Association of Two Nuclear Proteins, Npw38 and NpwBP, via the Interaction between the WW Domain and a Novel Proline-rich Motif Containing Glycine and Arginine

Abstract

We have previously reported a nuclear protein possessing a WW domain, Npw38 (Komuro, A., Saeki, M., and Kato, S. (1999) Nucleic Acids Res. 27, 1957–1965). Here we report a Npw38-binding protein, NpwBP, isolated from HeLa cell nuclear extracts and its characterization using a cloned cDNA. NpwBP contains two proline-rich regions that are capable of binding to the WW domain of Npw38. The binding analysis using an oligopeptide-immobilized membrane revealed that the WW domain of Npw38 preferentially recognizes a short proline-rich sequence, PPGPPP, surrounded by an arginine residue, so we named it a PGR motif. Localization analysis using green fluorescent protein fusion protein and immunostaining showed that Npw38 and NpwBP are colocalized in the same subnuclear region. Coimmunoprecipitation experiments confirmed the association between Npw38 and NpwBP, which were expressed as epitope-tagged forms in COS7 cells. Furthermore, the N-terminal region of NpwBP has binding ability for poly(rG) and G-rich single-stranded DNA. These results suggest that NpwBP is a physiological ligand of Npw38 and that the Npw38-NpwBP complex may function as a component of an mRNA factory in the nucleus. We have previously reported a nuclear protein possessing a WW domain, Npw38 (Komuro, A., Saeki, M., and Kato, S. (1999) Nucleic Acids Res. 27, 1957–1965). Here we report a Npw38-binding protein, NpwBP, isolated from HeLa cell nuclear extracts and its characterization using a cloned cDNA. NpwBP contains two proline-rich regions that are capable of binding to the WW domain of Npw38. The binding analysis using an oligopeptide-immobilized membrane revealed that the WW domain of Npw38 preferentially recognizes a short proline-rich sequence, PPGPPP, surrounded by an arginine residue, so we named it a PGR motif. Localization analysis using green fluorescent protein fusion protein and immunostaining showed that Npw38 and NpwBP are colocalized in the same subnuclear region. Coimmunoprecipitation experiments confirmed the association between Npw38 and NpwBP, which were expressed as epitope-tagged forms in COS7 cells. Furthermore, the N-terminal region of NpwBP has binding ability for poly(rG) and G-rich single-stranded DNA. These results suggest that NpwBP is a physiological ligand of Npw38 and that the Npw38-NpwBP complex may function as a component of an mRNA factory in the nucleus. glutathione S-transferase polymerase chain reaction polyacrylamide gel electrophoresis phosphate-buffered saline polyvinylidene difluoride hemagglutinin single-stranded WW domains are globular modules composed of 35–40 amino acids in which two tryptophans and one proline are conserved (1Bork P. Sudol M. Trends Biochem. Sci. 1994; 19: 531-533Abstract Full Text PDF PubMed Scopus (346) Google Scholar, 2André B. Springael J.-Y. Biochem. Biophys. Res. Commun. 1994; 205: 1201-1205Crossref PubMed Scopus (88) Google Scholar, 3Hofmann K. Bucher P. FEBS Lett. 1995; 358: 153-157Crossref PubMed Scopus (55) Google Scholar, 4Sudol M. Chen H.I. Bougeret C. Einbond A. Bork P. FEBS Lett. 1995; 369: 67-71Crossref PubMed Scopus (295) Google Scholar). WW domains have been revealed to be involved in mediating protein-protein interaction that resulted from a binding between the WW domain and a proline-rich motif in a corresponding ligand. Recent reports described various pairs of proteins mediated by WW domains, which have potential involvement in signaling processes that underlie human disease (5Staub O. Dho S. Henry P.C. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (734) Google Scholar, 6Rentschler S. Linn H. Deininger K. Bedford M.T. Espanel X. Sudol M. Biol. Chem. 1999; 380: 431-442Crossref PubMed Scopus (98) Google Scholar, 7Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 8Harvey K.F. Dinudom A. Komwatana P. Jolliffe C.N. Day M.L. Parasivam G. Cook D.I. Kumar S. J. Biol. Chem. 1999; 274: 12525-12530Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 9Sudol M. Prog. Biophys. Mol. Biol. 1996; 65: 113-132Crossref PubMed Scopus (250) Google Scholar). The analysis of the binding regions in these pairs revealed that the WW domains are classified into three classes by the proline-rich motifs of the ligands: a PY motif (10Chen H.I. Sudol M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7819-7823Crossref PubMed Scopus (484) Google Scholar), a PPLP motif (11Chan D.C. Bedford M.T. Leder P. EMBO J. 1996; 15: 1045-1054Crossref PubMed Scopus (193) Google Scholar, 12Bedford M.T. Chan D.C. Leder P. EMBO J. 1997; 16: 2376-2383Crossref PubMed Scopus (190) Google Scholar, 13Ermekova K.S. Zambrano N. Linn H. Minopoli G. Gertler F. Russo T. Sudol M. J. Biol. Chem. 1997; 272: 32869-32877Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), and a PGM motif (14Bedford M.T Reed R. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10602-10607Crossref PubMed Scopus (134) Google Scholar). Recently, a different class of motif has been found in phosphoproteins to which WW domains of the Pin1 and the ubiquitin ligase NEDD4 bind in a phosphorylation-dependent manner (15Lu P.-J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Crossref PubMed Scopus (585) Google Scholar).We have previously reported a novel WW domain-containing protein Npw38 (16Komuro A. Saeki M. Kato S. Nucleic Acids Res. 1999; 27: 1957-1965Crossref PubMed Scopus (49) Google Scholar). Npw38 is localized in the nucleus and has binding ability for poly(rG). The WW domain of Npw38 possesses eight hydrophobic amino acid residues in a relatively acidic region, while other WW domains have rather hydrophilic and more basic residues. These characteristics may contribute to the ligand binding specificity of Npw38. To characterize a binding motif in a Npw38 ligand, we tried to find a physiological ligand of the WW domain of Npw38 using a yeast two-hybrid screening system. However, we failed to identify the ligand because the WW domain itself caused a basal transcriptional activation (16Komuro A. Saeki M. Kato S. Nucleic Acids Res. 1999; 27: 1957-1965Crossref PubMed Scopus (49) Google Scholar).In this report, we describe the isolation of a Npw38-binding protein, NpwBP, from HeLa nuclear extracts using affinity purification and the characterization of NpwBP using its cDNA. NpwBP has been shown to have two proline-rich regions that are capable of interacting with the WW domain of Npw38. The precise binding motif was determined by a binding assay using an oligopeptide-immobilized membrane, demonstrating a novel WW domain-binding motif composed of proline, glycine, and arginine. Localization and immunoprecipitation analyses indicate that Npw38 and NpwBP associate with each other in the nucleus. Furthermore, NpwBP was shown to have nucleotide binding ability as does Npw38.DISCUSSIONWe have purified a nuclear protein associating Npw38 from HeLa nuclear extracts and have named it NpwBP. The cloning and characterization of the NpwBP cDNA revealed that NpwBP has two proline-rich regions. Two kinds of binding assays using GST fusion proteins and in vitro translated products and using the yeast two-hybrid system showed that NpwBP associates with Npw38 through interaction between the WW domain of Npw38 and the two proline-rich regions in NpwBP. The conserved two Trp and one Pro residues have been shown to be essential for this interaction by mutation analysis. Localization analysis showed that NpwBP is colocalized with Npw38 in the nucleus. Npw38 and NpwBP expressed in COS7 cells were coimmunoprecipitated. All these results indicate that NpwBP is a physiological ligand of the WW domain of Npw38.The binding assay using an oligopeptide-immobilized membrane demonstrated that the sequence motif interacting with the WW domain of Npw38 is PPGPPP. Npw38 has five sites containing a PPGPPP motif in which two sites (positions 199–204 and 459–464) are the most likely binding sites based on the results of the oligopeptide binding assay. It should be noted that these two PPGPPP sequences are flanked by Arg within three amino acids. The importance of the flanked Arg was confirmed by oligopeptide binding assay and site-directed mutation analysis. Thus we named it a PGR motif. The contribution of Arg to the interaction may be attributed to an acidic environment of the WW domain of Npw38 compared with the basic ones of other WW domains. The structural analysis of the complex between the WW domain of YAP and a peptide containing a PPXY motif revealed that the peptide binds to a hydrophobic surface formed by three amino acids, including the second Trp-39, Tyr-28, and Leu-30 (19Macias M.J. Hyvönen M. Baraldi E. Schultz J. Sudol M. Saraste M. Oschkinat H. Nature. 1996; 382: 646-649Crossref PubMed Scopus (359) Google Scholar), which correspond to Trp-75, Tyr-64, and Trp-66 in the WW domain of Npw38. This hydrophobic region of Npw38 is surrounded by three acidic amino acids (Asp-58, Asp-69, and Asp-71). These acidic residues may contribute to stabilize the binding of a PGR motif sequence to the hydrophobic pocket through ionic interaction between Arg and Asp.A protein data base search showed 21 sequences containing PPGPPP, in which the following three sequences have a PPGPPP flanked by Arg: (i) human hypothetical protein KIAA0755 (DDBJ/GenBankTM/EBI accession number AB018298), TRGMLPPGPPPPGPHQ (53–68); (ii) human atrophin-1 (Swiss-Prot accession number P54259), YKTASPPGPPPYGKRA (623–638);(iii) herpes simplex virus trans-acting transcriptional protein ICP4 (Swiss-Prot accession number P08392), PGRTPPPGPPPLSEAP (232–249). These sequences may not be a physiological ligand of Npw38, because Arg in KIAA0755 or Atrophin-1 is not located within three amino acids flanked by PPGPPP, and the trans-acting transcriptional protein ICP4 is flanked not only by upstream Arg but also by downstream Glu. Experiments are needed to confirm this.Previously, we demonstrated that the WW domain of Npw38 can activate a basal transcription in yeast and mammalian cells, suggesting that the WW domain may interact with transcriptional machinery. The single mutation among conserved two Trps and one Pro did not affect this transcription activation, whereas the same mutation abolished the interaction between Npw38 and NpwBP. These results imply that the transcriptional activation by the WW domain of Npw38 may not result from the interaction via a PGR motif and that the WW domain of Npw38 could have dual binding abilities for different ligands through which Npw38 may play dual roles in nuclear events. At present, it is not clear whether the interaction of the WW domain with transcriptional machinery is physiologically meaningful.NpwBP has been shown to have binding ability for poly(rG) and G-rich ssDNA, whereas Npw38 binds preferentially to poly(rG) but not ssDNA as described in the previous paper (16Komuro A. Saeki M. Kato S. Nucleic Acids Res. 1999; 27: 1957-1965Crossref PubMed Scopus (49) Google Scholar). The G-specific binding to AdMLP has been reported on hTAFII68, which is a component of a multiprotein complex composed of the TATA-binding protein and TATA-binding protein-associated factors (20Bertolotti A. Lutz Y. Heard D.J. Chambon P. Tora L. EMBO J. 1996; 15: 5022-5031Crossref PubMed Scopus (308) Google Scholar). Like this complex, the Npw38-NpwBP complex may play a role in mediating a link between DNA and RNA in transcriptional or posttranscriptional events. The identification of other proteins interacting with the Npw38-NpwBP complex will help elucidate the physiological role of this complex in these nuclear events. WW domains are globular modules composed of 35–40 amino acids in which two tryptophans and one proline are conserved (1Bork P. Sudol M. Trends Biochem. Sci. 1994; 19: 531-533Abstract Full Text PDF PubMed Scopus (346) Google Scholar, 2André B. Springael J.-Y. Biochem. Biophys. Res. Commun. 1994; 205: 1201-1205Crossref PubMed Scopus (88) Google Scholar, 3Hofmann K. Bucher P. FEBS Lett. 1995; 358: 153-157Crossref PubMed Scopus (55) Google Scholar, 4Sudol M. Chen H.I. Bougeret C. Einbond A. Bork P. FEBS Lett. 1995; 369: 67-71Crossref PubMed Scopus (295) Google Scholar). WW domains have been revealed to be involved in mediating protein-protein interaction that resulted from a binding between the WW domain and a proline-rich motif in a corresponding ligand. Recent reports described various pairs of proteins mediated by WW domains, which have potential involvement in signaling processes that underlie human disease (5Staub O. Dho S. Henry P.C. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (734) Google Scholar, 6Rentschler S. Linn H. Deininger K. Bedford M.T. Espanel X. Sudol M. Biol. Chem. 1999; 380: 431-442Crossref PubMed Scopus (98) Google Scholar, 7Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 8Harvey K.F. Dinudom A. Komwatana P. Jolliffe C.N. Day M.L. Parasivam G. Cook D.I. Kumar S. J. Biol. Chem. 1999; 274: 12525-12530Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 9Sudol M. Prog. Biophys. Mol. Biol. 1996; 65: 113-132Crossref PubMed Scopus (250) Google Scholar). The analysis of the binding regions in these pairs revealed that the WW domains are classified into three classes by the proline-rich motifs of the ligands: a PY motif (10Chen H.I. Sudol M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7819-7823Crossref PubMed Scopus (484) Google Scholar), a PPLP motif (11Chan D.C. Bedford M.T. Leder P. EMBO J. 1996; 15: 1045-1054Crossref PubMed Scopus (193) Google Scholar, 12Bedford M.T. Chan D.C. Leder P. EMBO J. 1997; 16: 2376-2383Crossref PubMed Scopus (190) Google Scholar, 13Ermekova K.S. Zambrano N. Linn H. Minopoli G. Gertler F. Russo T. Sudol M. J. Biol. Chem. 1997; 272: 32869-32877Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), and a PGM motif (14Bedford M.T Reed R. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10602-10607Crossref PubMed Scopus (134) Google Scholar). Recently, a different class of motif has been found in phosphoproteins to which WW domains of the Pin1 and the ubiquitin ligase NEDD4 bind in a phosphorylation-dependent manner (15Lu P.-J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Crossref PubMed Scopus (585) Google Scholar). We have previously reported a novel WW domain-containing protein Npw38 (16Komuro A. Saeki M. Kato S. Nucleic Acids Res. 1999; 27: 1957-1965Crossref PubMed Scopus (49) Google Scholar). Npw38 is localized in the nucleus and has binding ability for poly(rG). The WW domain of Npw38 possesses eight hydrophobic amino acid residues in a relatively acidic region, while other WW domains have rather hydrophilic and more basic residues. These characteristics may contribute to the ligand binding specificity of Npw38. To characterize a binding motif in a Npw38 ligand, we tried to find a physiological ligand of the WW domain of Npw38 using a yeast two-hybrid screening system. However, we failed to identify the ligand because the WW domain itself caused a basal transcriptional activation (16Komuro A. Saeki M. Kato S. Nucleic Acids Res. 1999; 27: 1957-1965Crossref PubMed Scopus (49) Google Scholar). In this report, we describe the isolation of a Npw38-binding protein, NpwBP, from HeLa nuclear extracts using affinity purification and the characterization of NpwBP using its cDNA. NpwBP has been shown to have two proline-rich regions that are capable of interacting with the WW domain of Npw38. The precise binding motif was determined by a binding assay using an oligopeptide-immobilized membrane, demonstrating a novel WW domain-binding motif composed of proline, glycine, and arginine. Localization and immunoprecipitation analyses indicate that Npw38 and NpwBP associate with each other in the nucleus. Furthermore, NpwBP was shown to have nucleotide binding ability as does Npw38. DISCUSSIONWe have purified a nuclear protein associating Npw38 from HeLa nuclear extracts and have named it NpwBP. The cloning and characterization of the NpwBP cDNA revealed that NpwBP has two proline-rich regions. Two kinds of binding assays using GST fusion proteins and in vitro translated products and using the yeast two-hybrid system showed that NpwBP associates with Npw38 through interaction between the WW domain of Npw38 and the two proline-rich regions in NpwBP. The conserved two Trp and one Pro residues have been shown to be essential for this interaction by mutation analysis. Localization analysis showed that NpwBP is colocalized with Npw38 in the nucleus. Npw38 and NpwBP expressed in COS7 cells were coimmunoprecipitated. All these results indicate that NpwBP is a physiological ligand of the WW domain of Npw38.The binding assay using an oligopeptide-immobilized membrane demonstrated that the sequence motif interacting with the WW domain of Npw38 is PPGPPP. Npw38 has five sites containing a PPGPPP motif in which two sites (positions 199–204 and 459–464) are the most likely binding sites based on the results of the oligopeptide binding assay. It should be noted that these two PPGPPP sequences are flanked by Arg within three amino acids. The importance of the flanked Arg was confirmed by oligopeptide binding assay and site-directed mutation analysis. Thus we named it a PGR motif. The contribution of Arg to the interaction may be attributed to an acidic environment of the WW domain of Npw38 compared with the basic ones of other WW domains. The structural analysis of the complex between the WW domain of YAP and a peptide containing a PPXY motif revealed that the peptide binds to a hydrophobic surface formed by three amino acids, including the second Trp-39, Tyr-28, and Leu-30 (19Macias M.J. Hyvönen M. Baraldi E. Schultz J. Sudol M. Saraste M. Oschkinat H. Nature. 1996; 382: 646-649Crossref PubMed Scopus (359) Google Scholar), which correspond to Trp-75, Tyr-64, and Trp-66 in the WW domain of Npw38. This hydrophobic region of Npw38 is surrounded by three acidic amino acids (Asp-58, Asp-69, and Asp-71). These acidic residues may contribute to stabilize the binding of a PGR motif sequence to the hydrophobic pocket through ionic interaction between Arg and Asp.A protein data base search showed 21 sequences containing PPGPPP, in which the following three sequences have a PPGPPP flanked by Arg: (i) human hypothetical protein KIAA0755 (DDBJ/GenBankTM/EBI accession number AB018298), TRGMLPPGPPPPGPHQ (53–68); (ii) human atrophin-1 (Swiss-Prot accession number P54259), YKTASPPGPPPYGKRA (623–638);(iii) herpes simplex virus trans-acting transcriptional protein ICP4 (Swiss-Prot accession number P08392), PGRTPPPGPPPLSEAP (232–249). These sequences may not be a physiological ligand of Npw38, because Arg in KIAA0755 or Atrophin-1 is not located within three amino acids flanked by PPGPPP, and the trans-acting transcriptional protein ICP4 is flanked not only by upstream Arg but also by downstream Glu. Experiments are needed to confirm this.Previously, we demonstrated that the WW domain of Npw38 can activate a basal transcription in yeast and mammalian cells, suggesting that the WW domain may interact with transcriptional machinery. The single mutation among conserved two Trps and one Pro did not affect this transcription activation, whereas the same mutation abolished the interaction between Npw38 and NpwBP. These results imply that the transcriptional activation by the WW domain of Npw38 may not result from the interaction via a PGR motif and that the WW domain of Npw38 could have dual binding abilities for different ligands through which Npw38 may play dual roles in nuclear events. At present, it is not clear whether the interaction of the WW domain with transcriptional machinery is physiologically meaningful.NpwBP has been shown to have binding ability for poly(rG) and G-rich ssDNA, whereas Npw38 binds preferentially to poly(rG) but not ssDNA as described in the previous paper (16Komuro A. Saeki M. Kato S. Nucleic Acids Res. 1999; 27: 1957-1965Crossref PubMed Scopus (49) Google Scholar). The G-specific binding to AdMLP has been reported on hTAFII68, which is a component of a multiprotein complex composed of the TATA-binding protein and TATA-binding protein-associated factors (20Bertolotti A. Lutz Y. Heard D.J. Chambon P. Tora L. EMBO J. 1996; 15: 5022-5031Crossref PubMed Scopus (308) Google Scholar). Like this complex, the Npw38-NpwBP complex may play a role in mediating a link between DNA and RNA in transcriptional or posttranscriptional events. The identification of other proteins interacting with the Npw38-NpwBP complex will help elucidate the physiological role of this complex in these nuclear events. We have purified a nuclear protein associating Npw38 from HeLa nuclear extracts and have named it NpwBP. The cloning and characterization of the NpwBP cDNA revealed that NpwBP has two proline-rich regions. Two kinds of binding assays using GST fusion proteins and in vitro translated products and using the yeast two-hybrid system showed that NpwBP associates with Npw38 through interaction between the WW domain of Npw38 and the two proline-rich regions in NpwBP. The conserved two Trp and one Pro residues have been shown to be essential for this interaction by mutation analysis. Localization analysis showed that NpwBP is colocalized with Npw38 in the nucleus. Npw38 and NpwBP expressed in COS7 cells were coimmunoprecipitated. All these results indicate that NpwBP is a physiological ligand of the WW domain of Npw38. The binding assay using an oligopeptide-immobilized membrane demonstrated that the sequence motif interacting with the WW domain of Npw38 is PPGPPP. Npw38 has five sites containing a PPGPPP motif in which two sites (positions 199–204 and 459–464) are the most likely binding sites based on the results of the oligopeptide binding assay. It should be noted that these two PPGPPP sequences are flanked by Arg within three amino acids. The importance of the flanked Arg was confirmed by oligopeptide binding assay and site-directed mutation analysis. Thus we named it a PGR motif. The contribution of Arg to the interaction may be attributed to an acidic environment of the WW domain of Npw38 compared with the basic ones of other WW domains. The structural analysis of the complex between the WW domain of YAP and a peptide containing a PPXY motif revealed that the peptide binds to a hydrophobic surface formed by three amino acids, including the second Trp-39, Tyr-28, and Leu-30 (19Macias M.J. Hyvönen M. Baraldi E. Schultz J. Sudol M. Saraste M. Oschkinat H. Nature. 1996; 382: 646-649Crossref PubMed Scopus (359) Google Scholar), which correspond to Trp-75, Tyr-64, and Trp-66 in the WW domain of Npw38. This hydrophobic region of Npw38 is surrounded by three acidic amino acids (Asp-58, Asp-69, and Asp-71). These acidic residues may contribute to stabilize the binding of a PGR motif sequence to the hydrophobic pocket through ionic interaction between Arg and Asp. A protein data base search showed 21 sequences containing PPGPPP, in which the following three sequences have a PPGPPP flanked by Arg: (i) human hypothetical protein KIAA0755 (DDBJ/GenBankTM/EBI accession number AB018298), TRGMLPPGPPPPGPHQ (53–68); (ii) human atrophin-1 (Swiss-Prot accession number P54259), YKTASPPGPPPYGKRA (623–638);(iii) herpes simplex virus trans-acting transcriptional protein ICP4 (Swiss-Prot accession number P08392), PGRTPPPGPPPLSEAP (232–249). These sequences may not be a physiological ligand of Npw38, because Arg in KIAA0755 or Atrophin-1 is not located within three amino acids flanked by PPGPPP, and the trans-acting transcriptional protein ICP4 is flanked not only by upstream Arg but also by downstream Glu. Experiments are needed to confirm this. Previously, we demonstrated that the WW domain of Npw38 can activate a basal transcription in yeast and mammalian cells, suggesting that the WW domain may interact with transcriptional machinery. The single mutation among conserved two Trps and one Pro did not affect this transcription activation, whereas the same mutation abolished the interaction between Npw38 and NpwBP. These results imply that the transcriptional activation by the WW domain of Npw38 may not result from the interaction via a PGR motif and that the WW domain of Npw38 could have dual binding abilities for different ligands through which Npw38 may play dual roles in nuclear events. At present, it is not clear whether the interaction of the WW domain with transcriptional machinery is physiologically meaningful. NpwBP has been shown to have binding ability for poly(rG) and G-rich ssDNA, whereas Npw38 binds preferentially to poly(rG) but not ssDNA as described in the previous paper (16Komuro A. Saeki M. Kato S. Nucleic Acids Res. 1999; 27: 1957-1965Crossref PubMed Scopus (49) Google Scholar). The G-specific binding to AdMLP has been reported on hTAFII68, which is a component of a multiprotein complex composed of the TATA-binding protein and TATA-binding protein-associated factors (20Bertolotti A. Lutz Y. Heard D.J. Chambon P. Tora L. EMBO J. 1996; 15: 5022-5031Crossref PubMed Scopus (308) Google Scholar). Like this complex, the Npw38-NpwBP complex may play a role in mediating a link between DNA and RNA in transcriptional or posttranscriptional events. The identification of other proteins interacting with the Npw38-NpwBP complex will help elucidate the physiological role of this complex in these nuclear events.