Title: Mutational Definition of RNA-binding and Protein-Protein Interaction Domains of Heterogeneous Nuclear RNP C1
Abstract: The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of ∼80–100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates. The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of ∼80–100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates. heterogeneous nuclear small nuclear RNA-binding domain C1-C1 interaction domain polymerase chain reaction glutathione S-transferase polyacrylamide gel electrophoresis The heterogeneous nuclear ribonucleoprotein (hnRNP)1 proteins are a group of about 20 abundant proteins that bind nascent RNA polymerase II transcripts and are involved in various aspects of pre-mRNA processing, mRNA transport, and mRNA metabolism (1Dreyfuss G. Matunis M.J. Pinol-Roma S. Burd C.G. Annu. Rev. Biochem. 1993; 62: 289-321Crossref PubMed Scopus (1317) Google Scholar, 2Kiledjian M. Burd C.G. Gorlach M. Portman D. Dreyfuss G. Mattaj I. Nagai K. RNA-Protein Interactions: Frontiers in Molecular Biology. Oxford University Press, Oxford, UK1994: 127-149Google Scholar, 3Nakielny S. Dreyfuss G. Curr. Opin. Cell Biol. 1997; 9: 420-429Crossref PubMed Scopus (191) Google Scholar, 4Krecic A.M. Swanson M.S. Curr. Opin. Cell Biol. 1999; 11: 363-371Crossref PubMed Scopus (704) Google Scholar). Among them, the hnRNP C1 protein is one of the most avid pre-mRNA-binding proteins, and it has been shown to preferentially bind to uridine-rich RNA sequences (5Swanson M.S. Dreyfuss G. EMBO J. 1988; 7: 3519-3529Crossref PubMed Scopus (176) Google Scholar, 6Swanson M.S. Dreyfuss G. Mol. Cell. Biol. 1988; 8: 2237-2241Crossref PubMed Scopus (251) Google Scholar, 7Wilusz J. Feig D.I. Shenk T. Mol. Cell. Biol. 1988; 8: 4477-4483Crossref PubMed Scopus (56) Google Scholar, 8Wilusz J. Shenk T. Mol. Cell. Biol. 1990; 10: 6397-6407Crossref PubMed Scopus (82) Google Scholar, 9Gorlach M. Wittekind M. Beckman R.A. Mueller L. Dreyfuss G. EMBO J. 1992; 11: 3289-3295Crossref PubMed Scopus (158) Google Scholar). In vitro selection/amplification from pools of random sequence RNA (SELEX procedure) demonstrated that C1 binds avidly to sequences containing a stretch of five or more uridines, its high affinity “winner sequence,” with an apparent dissociation constant (K d) of about 170 nm (10Gorlach M. Burd C.G. Dreyfuss G. J. Biol. Chem. 1994; 269: 23074-23078Abstract Full Text PDF PubMed Google Scholar). Recent studies have reported that C1 protein also binds specifically and with high affinity to several U snRNAs (11Temsamani J. Pederson T. J. Biol. Chem. 1996; 271: 24922-24926Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 12Shahied-Milam L. Soltaninassab S.R. Iyer G.V. LeStourgeon W.M. J. Biol. Chem. 1998; 273: 21359-21367Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). C1 also binds U6 snRNA, which contains an elongated uridylated stretch at the 3′ end and induces disruption of U4:U6 snRNA base pairing (13Forne T. Rossi F. Labourier E. Antoine E. Cathala G. Brunel C. Tazi J. J. Biol. Chem. 1995; 270: 16476-16481Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The RNA binding activity of hnRNP C1 has been thought to be mainly conferred by its single RNP motif RNA-binding domain (RBD) comprising the amino-terminal 94 amino acids (9Gorlach M. Wittekind M. Beckman R.A. Mueller L. Dreyfuss G. EMBO J. 1992; 11: 3289-3295Crossref PubMed Scopus (158) Google Scholar, 10Gorlach M. Burd C.G. Dreyfuss G. J. Biol. Chem. 1994; 269: 23074-23078Abstract Full Text PDF PubMed Google Scholar). The RBD (also referred to as RRM, which stands for RNA-recognition motif) is the most prevalent RNA-binding motif in eukaryotes (1Dreyfuss G. Matunis M.J. Pinol-Roma S. Burd C.G. Annu. Rev. Biochem. 1993; 62: 289-321Crossref PubMed Scopus (1317) Google Scholar, 14Swanson M.S. Lamond A.I. Pre-mRNA Processing. Springer Verlag, Berlin1995: 17-33Crossref Google Scholar, 15Kenan D.J. Query C.C. Keene J.D. Trends Biochem. Sci. 1991; 16: 214-220Abstract Full Text PDF PubMed Scopus (615) Google Scholar, 16McAfee J.G. Huang M. Soltaninassab S. Rech J.E. Iyengar S. LeStourgeon W.M. Krainer A.R. Eukaryotic mRNA Processing: Frontiers in Molecular Biology. Oxford University Press, Oxford, UK1997: 68-102Google Scholar, 17Query C.C. Bentley R.C. Keene J.D. Cell. 1989; 57: 89-101Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 18Mattaj I.W. Cell. 1989; 57: 1-3Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 19Bandziulis R.J. Swanson M.S. Dreyfuss G. Genes Dev. 1989; 3: 431-437Crossref PubMed Scopus (487) Google Scholar, 20Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1718) Google Scholar). It is an evolutionarily conserved domain present in pre-mRNA-, mRNA-, pre-rRNA-, and snRNA-binding proteins, including hnRNP proteins, splicing factors, and polyadenylation factors (1Dreyfuss G. Matunis M.J. Pinol-Roma S. Burd C.G. Annu. Rev. Biochem. 1993; 62: 289-321Crossref PubMed Scopus (1317) Google Scholar, 15Kenan D.J. Query C.C. Keene J.D. Trends Biochem. Sci. 1991; 16: 214-220Abstract Full Text PDF PubMed Scopus (615) Google Scholar). The RBD is comprised of ∼80–100 amino acids in which two consensus sequences, an octapeptide termed RNP1 and a hexapeptide termed RNP2, about 30 amino acids apart, and many other hydrophobic amino acids are particularly highly conserved (15Kenan D.J. Query C.C. Keene J.D. Trends Biochem. Sci. 1991; 16: 214-220Abstract Full Text PDF PubMed Scopus (615) Google Scholar, 17Query C.C. Bentley R.C. Keene J.D. Cell. 1989; 57: 89-101Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 18Mattaj I.W. Cell. 1989; 57: 1-3Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 20Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1718) Google Scholar). The RBD is folded into a compact domain structure of βαββαβ (21Wittekind M. Gorlach M. Friedrichs M. Dreyfuss G. Mueller L. Biochemistry. 1992; 31: 6254-6265Crossref PubMed Scopus (129) Google Scholar, 22Nagai K. Oubridge C. Jessen T.H. Li J. Evans P.R. Nature. 1990; 348: 515-520Crossref PubMed Scopus (549) Google Scholar, 23Hoffman D.W. Query C.C. Golden B.L. White S.W. Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2495-2499Crossref PubMed Scopus (166) Google Scholar). The four-stranded antiparallel β sheets are packed against the two perpendicularly oriented α helices. The highly conserved RNP1 and RNP2 consensus sequences are juxtaposed on the central β3and β1 strands, respectively. NMR studies of hnRNP C1 RBD bound with its high affinity RNA substrate suggest that the β sheets, the loops connecting the strands of the sheets, and the contiguous NH2- and COOH-terminal regions of the RBD together form an exposed platform for direct and specific RNA binding (9Gorlach M. Wittekind M. Beckman R.A. Mueller L. Dreyfuss G. EMBO J. 1992; 11: 3289-3295Crossref PubMed Scopus (158) Google Scholar). Crystal structure studies of U1A RBD complexed with its cognate U1 snRNA stem-loop II further support the view that the conserved RNP1 and RNP2 and the COOH-terminal extension of the RBD interact with RNA extensively (24Oubridge C. Ito N. Evans P.R. Teo C.H. Nagai K. Nature. 1994; 372: 432-438Crossref PubMed Scopus (776) Google Scholar). Additionally, site-directed mutagenesis has been carried out on RBDs of many RNA-binding proteins, and these studies have pinpointed several amino acids, particularly in the conserved RNP1 and RNP2, as essential residues for the RNA binding activity of this domain. For example, residues Asn9, Thr11, Tyr13, Gln54, Phe56, and Gln83 of U1A, which corresponds to Asn15, Arg17, Phe19, Phe52, Phe54, and Asn83, respectively, in the hnRNP C1, were identified as such essential amino acids (25Lutz-Freyermuth C. Query C.C. Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6393-6397Crossref PubMed Scopus (135) Google Scholar, 26Scherly D. Boelens W. van Venrooij W.J. Dathan N.A. Hamm J. Mattaj I.W. EMBO J. 1989; 8: 4163-4170Crossref PubMed Scopus (254) Google Scholar, 27Scherly D. Boelens W. Dathan N.A. van Venrooij W.J. Mattaj I.W. Nature. 1990; 345: 502-506Crossref PubMed Scopus (228) Google Scholar, 28Jessen T.H. Oubridge C. Teo C.H. Pritchard C. Nagai K. EMBO J. 1991; 10: 3447-3456Crossref PubMed Scopus (166) Google Scholar). More recent studies, however, have shown that C1 lacking the canonical RBD retained considerable RNA binding in vitro to U1, U2, and U6 snRNA as well as to its SELEX winner sequence (12Shahied-Milam L. Soltaninassab S.R. Iyer G.V. LeStourgeon W.M. J. Biol. Chem. 1998; 273: 21359-21367Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 29McAfee J.G. Shahied-Milam L. Soltaninassab S.R. LeStourgeon W.M. RNA. 1996; 2: 1139-1152PubMed Google Scholar). It was suggested that instead of the RBD, the major determinant for C1 RNA binding is a highly basic domain that consists of residues from Val140 to Asn161 and immediately precedes a leucine zipper motif. The zipper motif forms a coiled-coil structure that mediates C1 oligomerization. In general, the basic zipper motif present in C1 protein is reminiscent of the DNA-binding bZIP motifs found in many transcription factors. However, compared with the essential role of the RBD, the importance of bZIP motif in RNA binding by intact C1 has not been fully established, because site-directed mutagenesis failed to generate expressible proteins for functional assays (12Shahied-Milam L. Soltaninassab S.R. Iyer G.V. LeStourgeon W.M. J. Biol. Chem. 1998; 273: 21359-21367Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 29McAfee J.G. Shahied-Milam L. Soltaninassab S.R. LeStourgeon W.M. RNA. 1996; 2: 1139-1152PubMed Google Scholar). Here we describe a method to systematically identify, in the context of full-length C1 protein, amino acids that are essential for RNA binding. It is based on a functional screening of a randomly mutagenized C1 expression library constructed in phage. This screen identified many conserved and thus expected residues in the RBD to be essential for C1 RNA binding. It also implicated previously unidentified amino acids, particularly, residues in the C1-C1 interaction domain (CID), to influence its binding to RNA. Additionally, these identified mutations in CID resulted in a defect in C1-C1 interaction, indicating a connection between the ability of C1 to form oligomers and the RNA binding activity of this protein. This functional screening method should be generally applicable to any protein of interest to identify amino acids that are required for the binding to its cognate nucleic acid or protein ligands. To generate random point mutations in the coding region of the hnRNP C1 protein, error-prone PCR was performed using the standard protocol according to Leung et al. (30Leung D.W. Chen E. Goeddel D.V. Technique. 1989; 1: 11-15Google Scholar) with several modifications. The entire coding region of human hnRNP C1 cDNA (873 base pairs) was amplified by using the plasmid pHC12 (31Swanson M.S. Nakagawa T.Y. LeVan K. Dreyfuss G. Mol. Cell. Biol. 1987; 7: 1731-1739Crossref PubMed Scopus (165) Google Scholar) as the template and by using primers 5′-TCGAATTCGATGGCCAGCAACGTT-3′ and 5′-CAGGCTCGAGACCCCACTATGTGCTTAA-3′, which contain EcoRI or XhoI restriction enzyme site, respectively. Mutation frequency was estimated to be about 0.25–0.4% when using the following PCR conditions. Reaction mixtures (100 μl) contained 10 ng of template, 80 pmol of each primer, 1 mm of each dNTP, 16.6 mm(NH4)2SO4, 67 mmTris-HCl, pH 8.8, 6.1 mm MgCl2, 6.7 μm EDTA, pH 8.0, 0.17 mg/ml bovine serum albumin, 10% dimethyl sulfoxide, 10 mm β-mercaptoethanol, and 4 units of Taq DNA polymerase (PerkinElmer Life Sciences). Four identical but separate PCR reactions were subjected to 28 cycles of 95 °C for 1 min, 54 °C for 1 min, and 72 °C for 3 min. The pooled PCR products were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), ethanol precipitated, cut with EcoRI andXhoI, and purified on a 1% (w/v) agarose gel. The purifiedEcoRI-XhoI fragments were then ligated into theEcoRI-XhoI cleaved Uni-Zap XR vector arms (Stratagene). The ligation mixture was packaged using the Gigapack II Packaging Extract kit (Stratagene) according to the manufacturer's suggested conditions to generate a Uni-Zap λ phage library containing C1 cDNAs with random mutations. The titer of this library is ∼1 × 106 plaque-forming units/ml. To screen the mutant library, Escherichia coli XL-1 Blue bacteria were infected at ∼200 plaque-forming units/plate and plated onto 50 LB plates (100 mm) with top agarose. After incubation at 37 °C for 4 h, each plate was overlaid with a nitrocellulose filter impregnated with 10 mmisopropylthio-β-d-galactoside. Following incubation for 6–8 h at 37 °C, the filter was lifted, replaced with another isopropylthio-β-d-galactoside-treated nitrocellulose filter, and incubated at 37 °C overnight. The first set of filters was immunoblotted with the anti-C1 monoclonal antibody 4F4 and then with 125I-labeled goat anti-mouse F(ab′)2 as previously described (32Choi Y.D. Dreyfuss G. J. Cell Biol. 1984; 99: 1204-1997Crossref Scopus (145) Google Scholar). After washing, the immunoblotted filters were exposed to x-ray film. The second set of filters was incubated in screening buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 1 mm EDTA, and 10 mmdithiothreitol) for 30 min at room temperature with constant agitation, blocked with 3% nonfat dried milk and 0.02% sodium azide in phosphate-buffered saline without MgCl2 for 1 h at room temperature. After a brief wash with screening buffer, the filters were incubated in 100 ml of screening buffer containing denatured salmon sperm DNA (0.1 mg/ml) for 30 min at room temperature. Then, about 10 pmol of 32P-labeled oligo(dT)25(25-mer) was added to the screening buffer, and the incubation was continued for 1 h. After washing five times with screening buffer, the filters were exposed to x-ray films. Plaques scoring positive for 4F4 and negative for oligo(dT)25 were isolated and excised using ExAssist helper phage and SOLR E. coli strain as the host cell (Stratagene) according to the manufacturer's recommendations. The mutated bases within the C1 cDNA from these clones were identified by DNA sequencing using the Sequenase version 2.0 DNA sequencing kit (United States Biochemicals). Samples from each ddNTP terminated reaction were run as single base ladders on polyacrylamide gels to facilitate rapid identification of mutations. Wild type and mutant hnRNP C1 proteins were produced in vitro using TnT T7/T3 polymerase coupled rabbit reticulocyte lysate system (Promega) in the presence of [35S]methionine (Amersham Pharmacia Biotech) according to the manufacturer's protocols. The constructs used for TnT were either in pBluescript SK(−) vector (Stratagene) and transcribed by T3 RNA polymerase or in pcDNA3 vector (Invitrogen) and transcribed by T7 RNA polymerase. To produce recombinant proteins from E. coli, wild type and mutant C1 cDNA fragments were subcloned into EcoRI andXhoI cleaved pET-28b vector to produce His-tagged proteins (Novagen) or pGEX-5X-3 vector to produce GST-tagged proteins (Amersham Pharmacia Biotech). The resulting plasmids were transformed into BL21(DE3) E. coli (Novagen) for expression and purification of the fusion proteins according to the manufacturer's recommendations. The MT2N mutant was constructed using PCR to generate two partial COOH-terminal fragments. The more 5′ terminal of these fragments spanned the unique BsrGI site in C1. The 3′ primer for this fragment generated the V194N mutation and a silent mutation that formed an XhoI site at amino acids 198 and 199. The second fragment was amplified by PCR to contain the same engineered XhoI site, the L201N mutation, and the remaining COOH-terminal end of C1 followed by an XbaI site. These fragments were digested with BsrGI-XhoI andXhoI-XbaI, respectively, and both were subsequently inserted into BsrGI-XbaI cleaved pcDNA3-Myc-C1 plasmid (33Nakielny S. Dreyfuss G. J. Cell Biol. 1996; 134: 1365-1373Crossref PubMed Scopus (174) Google Scholar) that contained the amino-terminal portion of the C1 cDNA. The mutations were confirmed using the Sequenase version 2.0 DNA sequencing kit (United States Biochemicals). Binding ofin vitro transcribed and translated hnRNP C1 and mutants to AGpoly(U) type 6 beads (Amersham Pharmacia Biotech) was carried out as previously described (6Swanson M.S. Dreyfuss G. Mol. Cell. Biol. 1988; 8: 2237-2241Crossref PubMed Scopus (251) Google Scholar). 5 μl of in vitro produced proteins were used in each binding reaction. The poly(U)-binding buffer contains 10 mm Tris-HCl, pH 7.5, 2.5 mmMgCl2, 0.5% Triton X-100, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 0.5% aprotinin, and various concentrations of NaCl as indicated. In vitro protein interaction assays were carried out as previously described (34Zhang Y. O'Connor J.P. Siomi M.C. Srinivasan S. Dutra A. Nussbaum R.L. Dreyfuss G. EMBO J. 1995; 14: 5358-5366Crossref PubMed Scopus (271) Google Scholar). Briefly, purified GST or GST-C1 proteins (2 μg) bound to 30 μl of glutathione-Sepharose 4B resin (Amersham Pharmacia Biotech) were incubated with 5 μl of in vitro translated protein in 500 μl of binding buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 2 mm EDTA, 0.1% Nonidet P-40, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 0.5% aprotinin). Following incubation at 4 °C for 1 h, the resin was washed five times with 1 ml of binding buffer. Bound proteins were then eluted in SDS-PAGE sample buffer, separated by 12.5% SDS-PAGE, and visualized by fluorography. In vitro transcription and purification of U2 snRNA were done as previously described (35Fischer U. Sumpter V. Sekine M. Satoh T. Luhrmann R. EMBO J. 1993; 12: 573-583Crossref PubMed Scopus (143) Google Scholar). The32P-labeled and polyacrylamide gel-purified RNA probe (2 × 104 cpm) was incubated with the indicated recombinant His-tagged C1 proteins (25–250 nm) in 20 μl of reaction mixture containing 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 1 mg/ml yeast RNA (Sigma), and 5% glycerol at room temperature for 20 min. Reaction mixtures were loaded onto 4% nondenaturing polyacrylamide gels with ¼× TAE buffer (40 mm Tris-acetate, pH 7.5, 1 mm EDTA) that had been prerun at 100 V for 1 h. Gels were run at 100 V until bromphenol blue (loaded into an empty lane) migrated about 10 cm into the gel. The gel was then dried for autoradiography. For cross-linking, gel mobility shift reactions were prepared with cold RNA probe, but instead of loading onto gels, glutaraldehyde (final concentration, 0.01%) was added, and the reaction mixtures were incubated at room temperature for 30 min. Reactions were stopped by adding 110 volume of 1 m Tris-HCl, pH 7.5 (36Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar). Proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Western blot was carried out essentially as previously described (32Choi Y.D. Dreyfuss G. J. Cell Biol. 1984; 99: 1204-1997Crossref Scopus (145) Google Scholar), using 4F4 at a 1:1,000 dilution. To identify hnRNP C1 mutants that do not bind RNA, a λ phage expression library constructed from randomly mutated hnRNP C1 cDNA was screened with a high affinity nucleic acid probe. To generate random point mutations in C1, error-prone PCR was performed using the entire coding region of C1 as the template. Under the conditions used, this technique has been shown to produce mutation frequency of about 0.25–0.4% over 30 cycles of PCR amplification (30Leung D.W. Chen E. Goeddel D.V. Technique. 1989; 1: 11-15Google Scholar, 37Zhou Y.H. Zhang X.P. Ebright R.H. Nucleic Acids Res. 1991; 19: 6052Crossref PubMed Scopus (204) Google Scholar), based on ∼10−4errors/nucleotide synthesized by Taq DNA polymerase (38Saiki R.K. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13365) Google Scholar). To assure that library clones expressed full-length or near full-length C1 protein, a set of replica library filters was screened with 4F4 that binds to an epitope close to the carboxyl terminus of C1. 2G. Dreyfuss, unpublished data. Thus, clones not producing full-length C1 will not be detected with 4F4. Another replica set of library filters was probed with 32P-labeled oligo(dT)25. We used oligo(dT)25 instead of poly(U), the more typically used high affinity substrate for C1 (6Swanson M.S. Dreyfuss G. Mol. Cell. Biol. 1988; 8: 2237-2241Crossref PubMed Scopus (251) Google Scholar, 10Gorlach M. Burd C.G. Dreyfuss G. J. Biol. Chem. 1994; 269: 23074-23078Abstract Full Text PDF PubMed Google Scholar), because it is more stable and produces lower background than poly(U) (data not shown). Furthermore, in in vitro bead binding assay, oligo(dT)25 binds to C1 as well as poly(U) does. 3L. Wan and G. Dreyfuss, unpublished data. Fig.1 (A and B) shows filter duplicates probed with 4F4 and oligo(dT)25, respectively. Phage plaques that are positive with 4F4 but negative with oligo(dT)25 represent phage that likely express full-length C1 proteins but contain mutations that reduce RNA binding. After screening ∼10,000 clones, we obtained 60 clones that lost oligo(dT)25 binding capacity. The phagemids containing mutated hnRNP C1 coding regions were in vivo excised out of the λ phage, and the entire coding region of each clone was sequenced (39Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52239) Google Scholar). The sequencing gels were run as single nucleotide ladders to facilitate rapid identification of mutations. A portion of such a sequencing gel illustrating representative mutations found is shown in Fig. 1 C. For example, in clone 3, a guanine is mutated to a thymine, which results in Gln to His change at amino acid 56 (Q56H), and in clone 5, a cytosine is changed to a thymine resulting in a silent mutation at the amino acid level. The loss of oligo(dT)25 binding activity of this clone is attributed to the presence of a second mutation (data not shown). Among a total of 60 clones identified in the screening and sequenced, we found mutations at 29 amino acid positions. Mutations in a few positions were identified multiple times (data not shown). To verify that the mutants scored by the filter binding screening of the library indeed produced RNA binding defective hnRNP C1, the mutant C1 proteins were produced by in vitro transcription and translation and tested for binding to poly(U) beads at 0.1m NaCl. The results of the binding assays for several of these mutants are shown in Fig. 2. Compared with wild type C1 protein, mutants F19S and Q56H completely abolished poly(U) binding, and mutants S16F and G51Y significantly reduced binding. These results demonstrate that the filter binding screening successfully identified RNA binding defective mutants of hnRNP C1. The various C1 mutants we characterized, and their relative poly(U) binding avidity is listed in Fig.3 B. The positions of the mutations on the βαββαβ domain structure of the RBD are indicated in Fig. 3 A. Most of the mutations lie within the two RNP consensus motifs that form the two central β-sheets. The mutations within RNP consensus motifs, e.g. F19(S/L), G21V, F52L, and Q56H, showed no binding or severely impaired binding in the poly(U) bead binding assay. Mutations located in close proximity to the RNP consensus motifs, e.g. S16F and H49(Y/R/N), were less defective in binding to poly(U). A cluster of mutations near the COOH-terminal end of the RBD identified in the library screening (D81N, N83(D/Y), and E87G) had the same affinity as wild type C1 for poly(U) as measured by the in vitro bead binding assay. Amino acid residues Phe37, Gly41, and Ala66are well conserved in several RBD-containing proteins, and they are located in the two α-helices (Fig. 3 A). Mutations in these three positions were identified as RNA binding defective in the initial library screening. However, these mutants displayed wild type binding activity in the poly(U) bead binding assay (Fig. 3 B). For two reasons, the poly(U) bead binding assay is probably less sensitive than the library screening with oligo(dT)25 in detecting a slight decrease in binding affinity. First, the nucleic acid substrate used for screening, oligo(dT)25, is 25 nucleotides in length, whereas poly(U) beads present polyribouridylic acid chains of about 100 nucleotides. Second, the amount of poly(U) used (3 μm) was in excess over in vitro produced mutant C1 proteins (0.5 nm), whereas a relatively low concentration of oligo(dT)25 (0.1 nm) was used in the library screening. Nevertheless, the above mutations were generally expected in a successful library screening, because most of these residues are conserved in many RBD containing proteins (Fig.3 A). Furthermore, previous studies have indicated that residues located in the β-sheet (22Nagai K. Oubridge C. Jessen T.H. Li J. Evans P.R. Nature. 1990; 348: 515-520Crossref PubMed Scopus (549) Google Scholar, 26Scherly D. Boelens W. van Venrooij W.J. Dathan N.A. Hamm J. Mattaj I.W. EMBO J. 1989; 8: 4163-4170Crossref PubMed Scopus (254) Google Scholar, 28Jessen T.H. Oubridge C. Teo C.H. Pritchard C. Nagai K. EMBO J. 1991; 10: 3447-3456Crossref PubMed Scopus (166) Google Scholar) and in the flanking amino- and carboxyl-terminal regions (9Gorlach M. Wittekind M. Beckman R.A. Mueller L. Dreyfuss G. EMBO J. 1992; 11: 3289-3295Crossref PubMed Scopus (158) Google Scholar) are involved in RNA interaction. Our library screening identified amino acid changes that have not been previously predicted to be involved in the interaction with RNA. For example, multiple mutations were observed at Val28(V28(G/I)) and at Asp71 (D71(V/G)). These residues are located at the termini of the loop structures connecting the β-sheets and the α-helices and thus are likely to be critical for the global folding of the RBD. Of particular interest, we discovered three mutations affecting C1 binding to RNA that map outside of the RBD in the CID (Fig. 3 B). In isolated HeLa nuclear hnRNPs, the C1 and C2 proteins are present at a stoichiometry of (C1)3C2 possibly as tetramers (40Barnett S.F. Friedman D.L. LeStourgeon W.M. Mol. Cell. Biol. 1989; 9: 492-498Crossref PubMed Scopus (37) Google Scholar). Bacterially expressed C1 proteins spontaneously form C1 tetramers (41McAfee J.G. Soltaninassab S.R. Lindsay M.E. LeStourgeon W.M. Biochemistry. 1996; 35: 1212-1222Crossref PubMed Scopus (38) Google Scholar). CD spectra analysis of C1 deletion mutants revealed that a leucine-rich coiled-coil domain (Leu180–Glu207) mediates C1-C1 interaction (29McAfee J.G. Shahied-Milam L. Soltaninassab S.R. LeStourgeon W.M. RNA. 1996; 2: 1139-1152PubMed Google Scholar). Using the yeast two-hybrid system, we have also mapped CID to the same region. 4V. Pollard and G. Dreyfuss, unpublished data. However, it has not been previously known that in the context of full-length C1 protein, the coiled-coil motif is a determinant of C1 protein-protein interaction. Because three point mutations (L187Q, Q192P, and L201P) found in the search for RNA binding defective mutant C1 were located within the CID, we tested the effect of these mutations on C1-C1 interaction. CID is predicted to be comprised of four heptad repeats (Fig. 4 A). Hydrophobic residues at positions 1 and