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Isolation and characterization of a new 110 kDa human nuclear RNA-binding protein (p110nrb)
Biochimica et Biophysica Acta 1399 (1998) 1^9
Isolation and characterization of a new 110 kDa human nuclear RNA-binding protein (p110nrb ) Jian Gu a , Shigeki Shimba a
1;a
, Nobuo Nomura b , Ram Reddy
a;
*
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA b Kazusa DNA Research Institute, Kisarazu, Chiba 292, Japan Received 13 April 1998; accepted 4 May 1998
Abstract RNA-protein interactions play key roles in many fundamental cellular processes such as RNA processing, RNA transport, and RNA translation. During our attempts to isolate the human U6 small nuclear RNA capping enzyme, we identified a new 110 kDa nuclear RNA-binding protein, designated p110nrb . The full-length cDNA clone for p110nrb was characterized, and it encodes a 963 amino acid polypeptide. It is a highly acidic protein (pI 5.28) and the carboxyl terminal portion contains two conserved RNP motifs. A databank search found a putative C. elegans protein that might be the p110nrb homologue. The p110nrb was overexpressed as a glutathione S-transferase fusion protein in insect Sf9 cells, purified by affinity chromatography and injected into rabbits to produce specific polyclonal antibodies. Immunofluorescent staining showed that p110nrb is distributed evenly throughout the nucleoplasm. Northern blots showed that the mRNA is expressed in all tissues examined. An in vitro RNA-binding assay showed that p110nrb bound to RNA. These data suggest that p110nrb may play a role in the metabolism of nuclear RNA. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: RNA-binding protein; RNP motif; RNA-protein interaction
1. Introduction RNA-binding proteins are involved in various aspects of RNA metabolism. The cloning of cDNAs and the determination of amino acid sequences have identi¢ed several conserved motifs in RNAbinding proteins. The most common and best-characterized RNA-binding domain is the RNP motif, also referred to as RNA recognition motif (RRM) and RNP consensus sequence (RNP-CS). RNP motif proteins form a large and diverse family including * Corresponding author. Fax: +1 (713) 798-3145; E-mail: [email protected] 1 Current address: Department of Hygienic Chemistry, Nihon University, Chiba, Japan 274.
proteins that bind nuclear pre-mRNA (hnRNA), mature mRNA, small nuclear RNAs (snRNA), ribosomal RNA, as well as many RNAs transcribed by RNA polymerase III. The distinguishing characteristics of the RNP motif are two highly conserved short amino acid stretches which are separated by approximately 30 amino acids in this RNA-binding domain of about 90 amino acids. The ¢rst conserved amino acid stretch is a hydrophobic segment of six residues, which is called RNP2, and the second one is an octapeptide motif called RNP1 [1^4]. RNP motif proteins have diverse functions. Out of twenty hnRNP proteins that are known to associate with nascent hnRNA, at least nine of them belong to the category of RNP motif proteins [3]. RNP motif proteins play important roles in both constitutive
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and alternative splicing [5]. Several snRNP proteins contain RNP motifs, including U1-70K [1,6], U1A [7], and U2BQ protein [8]. Other splicing factors with RNP motifs include PTB [9], PSF [10,11] and several members of the SR protein family [12]. The well-characterized alternative splicing factors, Drosophila Sxl [13] and Tra-2 [14], are also RNP motif proteins. RNP motif proteins are also involved in both the 5P and 3P formation of mRNAs. Cap-binding proteins play important roles in splicing of pre-mRNAs and export of snRNAs containing trimethylguanosine cap structures. The m7 G cap-binding protein is a heterodimer of CBP20 and CBP80; the CBP20 contains a consensus RNP motif [15]. Several proteins involved in the polyadenylation of mRNAs, such as poly-A polymerase and the 64 kDa subunit of CstF, contain RNP motifs [16]. CPEB, which binds the cytoplasmic polyadenylation element (CPE) and mediates the cytoplasmic polyadenylation of maternal mRNA during early animal development in some species, contains an RNP motif [17]. Therefore, it is well established that proteins of the RNP motif family play important roles in numerous important cellular processes. During our attempts to isolate the enzyme for U6 snRNA 5P monomethylphosphate cap formation, we identi¢ed a new member of RNP motif protein family, designated p110nrb . The protein was expressed in baculovirus system, and polyclonal antibodies were raised against puri¢ed fusion protein GST-p110nrb . The protein is localized to the nucleoplasm and does not show capping activity in an in vitro capping assay. The mRNA of p110nrb is expressed in all tissues examined. p110nrb also binds RNA in an in vitro RNA-binding assay. The RNA substrate of p110nrb in vivo and its function remains to be established.
university microsequencing facility). The following six peptide sequences were obtained: (1) TEGLSED^DIAVQK; (2) LAEYQAYIDFEMK; (3) YANMWLEYYNLER; (4) AAA^QAENGPAAAPAV^APAATEA; (5) VGLHMTK; (6) TYGA(C)GK (dashes indicate amino acids that could not be identi¢ed; amino acid residues in brackets are probable, but not certain). 2.2. The cDNA cloning and expression of p110nrb We searched the databank and found our partial peptide sequences and cDNA sequences matched exactly to a recently sequenced human cDNA with continuous open reading frame. The complete cDNA for this protein was ampli¢ed by PCR and inserted into the baculovirus transfer vectors pVL1393 (for intact protein expression) and pAcGHLT-A (for GST fusion protein expression). Recombinant plasmids pVL1393/p110nrb and pAcGHLT-A/p110nrb and baculovirus gold DNA were cotransfected into Sf9 cells by calcium phosphate precipitation. The ampli¢cation of recombinant virus, expression of p110nrb protein in Sf9 cells and puri¢cation of GST-p110nrb fusion protein were done according to the Pharmagen protocol. 2.3. Preparation of anti-p110nrb polyclonal antibodies Rabbits were immunized with the puri¢ed recombinant GST-p110nrb produced in Sf9 cells. The antibody was produced and puri¢ed as previously described [19]. 2.4. SDS-PAGE and Western blotting
2. Materials and methods
Proteins were separated on 7.5% SDS-PAGE. For Western blotting, proteins were transferred to nitrocellulose and analyzed by immunoblotting as previously described [20].
2.1. Puri¢cation of p110nrb and sequencing of peptide
2.5. Immuno£uorescence staining
The sequential puri¢cation of p110nrb was described in detail by Shimba and Reddy [18]. Approximately 15 Wg of protein starting from the HeLa cell extract was obtained and subjected to cleavage and microsequencing of peptides (courtesy of Harvard
HeLa cells were grown on glass slides, ¢xed in 2% formaldehyde in phosphate-bu¡ered saline (PBS) for 20 min at room temperature, and permeabilized in acetone for 4 min at 320³C [21]. Cells were overlaid with a¤nity-puri¢ed anti-p110nrb antibodies, incu-
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bated for 1 h at 37³C, and washed three times in PBS (20 min, each wash). The second antibody (£uorescein-labeled anti-rabbit antibody) was added and after incubating the slides for 35 min at 37³C, washed three times with PBS (20 min, each wash). The slides were covered with coverslips after adding n-propyl gallate in glycerol/PBS. 2.6. Northern blot analysis The cDNA fragment of p110nrb was PCR ampli¢ed, gel isolated, labeled by random hexanucleotide priming, and used to probe a multiple tissue Northern blot of poly-A RNA (Clontech). The hybridization was done using ExpressHyb1 Hybridization Solution according to the protocol provided by Clontech. 2.7. Northwestern blot analysis The `Northwestern' blot RNA-binding assay was performed as described by Schi¡ et al. [22]. Samples of puri¢ed recombinant p110nrb and several control proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membrane. The membrane was blocked overnight at room temperature in Northwestern bu¡er (10 mM Tris-HCl, pH 6.8, 25 mM NaCl, 1 mM EDTA, 0.04% BSA, 0.04% Ficoll 400, 0.04% polyvinyl pyrrolidone-40). The probe used in this study was the mRNA of HBV-X (Hepatitis B Virus-X) protein which was obtained by in vitro transcription by T7 RNA polymerase; it was neither capped nor polyadenylated. The blots were probed at room temperature for 2 h. The blot was then washed three times (10 min, each wash) with Northwestern bu¡er to remove unbound or non-speci¢cally bound RNA. The dried nitrocellulose ¢lter was then subjected to autoradiography. 3. Results 3.1. Amino acid sequence of p110nrb The putative U6 snRNA capping enzyme was puri¢ed as described earlier [18]. The puri¢ed protein was subjected to sequencing and several peptide sequences were obtained as described in Section 2.
3
While the cDNA cloning by conventional methods (PCR using degenerate primers followed by cDNA library screening) was in progress, one of the cDNA clones that was sequenced at random by one of the authors (N. Nomura) contained perfect matches with our partial cDNA sequences. Analysis of the cDNA sequence showed an apparent full-length ORF of V2.9 kb, coding for 963 amino acids with a predicted molecular weight of 109 933 Da and a predicted pI of 5.28. In the carboxy terminal region, two highly conserved RNP motifs spanning amino acids 706^790 and 803^880 were found; no other motifs such as Gly-rich domain, Ser/Arg (SR) domain, or highly charged auxiliary domains, were identi¢ed (Fig. 1). A putative nuclear location signal was located in amino acids 601^616 (Fig. 1). We designated this protein p110nrb (110 kDa nuclear RNA-binding protein). 3.2. p110nrb is homologous to a C. elegans ORF A search of the sequence database and analysis by GAP program showed that the cDNA for p110nrb is 30% identical and 41% similar to a C. elegans ORF which codes for a protein of 836 amino acids. The C. elegans homologue also contains two RNP motifs at its C-terminal region (Fig. 1). Although no complete mouse homologue was found, a search of mouse EST library revealed that several long stretches of cDNA fragments were approximately 90% similar to human p110nrb cDNA. The carboxy terminal RNP motif region of the mouse homologue showed 94% identity to human p110nrb (data not shown). S. cerevisiae genome was searched and no yeast p110nrb homologue was identi¢ed. 3.3. Sequence comparison of p110nrb with other RNP proteins Most of the known RNP proteins also contain other auxiliary domains, such as Gly-rich domain, Ser/Arg (SR) domain, and highly charged auxiliary domains [23]. Thorough examination of p110nrb did not reveal any other previously characterized domains. However, it is notable that the amino terminal portion is more acidic than the carboxyl terminal region, where the predicted pI of amino acids 1^321 is 4.30, while the pI of amino acids 322^642 and 643^
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Fig. 1. Amino acid sequence of the protein p110nrb and comparison with its putative C. elegans homologue. The two RNA-binding domains are boxed, with the consensus RNP1 and RNP2 in each domain underlined. A putative nuclear localization sequence (NLS) is shown in bold and underlined. The GeneBank accession numbers for a p110nrb and its C. elegans homologue are D63879 and Z73102 (Chromosome III which contains the C. elegans homologue), respectively.
963 is 6.04 and 9.77, respectively. In the case of C. elegans homologue, the predicted pI of the full length (836 amino acids) is 6.04, while the pI of the amino terminal portion (amino acids 1^280) and the carboxyl terminal portion (amino acids 560^836) is 4.57 and 10.12, respectively. Fig. 2 shows the structural comparison of p110nrb with some other members of RNP motif proteins.
3.4. Expression and puri¢cation of p110nrb in insect cells To help understand the function(s) of p110nrb , we expressed recombinant p110nrb using the baculovirusSf9 expression system and GST tag puri¢cation methods. As shown in Fig. 3, p110nrb was overexpressed in Sf9 cells both in intact form (lane 4) and
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Fig. 2. Schematic representation of the modular structure of some representative RNP motif family proteins. The RNP motifs are indicated by roman numbering (I^IV). Auxiliary domains are indicated by their respective names. White boxes represent uncharacterized regions.
in the GST fusion form (lane 5). When glutathione beads were used to purify GST fusion protein, we obtained puri¢ed GST-p110nrb (lane 6). When the puri¢ed GST-p110nrb was used in an in vitro capping
5
Fig. 3. SDS-PAGE analysis of p110nrb proteins expressed in Sf9 insect cells. Lane 1, protein standards used as molecular weight markers; lane 2, mock-infected Sf9 cells ; lane 3, Sf9 cells infected with wild-type virus; lane 4, Sf9 cells infected with pVLp110nrb recombinant viruses (non-fusion protein); lane 5, Sf9 cells infected with pAcGHLT-p110nrb viruses (GST fusion protein); lane 6, GST-p110nrb fusion protein puri¢ed by glutathione beads from material analyzed in lane 5. The proteins were visualized by staining with Coomassie brilliant blue.
system, there was no detectable methylphosphate cap formation; and extracts from Sf9 cells infected with the recombinant baculovirus were not enriched in
Fig. 4. Western blot analysis of p110nrb protein expressed in Sf9 insect cells and in HeLa cells. Protein samples were fractionated by SDS-PAGE, transferred to nitrocellulose and analyzed by Western blotting with IgGs puri¢ed from anti-p110nrb serum. Panel A: Sf9 cells. Lane 1, protein standards used as molecular weight markers; lane 2, Sf9 cells infected with wild-type virus; lane 3, Sf9 cells infected with pVL-p110nrb viruses ; lane 4, Sf9 cells infected with pAcGHLT-p110nrb viruses ; lane 5, GST-p110nrb puri¢ed with GSH beads. Panel B: HeLa cells. Lane 1, protein standards; lane 2, HeLa S100 whole cell extracts.
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Fig. 5. Immuno£uorescence with anti-p110nrb antibodies. HeLa cells were grown on glass slides overlaid with puri¢ed anti-p110nrb antibody IgG fractions. The second antibody, £uorescein-labeled anti-rabbit antibody, was used to localize the anti-p110nrb antibody. Panel A: Preimmune serum. Panel B: Anti-p110nrb antibodies.
methylphosphate cap activity relative to the wildtype-infected cell extracts (data not shown). 3.5. Western blot analysis and immuno£uorescence staining Polyclonal antibodies were raised using puri¢ed GST-p110nrb as the antigen. Fig. 4 shows the specificity of anti-p110nrb antibodies. The polyclonal antibody against p110nrb was used to probe cell extracts. There was no signal in wild-type virus-infected cells, however, when Sf9 cells were infected with baculovirus transfer vector containing p110nrb and GSTp110nrb sequence, strong signals corresponding to p110nrb and GST-p110nrb were seen (Fig. 4A, lanes 3 and 4, respectively). After the ¢rst round of infection of the Sf9 cells, the enrichment of recombinant p110nrb was minimal and could not be visualized by Coomassie blue staining; even at this stage, the recombinant human p110nrb was readily detectable by Western blot (data not shown). When this antibody was used to probe the whole HeLa cell extracts, a prominent band of V110 kDa was seen (Fig. 4B, lane 2). When we did immunodepletion experiments using this polyclonal antibody, the V110 kDa protein was speci¢cally depleted, but the depleted HeLa cell extracts still possessed comparable methylphosphate capping activity (data not shown). Immuno£uorescence microscopy of HeLa cells with the anti-p110nrb demonstrates the nuclear localization of these proteins and their absence from cytoplasm (Fig. 5B). The pattern of staining is similar to many
hnRNP proteins in that they are uniformly stained throughout the nucleoplasm but excluded from nucleoli. This is in contrast with the immunostaining of the spliceosomal snRNP proteins, which typically show nucleoplasmic speckled pattern [24]. 3.6. Northern blot analysis Hybridization of poly-A RNA from di¡erent human tissues with p110nrb cDNA probe shows a V3.8 kb long positive band (Fig. 6). Although there are small variations on the expression level of p110nrb in di¡erent tissues, the mRNA is present in all the examined tissues and we did not observe any signi¢cant overexpression in any one single tissue. The extra length of mRNA was due to the long 3P untranslated sequences (data not shown, also see [25]). 3.7. p110nrb binds RNA in vitro In order to test the RNA-binding activity of p110nrb , we used a sensitive in vitro RNA-binding assay. Proteins were separated on SDS/PAGE and transferred to nitrocellulose paper. The nitrocellulose paper was then probed with a 32 P-labeled 0.8 kb long RNA transcript. Fig. 7A shows the Coomassie blue staining pattern of SDS-PAGE before transferring. C23 (nucleolin) and p120 were chosen as controls because C23 has four RNP motifs at its C-terminus and its RNP motifs are most closely related to those of p110nrb , and p120 was shown to be able to bind to rRNA by Gustafson et al. [26]. Fig. 7B shows the
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none of the molecular markers bind to RNA. These data demonstrate that p110nrb is indeed a nuclear RNA-binding protein. In our studies, RNA transcribed from a variety of DNA templates including plasmid DNA, ribosomal DNA, bound p110nrb protein (data not shown). These data suggest lack of substrate speci¢city in this in vitro assay. The cellular substrate of p110nrb remains to be elucidated. It is likely that p110nrb plays a general role in nuclear RNA metabolism, since it is expressed in all tissues examined by Northern blot (Fig. 6) and is distributed evenly throughout the nucleoplasm (Fig. 5B). 4. Discussion
Fig. 6. Expression of p110nrb in various human tissues. Human multiple tissue Northern blot (Clontech) was probed with p110nrb cDNA fragment probe (A). The blot was stripped of the probe after autoradiography and reprobed with a L-actin cDNA probe (B).
RNA-binding results. As expected, C23 and p120 wild-type proteins, but not the p120 mutant protein, bind to RNA (lanes 8, 9 and 10 respectively). p110nrb also showed clear RNA-binding activity (lane 7), but
We describe here the isolation and characterization of a new human nuclear RNA-binding protein, p110nrb . This protein contains two consensus RNP motifs and is capable of binding RNA in vitro. A putative C. elegans homologue of p110nrb has been identi¢ed by searching the databanks. Except for the two RNP motifs, these two proteins contain no other known protein motifs. Among all the characterized RNP motif proteins, very few of them are acidic. Both p110nrb and its C. elegans homologue are distinct acidic RNP motif proteins in that they have a large acidic region at the amino terminus which spans more than one-third of the molecule, while the one-third region at their carboxyl terminus is
Fig. 7. Northwestern blot of p110nrb to in vitro transcribed mRNA. Recombinant GST fusion proteins as indicated on top of each lane were electrophoresed on a 7.5% SDS-PAGE and (A) stained with Coomassie blue, (B) transferred to nitrocellulose and probed with 32 P-labeled 0.8 kb mRNA. The p120 wild-type used here is a partial sequence corresponding to amino acids 19^167 of p120, in the mutant, the sequence 46-RARKRAAKRRL-56 was mutated to 46-GAGILAANGGL-56. This mutation was shown previously to eliminate the RNA-binding activity of p120 [26].
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basic. The middle portions of both proteins are slightly acidic. This polar distribution of amino acids and the lack of Pro-, Gly-, or Ser/Arg-rich regions common to many other RNP motif proteins suggest that p110nrb and its homologue form a distinct subfamily of RNP motif proteins. In terms of the functional relevance of the polar distribution of amino acids, the basic carboxyl terminal region is responsible for RNA binding, while the amino terminal region may be involved in protein-protein interactions, and perhaps to have e¡ector functions. There are common epitopes between hnRNP proteins and antibodies raised against one RNP motif protein frequently cross-react with other proteins. For example, antibodies raised against hnRNP A0 protein cross-react with hnRNP A2/B1 [27]. The polyclonal antibodies raised against p110nrb protein did not show strong cross-reactivity with other proteins (Fig. 4). Therefore, it appears that p110nrb protein is not closely related in structure to other RNP motif proteins. Many of the nuclear RNA-binding proteins bind to pre-mRNAs or snRNAs and form large ribonucleoprotein complexes, called hnRNPs and snRNPs, respectively. Our data suggest that p110nrb is not in a large ribonucleoprotein complex. First, hnRNP proteins can be detected by immunoprecipitation with known abundant hnRNP proteins [3,28]. We did immunoprecipitation experiments with anti-hnRNP A1, C1 and U proteins. While the major hnRNP proteins were readily detectable in immunoprecipitates, the p110nrb protein was not detectable (data not shown). In addition, the uniform nucleoplasmic localization of p110nrb (Fig. 5B) is di¡erent from the nucleoplasmic speckles of snRNPs involved in pre-mRNA splicing [24]. Therefore, it is likely that p110nrb is not involved in hnRNP complexes and pre-mRNA processing. p110nrb is a novel nuclear RNA-binding protein, since it has two consensus RNP motifs and binds to RNA in our in vitro system. Although it is probably not part of nuclear splicing apparatus, it may still play general roles in nuclear RNA metabolism. Its mRNA is expressed in all tissues examined and it is localized uniformly in nucleoplasm. The cellular RNA substrate(s) and the ensuing function(s) of p110nrb remain to be elucidated.
Acknowledgements We thank T. Nagase for help in sequencing human p110nrb cDNA. We also thank M. Finley for HeLa cells, Dr. G. Dreyfuss of University of Pennsylvania for monoclonal antibodies against hnRNP proteins, Dr. L. Perlaky for help with immuno£uorescence staining, Y. Chen, K. Sinha and K. Perumal for helpful discussions. This work was supported by National Institute of Health Grant GM 38320. References [1] C.C. Query, R.C. Bentley, J.D. Keene, A common RNA recognition motif identi¢ed within a de¢ned U1 RNA binding domain of the 70K U1 snRNP protein, Cell 57 (1989) 89^101. [2] S.R. Haynes, The RNA motif protein family, New Biol. 4 (1992) 421^429. [3] G. Dreysuss, M.J. Matunis, S. Pinol-Roma, C.G. Burd, hnRNP Proteins and the biogenesis of mRNA, Annu. Rev. Biochem. 62 (1993) 289^321. [4] C.G. Burd, G. Dreyfuss, Conserved structures and diversity of functions of RNA-binding proteins, Science 265 (1994) 615^621. [5] A. Kramer, The structure and function of proteins involved in mammalian pre-mRNA splicing, Annu. Rev. Biochem. 65 (1996) 367^409. [6] J. Theissen, M. Etzerodt, R. Reuter, C. Schneider, F. Lottspeich, P. Argos, R. Luhrmann, L. Philipson, Cloning of the human cDNA for the U1 RNA-associated 70K protein, EMBO J. 5 (1986) 3209^3217. [7] C. Kambach, I.W. Mattaj, Intracellular distribution of the U1A protein depends on active transport and nuclear binding to U1 snRNA, J. Cell Biol. 118 (1992) 11^21. [8] W.J. Habets, P.T.G. Sillekens, M.H. Holt, J.A. Schalken, A.J.M. Roebroek, J.A.M. Leunissen, W.J.M. van de Ven, W.J. Van Venrooij, Analysis of a cDNA clone expressing a human autoimmune antigen: Full-length sequence of the U2 small nuclear RNA-associated BQ antigen, Proc. Natl. Acad. Sci. USA 84 (1987) 2421^2425. [9] J.G. Patton, S.A. Mayer, P. Tempst, B. Nadal-Ginard, Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing, Genes Dev. 5 (1991) 1237^1251. [10] J.G. Patton, E.B. Porro, J. Galceran, P. Tempst, B. NadalGinard, Cloning and characterization of PSF, a novel premRNA splicing factor, Genes Dev. 7 (1993) 393^406. [11] A. Gil, P.A. Sharp, S.F. Jamison, M.A. Garcia-Blanco, Characterization of cDNAs encoding the polypyrimidine tract-binding protein, Genes Dev. 5 (1911) 1224^1236. [12] J. Valcarcel, M.R. Green, The SR protein family: pleiotropic
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functions in pre-mRNA splicing, Trends Biol. Sci. 21 (1996) 296^301. T.J. Goralski, J.E. Edstrom, B.S. Baker, The sex determination locus transformer-2 of Drosophila encodes a polypeptide with similarity to RNA binding proteins, Cell 56 (1989) 1011^1018. J. Valcarcel, R. Singh, P.D. Zamore, M.R. Green, The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA, Nature 362 (1993) 171^175. E. Izaurralde, J. Lewis, C. Gamberi, A. Jarmolowski, C. McGuigan et al., A cap-binding protein complex mediating U snRNA export, Nature 376 (1995) 709^712. A. Sachs, E. Wahle, Poly(A) tail metabolism and function in eukaryotes, J. Biol. Chem. 268 (1993) 22955^22958. L.E. Hake, J.D. Richter, CPEB is a speci¢city factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation, Cell 79 (1994) 617^627. S. Shimba, R. Reddy, Puri¢cation of human U6 small nuclear RNA capping enzyme, J. Biol. Chem. 269 (1994) 12419^12423. S. Gupta, R. Busch, R. Singh, R. Reddy, Characterization of U6 small nuclear RNA cap-speci¢c antibodies. Identi¢cation of gamma-monomethyl-GTP cap structure in 7SK and several other human small RNAs, J. Biol. Chem. 265 (1990) 19137^19142. J. Towbin, T. Stahelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets procedure and some applications, Proc. Natl. Acad. Sci. USA 76 (1979) 4350^4354.
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[21] J.W. Freeman, R.K. Busch, F. Gyorkey, P. Gyorkey, B.E. Ross, H. Busch, Identi¢cation and characterization of a human proliferation-associated nucleolar antigen with a molecular weight of 120,000 expressed in early G1 phase, Cancer Res. 48 (1988) 1244^1251. [22] L.A. Schi¡, M.L. Nivert, M.S. Co, E.G. Brown, B.N. Fields, Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein c3 , Mol. Cell. Biol. 8 (1988) 273^283. [23] G. Biamonti, S. Riva, New insights into the auxiliary domains of eukaryotic RNA binding proteins, FEBS lett. 340 (1994) 1^8. [24] S. Huang, D.L. Spector, Dynamic organization of premRNA splicing factors, J. Cell. Biochem. 62 (1996) 191^197. [25] T. Nagase, N. Seki, A. Tanaka, K. Ishikawa, N. Nomura, Prediction of the coding sequences of 40 new genes (KIAA0121-KIAA0160) deduced by analysis of cDNA clones from human cell line KG-1, DNA Res. 2 (1995) 167^174. [26] Gustafson, W.C., Taylor, C.W., Valdez, B.C., Henning, D., Phippard, A., Ren, Y., Busch, H. and Durban, E. (1998) Nucleolar protein p120 contains an arginine-rich domain that binds to ribosomal RNA. Biochem. J., in press. [27] V.E. Myer, J.A. Steitz, Isolation and characterization of novel low abundance hnRNP protein: A0, RNA 1 (1995) 171^182. [28] S. Pinol-Roma, Y.D. Choi, M.J. Matunis, G. Dreyfuss, Immunopuri¢cation of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins, Genes Dev. 2 (1988) 215^227.
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Isolation and characterization of a new 110 kDa human nuclear RNA-binding protein (p110nrb ) Jian Gu a , Shigeki Shimba a
1;a
, Nobuo Nomura b , Ram Reddy
a;
*
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA b Kazusa DNA Research Institute, Kisarazu, Chiba 292, Japan Received 13 April 1998; accepted 4 May 1998
Abstract RNA-protein interactions play key roles in many fundamental cellular processes such as RNA processing, RNA transport, and RNA translation. During our attempts to isolate the human U6 small nuclear RNA capping enzyme, we identified a new 110 kDa nuclear RNA-binding protein, designated p110nrb . The full-length cDNA clone for p110nrb was characterized, and it encodes a 963 amino acid polypeptide. It is a highly acidic protein (pI 5.28) and the carboxyl terminal portion contains two conserved RNP motifs. A databank search found a putative C. elegans protein that might be the p110nrb homologue. The p110nrb was overexpressed as a glutathione S-transferase fusion protein in insect Sf9 cells, purified by affinity chromatography and injected into rabbits to produce specific polyclonal antibodies. Immunofluorescent staining showed that p110nrb is distributed evenly throughout the nucleoplasm. Northern blots showed that the mRNA is expressed in all tissues examined. An in vitro RNA-binding assay showed that p110nrb bound to RNA. These data suggest that p110nrb may play a role in the metabolism of nuclear RNA. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: RNA-binding protein; RNP motif; RNA-protein interaction
1. Introduction RNA-binding proteins are involved in various aspects of RNA metabolism. The cloning of cDNAs and the determination of amino acid sequences have identi¢ed several conserved motifs in RNAbinding proteins. The most common and best-characterized RNA-binding domain is the RNP motif, also referred to as RNA recognition motif (RRM) and RNP consensus sequence (RNP-CS). RNP motif proteins form a large and diverse family including * Corresponding author. Fax: +1 (713) 798-3145; E-mail: [email protected] 1 Current address: Department of Hygienic Chemistry, Nihon University, Chiba, Japan 274.
proteins that bind nuclear pre-mRNA (hnRNA), mature mRNA, small nuclear RNAs (snRNA), ribosomal RNA, as well as many RNAs transcribed by RNA polymerase III. The distinguishing characteristics of the RNP motif are two highly conserved short amino acid stretches which are separated by approximately 30 amino acids in this RNA-binding domain of about 90 amino acids. The ¢rst conserved amino acid stretch is a hydrophobic segment of six residues, which is called RNP2, and the second one is an octapeptide motif called RNP1 [1^4]. RNP motif proteins have diverse functions. Out of twenty hnRNP proteins that are known to associate with nascent hnRNA, at least nine of them belong to the category of RNP motif proteins [3]. RNP motif proteins play important roles in both constitutive
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and alternative splicing [5]. Several snRNP proteins contain RNP motifs, including U1-70K [1,6], U1A [7], and U2BQ protein [8]. Other splicing factors with RNP motifs include PTB [9], PSF [10,11] and several members of the SR protein family [12]. The well-characterized alternative splicing factors, Drosophila Sxl [13] and Tra-2 [14], are also RNP motif proteins. RNP motif proteins are also involved in both the 5P and 3P formation of mRNAs. Cap-binding proteins play important roles in splicing of pre-mRNAs and export of snRNAs containing trimethylguanosine cap structures. The m7 G cap-binding protein is a heterodimer of CBP20 and CBP80; the CBP20 contains a consensus RNP motif [15]. Several proteins involved in the polyadenylation of mRNAs, such as poly-A polymerase and the 64 kDa subunit of CstF, contain RNP motifs [16]. CPEB, which binds the cytoplasmic polyadenylation element (CPE) and mediates the cytoplasmic polyadenylation of maternal mRNA during early animal development in some species, contains an RNP motif [17]. Therefore, it is well established that proteins of the RNP motif family play important roles in numerous important cellular processes. During our attempts to isolate the enzyme for U6 snRNA 5P monomethylphosphate cap formation, we identi¢ed a new member of RNP motif protein family, designated p110nrb . The protein was expressed in baculovirus system, and polyclonal antibodies were raised against puri¢ed fusion protein GST-p110nrb . The protein is localized to the nucleoplasm and does not show capping activity in an in vitro capping assay. The mRNA of p110nrb is expressed in all tissues examined. p110nrb also binds RNA in an in vitro RNA-binding assay. The RNA substrate of p110nrb in vivo and its function remains to be established.
university microsequencing facility). The following six peptide sequences were obtained: (1) TEGLSED^DIAVQK; (2) LAEYQAYIDFEMK; (3) YANMWLEYYNLER; (4) AAA^QAENGPAAAPAV^APAATEA; (5) VGLHMTK; (6) TYGA(C)GK (dashes indicate amino acids that could not be identi¢ed; amino acid residues in brackets are probable, but not certain). 2.2. The cDNA cloning and expression of p110nrb We searched the databank and found our partial peptide sequences and cDNA sequences matched exactly to a recently sequenced human cDNA with continuous open reading frame. The complete cDNA for this protein was ampli¢ed by PCR and inserted into the baculovirus transfer vectors pVL1393 (for intact protein expression) and pAcGHLT-A (for GST fusion protein expression). Recombinant plasmids pVL1393/p110nrb and pAcGHLT-A/p110nrb and baculovirus gold DNA were cotransfected into Sf9 cells by calcium phosphate precipitation. The ampli¢cation of recombinant virus, expression of p110nrb protein in Sf9 cells and puri¢cation of GST-p110nrb fusion protein were done according to the Pharmagen protocol. 2.3. Preparation of anti-p110nrb polyclonal antibodies Rabbits were immunized with the puri¢ed recombinant GST-p110nrb produced in Sf9 cells. The antibody was produced and puri¢ed as previously described [19]. 2.4. SDS-PAGE and Western blotting
2. Materials and methods
Proteins were separated on 7.5% SDS-PAGE. For Western blotting, proteins were transferred to nitrocellulose and analyzed by immunoblotting as previously described [20].
2.1. Puri¢cation of p110nrb and sequencing of peptide
2.5. Immuno£uorescence staining
The sequential puri¢cation of p110nrb was described in detail by Shimba and Reddy [18]. Approximately 15 Wg of protein starting from the HeLa cell extract was obtained and subjected to cleavage and microsequencing of peptides (courtesy of Harvard
HeLa cells were grown on glass slides, ¢xed in 2% formaldehyde in phosphate-bu¡ered saline (PBS) for 20 min at room temperature, and permeabilized in acetone for 4 min at 320³C [21]. Cells were overlaid with a¤nity-puri¢ed anti-p110nrb antibodies, incu-
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bated for 1 h at 37³C, and washed three times in PBS (20 min, each wash). The second antibody (£uorescein-labeled anti-rabbit antibody) was added and after incubating the slides for 35 min at 37³C, washed three times with PBS (20 min, each wash). The slides were covered with coverslips after adding n-propyl gallate in glycerol/PBS. 2.6. Northern blot analysis The cDNA fragment of p110nrb was PCR ampli¢ed, gel isolated, labeled by random hexanucleotide priming, and used to probe a multiple tissue Northern blot of poly-A RNA (Clontech). The hybridization was done using ExpressHyb1 Hybridization Solution according to the protocol provided by Clontech. 2.7. Northwestern blot analysis The `Northwestern' blot RNA-binding assay was performed as described by Schi¡ et al. [22]. Samples of puri¢ed recombinant p110nrb and several control proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membrane. The membrane was blocked overnight at room temperature in Northwestern bu¡er (10 mM Tris-HCl, pH 6.8, 25 mM NaCl, 1 mM EDTA, 0.04% BSA, 0.04% Ficoll 400, 0.04% polyvinyl pyrrolidone-40). The probe used in this study was the mRNA of HBV-X (Hepatitis B Virus-X) protein which was obtained by in vitro transcription by T7 RNA polymerase; it was neither capped nor polyadenylated. The blots were probed at room temperature for 2 h. The blot was then washed three times (10 min, each wash) with Northwestern bu¡er to remove unbound or non-speci¢cally bound RNA. The dried nitrocellulose ¢lter was then subjected to autoradiography. 3. Results 3.1. Amino acid sequence of p110nrb The putative U6 snRNA capping enzyme was puri¢ed as described earlier [18]. The puri¢ed protein was subjected to sequencing and several peptide sequences were obtained as described in Section 2.
3
While the cDNA cloning by conventional methods (PCR using degenerate primers followed by cDNA library screening) was in progress, one of the cDNA clones that was sequenced at random by one of the authors (N. Nomura) contained perfect matches with our partial cDNA sequences. Analysis of the cDNA sequence showed an apparent full-length ORF of V2.9 kb, coding for 963 amino acids with a predicted molecular weight of 109 933 Da and a predicted pI of 5.28. In the carboxy terminal region, two highly conserved RNP motifs spanning amino acids 706^790 and 803^880 were found; no other motifs such as Gly-rich domain, Ser/Arg (SR) domain, or highly charged auxiliary domains, were identi¢ed (Fig. 1). A putative nuclear location signal was located in amino acids 601^616 (Fig. 1). We designated this protein p110nrb (110 kDa nuclear RNA-binding protein). 3.2. p110nrb is homologous to a C. elegans ORF A search of the sequence database and analysis by GAP program showed that the cDNA for p110nrb is 30% identical and 41% similar to a C. elegans ORF which codes for a protein of 836 amino acids. The C. elegans homologue also contains two RNP motifs at its C-terminal region (Fig. 1). Although no complete mouse homologue was found, a search of mouse EST library revealed that several long stretches of cDNA fragments were approximately 90% similar to human p110nrb cDNA. The carboxy terminal RNP motif region of the mouse homologue showed 94% identity to human p110nrb (data not shown). S. cerevisiae genome was searched and no yeast p110nrb homologue was identi¢ed. 3.3. Sequence comparison of p110nrb with other RNP proteins Most of the known RNP proteins also contain other auxiliary domains, such as Gly-rich domain, Ser/Arg (SR) domain, and highly charged auxiliary domains [23]. Thorough examination of p110nrb did not reveal any other previously characterized domains. However, it is notable that the amino terminal portion is more acidic than the carboxyl terminal region, where the predicted pI of amino acids 1^321 is 4.30, while the pI of amino acids 322^642 and 643^
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Fig. 1. Amino acid sequence of the protein p110nrb and comparison with its putative C. elegans homologue. The two RNA-binding domains are boxed, with the consensus RNP1 and RNP2 in each domain underlined. A putative nuclear localization sequence (NLS) is shown in bold and underlined. The GeneBank accession numbers for a p110nrb and its C. elegans homologue are D63879 and Z73102 (Chromosome III which contains the C. elegans homologue), respectively.
963 is 6.04 and 9.77, respectively. In the case of C. elegans homologue, the predicted pI of the full length (836 amino acids) is 6.04, while the pI of the amino terminal portion (amino acids 1^280) and the carboxyl terminal portion (amino acids 560^836) is 4.57 and 10.12, respectively. Fig. 2 shows the structural comparison of p110nrb with some other members of RNP motif proteins.
3.4. Expression and puri¢cation of p110nrb in insect cells To help understand the function(s) of p110nrb , we expressed recombinant p110nrb using the baculovirusSf9 expression system and GST tag puri¢cation methods. As shown in Fig. 3, p110nrb was overexpressed in Sf9 cells both in intact form (lane 4) and
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Fig. 2. Schematic representation of the modular structure of some representative RNP motif family proteins. The RNP motifs are indicated by roman numbering (I^IV). Auxiliary domains are indicated by their respective names. White boxes represent uncharacterized regions.
in the GST fusion form (lane 5). When glutathione beads were used to purify GST fusion protein, we obtained puri¢ed GST-p110nrb (lane 6). When the puri¢ed GST-p110nrb was used in an in vitro capping
5
Fig. 3. SDS-PAGE analysis of p110nrb proteins expressed in Sf9 insect cells. Lane 1, protein standards used as molecular weight markers; lane 2, mock-infected Sf9 cells ; lane 3, Sf9 cells infected with wild-type virus; lane 4, Sf9 cells infected with pVLp110nrb recombinant viruses (non-fusion protein); lane 5, Sf9 cells infected with pAcGHLT-p110nrb viruses (GST fusion protein); lane 6, GST-p110nrb fusion protein puri¢ed by glutathione beads from material analyzed in lane 5. The proteins were visualized by staining with Coomassie brilliant blue.
system, there was no detectable methylphosphate cap formation; and extracts from Sf9 cells infected with the recombinant baculovirus were not enriched in
Fig. 4. Western blot analysis of p110nrb protein expressed in Sf9 insect cells and in HeLa cells. Protein samples were fractionated by SDS-PAGE, transferred to nitrocellulose and analyzed by Western blotting with IgGs puri¢ed from anti-p110nrb serum. Panel A: Sf9 cells. Lane 1, protein standards used as molecular weight markers; lane 2, Sf9 cells infected with wild-type virus; lane 3, Sf9 cells infected with pVL-p110nrb viruses ; lane 4, Sf9 cells infected with pAcGHLT-p110nrb viruses ; lane 5, GST-p110nrb puri¢ed with GSH beads. Panel B: HeLa cells. Lane 1, protein standards; lane 2, HeLa S100 whole cell extracts.
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Fig. 5. Immuno£uorescence with anti-p110nrb antibodies. HeLa cells were grown on glass slides overlaid with puri¢ed anti-p110nrb antibody IgG fractions. The second antibody, £uorescein-labeled anti-rabbit antibody, was used to localize the anti-p110nrb antibody. Panel A: Preimmune serum. Panel B: Anti-p110nrb antibodies.
methylphosphate cap activity relative to the wildtype-infected cell extracts (data not shown). 3.5. Western blot analysis and immuno£uorescence staining Polyclonal antibodies were raised using puri¢ed GST-p110nrb as the antigen. Fig. 4 shows the specificity of anti-p110nrb antibodies. The polyclonal antibody against p110nrb was used to probe cell extracts. There was no signal in wild-type virus-infected cells, however, when Sf9 cells were infected with baculovirus transfer vector containing p110nrb and GSTp110nrb sequence, strong signals corresponding to p110nrb and GST-p110nrb were seen (Fig. 4A, lanes 3 and 4, respectively). After the ¢rst round of infection of the Sf9 cells, the enrichment of recombinant p110nrb was minimal and could not be visualized by Coomassie blue staining; even at this stage, the recombinant human p110nrb was readily detectable by Western blot (data not shown). When this antibody was used to probe the whole HeLa cell extracts, a prominent band of V110 kDa was seen (Fig. 4B, lane 2). When we did immunodepletion experiments using this polyclonal antibody, the V110 kDa protein was speci¢cally depleted, but the depleted HeLa cell extracts still possessed comparable methylphosphate capping activity (data not shown). Immuno£uorescence microscopy of HeLa cells with the anti-p110nrb demonstrates the nuclear localization of these proteins and their absence from cytoplasm (Fig. 5B). The pattern of staining is similar to many
hnRNP proteins in that they are uniformly stained throughout the nucleoplasm but excluded from nucleoli. This is in contrast with the immunostaining of the spliceosomal snRNP proteins, which typically show nucleoplasmic speckled pattern [24]. 3.6. Northern blot analysis Hybridization of poly-A RNA from di¡erent human tissues with p110nrb cDNA probe shows a V3.8 kb long positive band (Fig. 6). Although there are small variations on the expression level of p110nrb in di¡erent tissues, the mRNA is present in all the examined tissues and we did not observe any signi¢cant overexpression in any one single tissue. The extra length of mRNA was due to the long 3P untranslated sequences (data not shown, also see [25]). 3.7. p110nrb binds RNA in vitro In order to test the RNA-binding activity of p110nrb , we used a sensitive in vitro RNA-binding assay. Proteins were separated on SDS/PAGE and transferred to nitrocellulose paper. The nitrocellulose paper was then probed with a 32 P-labeled 0.8 kb long RNA transcript. Fig. 7A shows the Coomassie blue staining pattern of SDS-PAGE before transferring. C23 (nucleolin) and p120 were chosen as controls because C23 has four RNP motifs at its C-terminus and its RNP motifs are most closely related to those of p110nrb , and p120 was shown to be able to bind to rRNA by Gustafson et al. [26]. Fig. 7B shows the
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none of the molecular markers bind to RNA. These data demonstrate that p110nrb is indeed a nuclear RNA-binding protein. In our studies, RNA transcribed from a variety of DNA templates including plasmid DNA, ribosomal DNA, bound p110nrb protein (data not shown). These data suggest lack of substrate speci¢city in this in vitro assay. The cellular substrate of p110nrb remains to be elucidated. It is likely that p110nrb plays a general role in nuclear RNA metabolism, since it is expressed in all tissues examined by Northern blot (Fig. 6) and is distributed evenly throughout the nucleoplasm (Fig. 5B). 4. Discussion
Fig. 6. Expression of p110nrb in various human tissues. Human multiple tissue Northern blot (Clontech) was probed with p110nrb cDNA fragment probe (A). The blot was stripped of the probe after autoradiography and reprobed with a L-actin cDNA probe (B).
RNA-binding results. As expected, C23 and p120 wild-type proteins, but not the p120 mutant protein, bind to RNA (lanes 8, 9 and 10 respectively). p110nrb also showed clear RNA-binding activity (lane 7), but
We describe here the isolation and characterization of a new human nuclear RNA-binding protein, p110nrb . This protein contains two consensus RNP motifs and is capable of binding RNA in vitro. A putative C. elegans homologue of p110nrb has been identi¢ed by searching the databanks. Except for the two RNP motifs, these two proteins contain no other known protein motifs. Among all the characterized RNP motif proteins, very few of them are acidic. Both p110nrb and its C. elegans homologue are distinct acidic RNP motif proteins in that they have a large acidic region at the amino terminus which spans more than one-third of the molecule, while the one-third region at their carboxyl terminus is
Fig. 7. Northwestern blot of p110nrb to in vitro transcribed mRNA. Recombinant GST fusion proteins as indicated on top of each lane were electrophoresed on a 7.5% SDS-PAGE and (A) stained with Coomassie blue, (B) transferred to nitrocellulose and probed with 32 P-labeled 0.8 kb mRNA. The p120 wild-type used here is a partial sequence corresponding to amino acids 19^167 of p120, in the mutant, the sequence 46-RARKRAAKRRL-56 was mutated to 46-GAGILAANGGL-56. This mutation was shown previously to eliminate the RNA-binding activity of p120 [26].
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basic. The middle portions of both proteins are slightly acidic. This polar distribution of amino acids and the lack of Pro-, Gly-, or Ser/Arg-rich regions common to many other RNP motif proteins suggest that p110nrb and its homologue form a distinct subfamily of RNP motif proteins. In terms of the functional relevance of the polar distribution of amino acids, the basic carboxyl terminal region is responsible for RNA binding, while the amino terminal region may be involved in protein-protein interactions, and perhaps to have e¡ector functions. There are common epitopes between hnRNP proteins and antibodies raised against one RNP motif protein frequently cross-react with other proteins. For example, antibodies raised against hnRNP A0 protein cross-react with hnRNP A2/B1 [27]. The polyclonal antibodies raised against p110nrb protein did not show strong cross-reactivity with other proteins (Fig. 4). Therefore, it appears that p110nrb protein is not closely related in structure to other RNP motif proteins. Many of the nuclear RNA-binding proteins bind to pre-mRNAs or snRNAs and form large ribonucleoprotein complexes, called hnRNPs and snRNPs, respectively. Our data suggest that p110nrb is not in a large ribonucleoprotein complex. First, hnRNP proteins can be detected by immunoprecipitation with known abundant hnRNP proteins [3,28]. We did immunoprecipitation experiments with anti-hnRNP A1, C1 and U proteins. While the major hnRNP proteins were readily detectable in immunoprecipitates, the p110nrb protein was not detectable (data not shown). In addition, the uniform nucleoplasmic localization of p110nrb (Fig. 5B) is di¡erent from the nucleoplasmic speckles of snRNPs involved in pre-mRNA splicing [24]. Therefore, it is likely that p110nrb is not involved in hnRNP complexes and pre-mRNA processing. p110nrb is a novel nuclear RNA-binding protein, since it has two consensus RNP motifs and binds to RNA in our in vitro system. Although it is probably not part of nuclear splicing apparatus, it may still play general roles in nuclear RNA metabolism. Its mRNA is expressed in all tissues examined and it is localized uniformly in nucleoplasm. The cellular RNA substrate(s) and the ensuing function(s) of p110nrb remain to be elucidated.
Acknowledgements We thank T. Nagase for help in sequencing human p110nrb cDNA. We also thank M. Finley for HeLa cells, Dr. G. Dreyfuss of University of Pennsylvania for monoclonal antibodies against hnRNP proteins, Dr. L. Perlaky for help with immuno£uorescence staining, Y. Chen, K. Sinha and K. Perumal for helpful discussions. This work was supported by National Institute of Health Grant GM 38320. References [1] C.C. Query, R.C. Bentley, J.D. Keene, A common RNA recognition motif identi¢ed within a de¢ned U1 RNA binding domain of the 70K U1 snRNP protein, Cell 57 (1989) 89^101. [2] S.R. Haynes, The RNA motif protein family, New Biol. 4 (1992) 421^429. [3] G. Dreysuss, M.J. Matunis, S. Pinol-Roma, C.G. Burd, hnRNP Proteins and the biogenesis of mRNA, Annu. Rev. Biochem. 62 (1993) 289^321. [4] C.G. Burd, G. Dreyfuss, Conserved structures and diversity of functions of RNA-binding proteins, Science 265 (1994) 615^621. [5] A. Kramer, The structure and function of proteins involved in mammalian pre-mRNA splicing, Annu. Rev. Biochem. 65 (1996) 367^409. [6] J. Theissen, M. Etzerodt, R. Reuter, C. Schneider, F. Lottspeich, P. Argos, R. Luhrmann, L. Philipson, Cloning of the human cDNA for the U1 RNA-associated 70K protein, EMBO J. 5 (1986) 3209^3217. [7] C. Kambach, I.W. Mattaj, Intracellular distribution of the U1A protein depends on active transport and nuclear binding to U1 snRNA, J. Cell Biol. 118 (1992) 11^21. [8] W.J. Habets, P.T.G. Sillekens, M.H. Holt, J.A. Schalken, A.J.M. Roebroek, J.A.M. Leunissen, W.J.M. van de Ven, W.J. Van Venrooij, Analysis of a cDNA clone expressing a human autoimmune antigen: Full-length sequence of the U2 small nuclear RNA-associated BQ antigen, Proc. Natl. Acad. Sci. USA 84 (1987) 2421^2425. [9] J.G. Patton, S.A. Mayer, P. Tempst, B. Nadal-Ginard, Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing, Genes Dev. 5 (1991) 1237^1251. [10] J.G. Patton, E.B. Porro, J. Galceran, P. Tempst, B. NadalGinard, Cloning and characterization of PSF, a novel premRNA splicing factor, Genes Dev. 7 (1993) 393^406. [11] A. Gil, P.A. Sharp, S.F. Jamison, M.A. Garcia-Blanco, Characterization of cDNAs encoding the polypyrimidine tract-binding protein, Genes Dev. 5 (1911) 1224^1236. [12] J. Valcarcel, M.R. Green, The SR protein family: pleiotropic
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[21] J.W. Freeman, R.K. Busch, F. Gyorkey, P. Gyorkey, B.E. Ross, H. Busch, Identi¢cation and characterization of a human proliferation-associated nucleolar antigen with a molecular weight of 120,000 expressed in early G1 phase, Cancer Res. 48 (1988) 1244^1251. [22] L.A. Schi¡, M.L. Nivert, M.S. Co, E.G. Brown, B.N. Fields, Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein c3 , Mol. Cell. Biol. 8 (1988) 273^283. [23] G. Biamonti, S. Riva, New insights into the auxiliary domains of eukaryotic RNA binding proteins, FEBS lett. 340 (1994) 1^8. [24] S. Huang, D.L. Spector, Dynamic organization of premRNA splicing factors, J. Cell. Biochem. 62 (1996) 191^197. [25] T. Nagase, N. Seki, A. Tanaka, K. Ishikawa, N. Nomura, Prediction of the coding sequences of 40 new genes (KIAA0121-KIAA0160) deduced by analysis of cDNA clones from human cell line KG-1, DNA Res. 2 (1995) 167^174. [26] Gustafson, W.C., Taylor, C.W., Valdez, B.C., Henning, D., Phippard, A., Ren, Y., Busch, H. and Durban, E. (1998) Nucleolar protein p120 contains an arginine-rich domain that binds to ribosomal RNA. Biochem. J., in press. [27] V.E. Myer, J.A. Steitz, Isolation and characterization of novel low abundance hnRNP protein: A0, RNA 1 (1995) 171^182. [28] S. Pinol-Roma, Y.D. Choi, M.J. Matunis, G. Dreyfuss, Immunopuri¢cation of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins, Genes Dev. 2 (1988) 215^227.
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