplified tumors. Similarly, Squire et al. (38Squire J.A. Thorner P.S. Weitzman S. Maggi J.D. Dirks P. Doyle J. Hale M. Godbout R. Oncogene. 1995; 10: 1417-1422PubMed Google Scholar) observed a trend toward a worse clinical prognosis when both genes were amplified in the tumors of NB patients. To date, there have been no reports of a tumor amplified only for DDX1, and the role that this gene plays in cancer formation and progression is not known. Because of the high rate of rearrangements in amplified DNA (31Akiyama K. Nishi Y. Nucleic Acids Res. 1991; 19: 6887-6894Crossref PubMed Scopus (12) Google Scholar, 40Reiter J.L. Brodeur G.M. Genomics. 1996; 32: 97-103Crossref PubMed Scopus (33) Google Scholar), it is unlikely that a gene located ∼400 kb from the MYCNgene will be consistently amplified as an intact unit unless its product provides a growth advantage to the cell. Based on Southern blot analysis, the DDX1 gene extends over more than 30 kb, and there are no gross rearrangements of this gene inDDX1-amplified tumors (21Godbout R. Squire J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7578-7582Crossref PubMed Scopus (99) Google Scholar, 38Squire J.A. Thorner P.S. Weitzman S. Maggi J.D. Dirks P. Doyle J. Hale M. Godbout R. Oncogene. 1995; 10: 1417-1422PubMed Google Scholar). Furthermore, there is a good correlation between DDX1 transcript levels and gene copy number in the tumors analyzed to date. However, we need to show that DDX1 protein is overexpressed in DDX1-amplified tumors if we are to entertain the possibility that this protein plays a role in the tumorigenic process. Here, we isolate and characterize the 5′-end ofDDX1 mRNA and extend the DDX1 cDNA sequence by ∼300 nt. We identify the predicted initiation codon of DDX1 and generate antisera that specifically recognize DDX1 protein. We analyze levels of DDX1 protein in both DDX1-amplified and nonamplified RB and NB tumors and study the subcellular location of this protein in the cell. A human fetal brain cDNA library (Stratagene) was screened using a 320-bp DNA fragment from the 5′-end of the 2.4-kb DDX1 cDNA previously described (23Godbout R. Hale M. Bisgrove D. Gene ( Amst. ). 1994; 138: 243-245Crossref PubMed Scopus (20) Google Scholar). Phagemids containing positive inserts were excised from λ ZAP II following the supplier's directions. The ends of the cDNA inserts were sequenced using the dideoxynucleotide chain termination method with T7 DNA polymerase (Amersham Pharmacia Biotech). A human placenta genomic library (CLONTECH) was screened with the 5′-end of DDX1 cDNA. Positive plaques were purified, and the genomic DNA was analyzed using restriction enzymes and Southern blotting. EcoRI-digested DNA fragments from these clones were subcloned into pBluescript and digested with exonuclease III and mung bean nuclease to obtain sequentially deleted clones. The exon/intron map of the 5′ portion of the DDX1gene was obtained by comparing the sequence of DDX1 cDNA with that of the genomic DNA. We used the AmpliFINDER RACE kit (CLONTECH) to extend the 5′-end of DDX1 cDNA. Briefly, two μg of poly(A)+ RNA isolated from RB522A was reverse transcribed at 52 °C using either primer P1 or P3 (Fig. 1 A). The RNA template was hydrolyzed, and excess primer was removed. A single-stranded AmpliFINDER anchor containing an EcoRI site was ligated to the 3′-end of the cDNA using T4 RNA ligase. The cDNA was amplified using either primer P2 or P4 (Fig.1 A) and AmpliFINDER anchor primer. RACE products were cloned into pBluescript. Poly(A)+ RNAs were isolated from RB and NB cell lines as described previously (21Godbout R. Squire J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7578-7582Crossref PubMed Scopus (99) Google Scholar, 38Squire J.A. Thorner P.S. Weitzman S. Maggi J.D. Dirks P. Doyle J. Hale M. Godbout R. Oncogene. 1995; 10: 1417-1422PubMed Google Scholar). The 21-nt primers 5′-TTCGTTCTGGGCACCATGTGT-3′ (primer P4 in Fig. 1 A) and 5′-TGGGACCTAGGGCTTCTGGAC-3′ (primer P3 in Fig. 1 A) were end-labeled with [γ-32P]ATP (3000 Ci/mmol; Mandel Scientific) and T4 polynucleotide kinase. Each of the labeled primers was annealed to 2 μg of poly(A)+ RNA at 45 °C for 90 min, and the cDNA was extended at 42 °C for 60 min using avian myeloblastosis virus reverse transcriptase (Promega). The primer extension products were heat-denatured and run on a 8% polyacrylamide gel containing 7 m urea in 1× TBE buffer. A G + A sequencing ladder served as the size standard. The S1 nuclease protection assay to map the transcription initiation site of DDX1 was performed as described by Favaloro et al. (41Favaloro J. Treisman R. Kamen R. Methods Enzymol. 1980; 65: 718-745Crossref PubMed Scopus (785) Google Scholar). The DNA probe was prepared by digesting genomic DNA spanning the upstream region of DDX1 and exon 1 with AvaI, labeling the ends with [γ-32P]ATP (3000 Ci/mmol) and polynucleotide kinase, and removing the label from one of the ends by digesting the DNA with SphI (Fig. 4). The RNA samples were resuspended in a hybridization mixture containing 80% formamide, 40 mmPIPES, 400 mm NaCl, 1 mm EDTA, and the heat-denatured SphI–AvaI probe labeled at theAvaI site. The samples were incubated at 45 °C for 16 h and digested with 3000 units/ml S1 nuclease (Boehringer Mannheim) for 60 min at 37 °C. The samples were precipitated with ethanol; resuspended in 80% formaldehyde, TBE buffer, 0.1% bromphenol blue, xylene cyanol; denatured at 90 °C for 2 min; and electrophoresed in a 7 m urea, 8% polyacrylamide gel in TBE buffer. Poly(A)+RNAs were isolated from RB and NB cell lines as described previously (21Godbout R. Squire J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7578-7582Crossref PubMed Scopus (99) Google Scholar, 38Squire J.A. Thorner P.S. Weitzman S. Maggi J.D. Dirks P. Doyle J. Hale M. Godbout R. Oncogene. 1995; 10: 1417-1422PubMed Google Scholar). Two μg of poly(A)+ RNA/lane were electrophoresed in a 6% formaldehyde, 1.5% agarose gel in MOPS buffer (20 mm MOPS, 5 mm sodium acetate, 1 mm EDTA, pH 7.0) and transferred to nitrocellulose filter in 3 m sodium chloride, 0.3 m sodium citrate. The filters were hybridized to the following DNA probes,32P-labeled by nick translation: (i) a 1.6-kbEcoRI insert from DDX1 cDNA clone 1042 (21Godbout R. Squire J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7578-7582Crossref PubMed Scopus (99) Google Scholar), (ii) a 260-bp cDNA fragment spanning the 3′-end of DDX1exon 1 as well as exons 2 and 3, (iii) a 160-bp fragment derived from the 5′-end of DDX1 exon 1, and (iv) α-actin cDNA to control for lane to lane variation in RNA levels. Filters were hybridized and washed under high stringency. Southern blot analysis was as described previously (21Godbout R. Squire J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7578-7582Crossref PubMed Scopus (99) Google Scholar). To prepare antiserum to the C terminus of the DDX1 protein, we inserted a 1.8-kbEcoRI fragment from bp 848 to 2668 ofDDX1 cDNA (Fig. 1 B) intoEcoRI-digested pMAL-c2 expression vector (New England Biolabs). DH5α cells transformed with this vector were grown to mid-log phase and induced with 0.1 mmisopropyl-1-thio-β-d-thiogalactoside. The cells were harvested 3–4 h postinduction and lysed by sonication. Soluble maltose binding protein-DDX1 fusion protein was affinity-purified using amylose resin, and the maltose-binding protein was cleaved with factor Xa. The DDX1 protein was purified on a SDS-PAGE gel, electroeluted, and concentrated. Approximately 100 μg of protein was injected into rabbits at 4–6-week intervals. For the initial injection, the protein was dispersed in complete Freund's adjuvant (Sigma), while subsequent injections were prepared in Freund's incomplete adjuvant. Blood was collected from each rabbit 10 days after injection, and the specificity of the antiserum was tested using cell extracts from RB522A. To prepare antiserum to the N terminus of DDX1 protein, a DDX1 cDNA fragment from bp 268 to 851 (Fig. 1 B) was inserted into pGEX-4T2 (Amersham Pharmacia Biotech). The recombinant protein produced from this construct contains the first 186 amino acids of the predicted DDX1 sequence. Soluble glutathione S-transferase-DDX1 fusion protein was purified with glutathione-Sepharose 4B (Amersham Pharmacia Biotech). The glutathione S-transferase component of the fusion protein was cleaved with thrombin. We used two different procedures for subcellular fractionations. First, we isolated nuclear and S100 (soluble cytoplasmic) fractions from RB522A, IMR-32, Y79, RB(E)-2, HeLa, and HL60 using the procedure of Dignam (42Dignam J.D. Methods Enzymol. 1990; 182: 194-203Crossref PubMed Scopus (223) Google Scholar). On average, we obtained 5–6 times more protein in the cytosolic fractions than in the nuclear fractions. Second, 108 RB522A cells were lysed and fractionated into S4 (soluble cytoplasmic components), P2 (heavy mitochondria, plasma membrane fragments), P3 (mitochondria, lysozymes, peroxisomes, and Golgi membranes), and P4 fractions (membrane vesicles from rough and smooth endoplasmic reticulum, Golgi, and plasma membrane) by differential centrifugation (43Graham J. Rickwood D. Centrifugation: A Practical Approach. 2nd Ed. IRL Press, Oxford1984: 161-182Google Scholar). We obtained 8 mg of protein in the S4 fraction, 1 mg in P2, 0.5 mg in P3, and 2 mg in P4 fraction. The procedures related to the immunoelectron microscopy have been previously described (44Godbout R. Marusyk H. Bisgrove D. Dabbagh L. Poppema S. Exp. Eye Res. 1995; 60: 645-657Crossref PubMed Scopus (19) Google Scholar). For Western blot analysis, proteins were electrophoresed in polyacrylamide-SDS gels and electroblotted onto nitrocellulose using the standard protocol for protein transfer described by Schleicher and Schuell. The filters were incubated with a 1:5000 dilution of DDX1 antiserum, a 1:200 dilution of anti-MYCN monoclonal antibody (Boehringer Mannheim), or a 1:200 dilution of anti-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For the colorimetric analysis, antigen-antibody interactions were visualized using either alkaline phosphatase-linked goat anti-rabbit IgG (for DDX1) or goat anti-mouse IgG (for MYCN) at a 1:3000 dilution. For the ECL Western blotting analysis (Amersham Pharmacia Biotech), we used a 1:100,000 dilution of peroxidase-linked secondary anti-rabbit IgG antibody (for DDX1) or secondary anti-goat IgG antibody (Jackson ImmunoResearch Laboratories). We have previously reported the sequence of DDX1 cDNA isolated from an RB cDNA library (21Godbout R. Squire J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7578-7582Crossref PubMed Scopus (99) Google Scholar, 23Godbout R. Hale M. Bisgrove D. Gene ( Amst. ). 1994; 138: 243-245Crossref PubMed Scopus (20) Google Scholar). This 2.4-kb DDX1cDNA contains an open reading frame spanning positions 1–2201 with a methionine encoded by the first three nucleotides (Fig.1 A). There is a polyadenylation signal and poly(A) tail in the 3′-untranslated region, indicating that the sequence is complete at the 3′-end. Manoharet al. (37Manohar C.F. Salwen H.R. Brodeur G.M. Cohn S.L. Genes Chromosomes Cancer. 1995; 14: 196-203Crossref PubMed Scopus (55) Google Scholar) have also isolated DDX1 cDNA from the NB cell line LA-N-5. Their cDNA extended the 5′-end of our sequence by 42 bp and included an additional in frame methionine (double underlined in Fig. 1 A). The possibility of additional in frame methionines located further upstream could not be excluded, because there were no predicted stop codons in the upstream region of the cDNA. Northern blot analysis indicated a DDX1 transcript size of ∼2800 nt, suggesting that the DDX1 cDNAs isolated to date were lacking ∼300–350 bp of 5′ sequence. We have used different approaches to identify the transcription start site of DDX1. First, we exhaustively screened a commercial fetal brain cDNA library with the 5′-end of DDX1 cDNA. Although numerous clones were analyzed, only one extended the sequence (by 35 bp) beyond that published by Manohar et al. (37Manohar C.F. Salwen H.R. Brodeur G.M. Cohn S.L. Genes Chromosomes Cancer. 1995; 14: 196-203Crossref PubMed Scopus (55) Google Scholar) (Fig.1 A). We next used the RACE procedure in an attempt to isolate additional 5′ sequence. The nested primers used to amplify the 5′-end of theDDX1 transcript are labeled as primers P1 and P2 in Fig.1 A and are located downstream of the three in frame methionines (double underlined in Fig. 1 A). Poly(A)+ RNA from RB522A was reverse transcribed at 52 °C using primer P1, and the reverse transcribed cDNA was amplified using the nested primer P2 and the 5′-RACE primer. Using this approach, we generated a product that was 230 bp longer than any of the cDNAs obtained by screening libraries (Fig. 1 A). Sequencing of this 230-bp cDNA revealed an in frame stop codon (boldface double underline in Fig. 1 A) located 123 bp upstream of the predicted translation initiation site. We then prepared primers P3 and P4, located near the 5′-end of the RACE cDNA (Fig. 1 A) and repeated the RACE procedure to see if additional 5′ sequences could be obtained. The resulting RACE products did not extend the DDX1 cDNA sequence further. The location of the DDX1 transcription initiation site was verified by primer extension. Poly(A)+ RNA was prepared from the following two cell lines: DDX1-amplified RB cell line RB522A and a nonamplified RB cell line RB(E)-2. RB522A has elevated levels of DDX1 mRNA, while RB(E)-2 has at least 20-fold lower levels of this transcript. Three products of 40, 43, and 46 nt (with a weak signal at 45 nt) were detected in RB522A using primer P4 (Figs. 1 A and 2). The 40-nt product corresponded exactly with the 5′-end of the RACE-derived cDNA while the 43- and 46-nt products extended the predicted size of the DDX1 transcript by 3 and 6 nt, respectively. None of these products were observed in RB(E)-2. Bands of identical sizes to those obtained with RB522A mRNA were also observed in the DDX1-amplified NB cell line BE(2)-C but not in the DDX1-amplified NB cell line IMR-32 (data not shown). The same predicted DDX1 transcription initiation site was identified with primer P3 except that the bands were of weaker intensity (data not shown). We have designated the transcription start site identified by primer extension as +1 (Fig. 1 A). The sequence of the 6 nt extending beyond the RACE cDNA was obtained by comparison of the cDNA sequence with that ofDDX1 genomic DNA. Bacteriophages containing DDX1genomic DNA were isolated by screening a human placenta library with 5′DDX1 cDNA. Eighteen kb of DNA were sequenced from two bacteriophages with overlapping DDX1 genomic DNA. Thirteen exons were identified within this 18-kb region (Fig.3) corresponding to cDNA sequences from position 1 to 1249. The 310-bp exon 1 was by far the longest of the 13 exons sequenced, corresponding to the entire 5′-untranslated region of DDX1 as well as the first in frame methionine. The sequences transcribed from exons 1, 2, and 3 are indicated in Fig.1 A. Knowledge of the genomic structure of DDX1 allowed us to use the S1 protection assay, a technique that is independent of reverse transcriptase, to further define the 5′-end of the DDX1transcript. Poly(A)+ RNAs from sixDDX1-amplified lines (RB lines: Y79 and RB522A; NB lines: BE(2)-C, IMR-32, LA-N-1, and LA-N-5) and six nonamplified lines (RB lines: RB(E)-2 and RB412; NB lines, GOTO, NB-1, NUB-7, and SK-N-MC) were hybridized to a DNA probe that extended from position −745 in the 5′-flanking DDX1 DNA to position +164 in exon 1. This DNA probe was labeled at position +164 as indicated in Fig.4. Nonhybridized DNA was digested with S1 nuclease, and the sizes of the protected fragments were analyzed on a denaturing polyacrylamide gel. Bands of 150–153 nt were observed inlane 2 (RB522A), lane 5(BE(2)-C), and lane 8 (LA-N-1) with bands of much weaker intensity in lane 7 (IMR-32) (Fig. 4). Specific bands were not detected in either DDX1-amplified Y79 and LA-N-5 or the nonamplified lines. Although the sizes of the S1 protected bands in RB522A, BE(2)-C, and LA-N-1 were 5 and 11 nt shorter than predicted based on RACE and primer extensions, respectively, there was general agreement with all three techniques regarding the location of the DDX1 transcription initiation site (Fig.1 A). The smaller S1 nuclease protected products could have arisen as the result of S1 digestion of the 5′-end of the RNA:DNA heteroduplex because of its relatively high rU:dA content (45Miller K.G. Sollner-Webb B. Cell. 1981; 27: 165-174Abstract Full Text PDF PubMed Scopus (155) Google Scholar). Identification of the same transcription initiation site in threeDDX1-amplified lines suggests that this represents the bona fide start site of DDX1 transcription. However, it was not clear why this start site was either very weak or not detected in three other amplified lines. To determine whether the 5′-end of exon 1 is transcribed in all DDX1-amplified lines, we carried out a direct analysis of the 5′-end of the DDX1 transcript by Northern blotting. Two probes were used for this analysis: the 5′ probe contained a 160-bp fragment from bp 1 to 160 (5′-half of exon 1), and the 3′ probe contained a 260-bp fragment from bp 160 to 420 (3′-half of exon 1 as well exons 2 and 3) (Fig. 1 A). With the 3′ probe, we obtained bands of similar size and intensity in fourDDX1-amplified lines (RB522A, BE(2)-C, IMR-32, and LA-N-5). Band intensity was somewhat weaker in Y79 and stronger in LA-N-1 in comparison with the other lines (Fig. 5). No signal was detected in the non-DDX1-amplified line RB412. With the 5′ probe, a relatively strong signal was observed in RB522A, BE(2)-C, and LA-N-1, while a considerably weaker but readily apparent signal was detected in Y79, IMR-32, and LA-N-5. The signal obtained with actin indicates that, with the exception of LA-N-1,
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