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Human Ribosomal Protein L7 Carries Two Nucleic Acid-Binding Domains with Distinct Specificities
Biochemical and Biophysical Research Communications 258, 530 –536 (1999) Article ID bbrc.1999.0682, available online at http://www.idealibrary.com on
Human Ribosomal Protein L7 Carries Two Nucleic Acid-Binding Domains with Distinct Specificities Anna von Mikecz,* ,† Elli Neu,* Ulrich Krawinkel,* and Peter Hemmerich* ,‡ ,1 *Department of Immunology, University of Konstanz, Konstanz, Germany; †Medical Institute for Environmental Hygiene, Du¨sseldorf, Germany; and ‡Institute for Molecular Biotechnology, Jena, Germany
Received April 12, 1999
Protein L7 is associated with the large subunit of eukaryotic ribosomes that can act as a co-regulator of nuclear receptor-mediated transcription. In this study we show that L7 carries in addition to the known N-terminal nucleic acid-binding domain (NBD 1) a second one (NBD 2) which maps to the 50 C-terminal amino acids of the protein. The amino acid sequence of this region does not contain any of the known nucleic acid binding motifs; thus, NBD 2 may represent a new class of nucleic acid-binding protein motifs. NBD 2 is conserved in all known eukaryotic L7 homologs, whereas NBD 1 is only present in mammalian L7. Binding studies show that NBD 2 is functionally different from NBD 1 in that it binds preferentially to 28S rRNA, suggesting that NBD 2 is involved in the attachment of protein L7 to the large ribosomal subunit. Potential functions of NBD 1 and NBD 2 in translational and nuclear receptor-mediated transcriptional control are discussed. © 1999 Academic Press
Human and rodent protein L7 have been shown to be an integral part of the large subunit of ribosomes [1, and references therein]. The human and mouse L7genes share characteristic structural features with genes of other ribosomal proteins [2, 3]. There is no bacterial homologue of eukaryotic protein L7 [4]. Rodent and human L7 proteins carry in their N-terminal region a motif which is similar to the basic-regionleucine-zipper (bzip) domain of DNA-binding eukaryotic transcription factors [5]. Recombinant L7-peptides containing the bzip-like region bind to RNA and DNA as homodimers, as has been demonstrated by filterbinding, cross-linking analyses, and mass spectrometry studies [5–7]. A 22 amino acid long, alpha-helical 1 To whom correspondence should be addressed at Institute for Molecular Biotechnology, Beutenbergstr. 11, 07745 Jena, Germany. Fax: 149 3641 656225. E-mail: [email protected]. Abbreviations used: His; histidine; GST; glutathione-S-transferase; bzip; basic region leucine zipper; K d, apparent dissociation constant; aa, amino acid(s); NBD, nucleic acid-binding domain.
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arginine- and lysine-rich peptide derived from the bziplike region is sufficient for DNA-binding and specific RNA-binding [8]. In vitro and in vivo studies together with its ribosomal association [1] suggest that L7 is involved in translational regulation of gene expression [7, 9]. The recently reported interaction of L7 with ribosomal protein S7 and the multi-zinc finger protein ZNF7 in vivo may also be relevant for this function [10]. In addition to its role in the translation apparatus L7 has been demonstrated to be a co-regulator of nuclear receptor-mediated transcription. L7 can function as a co-activator that strongly enhances transcription of RU486-occupied human progesterone receptor (hPR) or glucocorticoid receptor (GR), and tamoxifenoccupied human oestrogen receptor (hER) [11]. L7 also co-regulates vitamin D receptor-retinoid X receptor (VDR-RXR) mediated transactivation [12]. Deletion analyses indicated that the N-terminus of L7, which harbours the bzip-like domain, mediates the physical interaction with the vitamin D receptor in vitro and in vivo [12]. Thus, L7 seems to be involved in the control of eukaryotic gene expression at the level of both, transcription and translation. In addition, we have described protein L7 as a major autoantigen in patients with systemic rheumatic diseases. Quantitative and qualitative changes of the anti-L7 autoantibody response appear to correlate with acute disease manifestation [13–17], thus indicating that L7 is involved in as yet unknown pathomechanisms. Ribonucleoprotein particles and ribosomal components are main targets of systemic autoimmune responses [18, 19]. Since the study of the structure and function of autoantigens is believed to provide insights into the cellular and molecular basis of autoimmune processes [18, 19] we further analysed the structurefunction relationship of protein L7. It has been unclear as yet how protein L7 interacts with ribosomal components. In this report we demonstrate that L7 carries in addition to the N-terminal nucleic acid-binding domain (NBD 1) a second one (NBD 2) which resides in the fifty C-terminal amino
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acids. The two NBDs of protein L7 appear to be functionally distinct in that NBD 1 binds 28S rRNA and mRNA with similar affinity, whereas NBD 2 clearly prefers 28S rRNA over all other ligands. In addition, NBD 1 also binds to dsDNA whereas NBD 2 seems to be specific for RNA-binding. MATERIALS AND METHODS Standard procedures. Molecular cloning procedures, analytical and preparative nucleic acid and protein gel electrophoresis, and polymerase chain reactions were performed as described by Ausubel et al. [20]. Construction and expression of L7 fusion proteins. Plasmid pHL7.14 [5] was used as a template to PCR-amplify a DNA-fragment containing the open reading frame of human ribosomal protein L7 with forward primer rat1 (59-CCGGATCCGGCTGGAACCATGGAGGCTG-39, BamHI site underlined) and reverse primer amb4 (59GGGAATTCGCAGGTACTGTTTATTAACCC-39, EcoRI site underlined) lacking the poly-A tail of L7 cDNA. The resulting fragment was cloned into BamHI/EcoRI prepared pBluescript II KS (Stratagene, San Diego, CA, USA) to yield pL7.14A 2. The BamHI/EcoRI fragment and a PstI/EcoRI fragment (coding for aa 87-248 of human protein L7) was further subcloned into expression vector pRSET-B (Invitrogen, San Diego, CA, USA) to obtain pRSET-L7 and pRSET-L7Dbzip. All plasmid inserts were verified by sequencing. His-tagged fusion proteins were expressed from these plasmids in E. coli BL21(DE)3 according to the manufacturers instructions (Invitrogen) and purified by affinity chromatography on Ni 21-NTA beads (Quiagen, Hilden, Germany). The construction and expression of GST-fusion proteins EP6, EP7, EP8, and EP9 has been described previously [14]. Nucleic acids, saturation filter binding assays. The synthesis of S-labeled L7 mRNA, saturation filter binding assays, the synthesis and isolation of unlabeled competitor RNAs and DNAs, and filter binding competition assays have been described in detail in previous reports [5, 7, 8]. Apparent dissociation constants (K d) were graphically determined from the slope of graphs obtained by plotting the number of moles of complexed RNA per mole of total RNA (1/r) versus the reciprocal of the concentration of free protein L7 (1/c) (double reciprocal plot). 35
Solid phase binding competition assay. PolyA 2- and polyA 1fractions of total RNA from peripheral blood mononuclear cells were prepared by affinity chromatography on oligo dT-cellulose. The 28S rRNA in the polyA 2-fraction was subsequently purified by preparative agarose gel electrophoresis, filtration through Spin-X columns (Sigma, Deisenhofen, Germany), and ethanol precipitation. Purified 28S rRNA was then coupled to suspended cyanogen-bromideactivated sepharose (Pharmacia, Uppsala, Sweden) according to the manufacturer’s instructions (10 mg per 100 ml beads). 35S-radiolabeled GST-L7 fragments were obtained by coupled cell free transcription/translation of polymerase chain reaction (PCR) products in a reticulocyte lysate (Promega, Madison, Wisconsin, USA). PCRfragments encoding GST-fused peptide domIII (represents NBD 1, aa 27– 48 of L7) and peptide EP9 (represents NBD 2, aa 199 –248 of L7) were generated using forward primer T3-ATG/GST and reverse primer pGEXrev from plasmids pGEX-L7domIII [8], and pGEX-EP9 [14], respectively. Primer T3-ATG/GST contained the T3 RNApolymerase recognition sequence for transcription, an ATG start codon followed by an in frame sequence complementary to nucleotide position 12 to 29 of Sj26 cDNA [21] and primer pGEXrev is reverse complementary to the 18 nucleotides downstream from the multiple cloning site of pGEX vectors [22]. 50 ml reticulocyte lysate were diluted 1:10 in phosphate buffered saline and GST-fused polypeptides were purified on glutathione-agarose beads. 35S-labeled L7fragments were then incubated for 20 min at room temperature with 10 ml of 28S rRNA-immobilised sepharose beads in 100 ml binding
buffer (10 mM TRIS/HCl, pH 7.0, 100 mM NaCl, 1 mM EDTA, 0.1 mg/ml bovine serum albumin), washed three times, and incubated for another 20 min with increasing concentrations of competitor nucleic acids in 100 ml binding buffer. The beads were then washed again three times in binding buffer, precipitated by centrifugation, resuspended in 10 ml reducing SDS-gel loading buffer, boiled for 2 min, and subjected to SDS-gel electrophoresis. Finally, the gels were soaked in “Amplify” (Amersham, Amersham, UK), dried and bands were visualised by autoradiography.
RESULTS Protein L7 Carries Two Nucleic Acid-Binding Domains The N-terminal region of protein L7, designated L7bzip (see Fig. 1A), carries a nucleic acid-binding domain (NBD 1) which is bzip-like and can bind as a homodimer with an arginine-lysine-rich sequence to dsDNA, and cognate sites on mRNAs [5, 8]. As protein L7 is capable of interfering with the translation from distinct mRNAs in vitro and in vivo [7] we hypothesised that mRNAs of as yet unspecified cellular proteins are the physiological targets of protein L7. In model systems, we demonstrated high affinity binding to L7 mRNA (K d 5 40 nM) and rev-responsive-element (RRE)-RNA of human immunodeficiency virus 1 (K d 5 10 nM) [8]. Upon analysis of saturation filter binding of histidine (His)-tagged complete protein L7 to L7 mRNA we found that it bound to this ligand with an at least twofold higher affinity (K d 5 20 nM) than Histagged or GST-fused L7bzip [7, and data not shown]. These results suggested that protein L7 carries sequences C-terminal to NBD 1 which contribute to the strength of mRNA-binding. Surprisingly, the Histagged C-terminal fragment of protein L7 showed a L7 mRNA-binding curve which was indistinguishable from the one of the complete protein (Fig. 1). This clearly indicated that protein L7 carries C-terminal to NBD 1 a second nucleic acid-binding domain (NBD 2) which is responsible for high affinity binding to L7 mRNA. In contrast to complete protein L7 and L7bzip, there is only weak, but measurable binding of L7Dbzip to double-stranded DNA (data not shown, see also Fig. 3). Mapping of NBD 2 To map NBD 2 within fragment L7Dbzip we performed saturation filter binding analyses with four GST-fused overlapping subfragments and His-L7Dbzip as a control, using radiolabeled L7 mRNA as a ligand (Fig. 2A). Fragment EP9 and L7Dbzip showed identical binding curves, fragment EP8 showed at least 30fold weaker but still significant binding, whereas binding by fragments EP6 and EP7 was hardly above background binding compared to GST alone (Fig. 2B). One can thus locate NBD 2 to EP9, that is to the 50 C-terminal amino acids of protein L7. The 10
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FIG. 1. Protein L7 carries a second NBD. (A) Schematic depiction of human L7 protein and constructs used for filter-binding assays. The positions of the previously characterized bzip-like region and the arginine-rich NBD [8] are indicated. (B) L7 full-length protein (lane-1) and L7Dbzip (lane-2) were expressed as His-tagged fusion proteins, affinity-purified and analyzed in SDS-gels. (C) Filter-binding curves of His-L7 and His-L7Dbzip to radiolabeled L7 mRNA.
N-terminal amino acids of EP9 overlap with the 10 C-terminal ones of EP8 (positions 198-207). These residues may contribute to RNA-binding as the N-terminal part of NBD 2, and could be responsible for the weak binding activity of EP8. Specificity of NBD 2 To functionally discriminate between NBD 1 and NBD 2 we performed competition analyses of RNA
binding. Firstly, we competed binding of radiolabeled L7 mRNA to His-L7Dbzip protein, that is to NBD 2, with various unlabeled nucleic acids on a mass basis (Fig. 3). In this analysis 28S rRNA and poly G compete significantly more efficient than the autologous competitor L7 mRNA. Transfer RNA (tRNA), linearised plasmid pBluescript (dsDNA), and poly dI:dC compete weakly. No competition is observed with synthetic ribohomopolymers poly A, poly U, poly C, and synthetic
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diolabeled L7-polypeptides was monitored by SDS gel electrophoresis (Fig. 4B). One thousand nanograms 28S rRNA per sample are sufficient to compete significantly with the binding of EP9 (NBD 2) to immobilized 28S rRNA, whereas more than 5000 nanograms of L7 mRNA, tRNA, unrelated RNA (ssRNA) or DNA are required to do so. For significant competition with the binding of peptide domIII (NBD 1) to immobilized 28S rRNA, 1000 nanograms of 28S rRNA, L7 mRNA, or DNA per sample are sufficient, and 5000 nanograms tRNA compete weakly. In summary, the two NBDs of protein L7 appear to be functionally distinct in that NBD 1 binds 28S rRNA and L7mRNA with similar affinity, whereas NBD 2 clearly prefers 28S rRNA over L7 mRNA. In addition, NBD 1 also binds to dsDNA whereas NBD 2 seems to be specific for RNA-binding. Conservation of NBD 1 and NBD 2 in L7 Proteins
FIG. 2. NBD 2 maps to the 50 C-terminal amino acids of protein L7. Overlapping fragments of protein L7 were expressed as GSTfusion proteins (schematically shown in A), and their mRNA-binding activity was analyzed by saturation filter binding to radiolabeled L7 mRNA (B).
deoxyribohomopolymers. GST-EP9 exhibited exactly the same profile of RNA-competition as His-L7Dbzip indicating that this function is fully retained in the fifty C-terminal aa of L7 (data not shown). The pattern of competitor efficiencies, namely poly G, 28S rRNA . L7 mRNA . tRNA . dsDNA, defines the relative affinities of a set of natural and synthetic ligands for NBD 2. It is different from the pattern poly G . L7 mRNA, 28S rRNA . dsDNA . tRNA, which we have determined previously by competition of L7 mRNAbinding to NBD 1 [5, 7, 8]. Significantly, calculated on a molar basis, the competitive efficiency of 28S rRNA binding to NBD2 is about twentyfold better than that of L7 mRNA, possibly indicating high affinity binding site(s) of NBD 2 on 28S rRNA. For a direct comparison of specificities, the binding of the 35S-labeled GST-fused polypeptides domIII [8] and EP9, which carry NBD 1 or NBD 2, respectively to 28S rRNA immobilised on agarose beads were competed with unlabeled 28S rRNA, L7 mRNA, tRNA, unrelated RNA (ssRNA, which represents the in vitro transcribed polylinker of plasmid pBluescript), and DNA (linearised pBluescript). The amount of agarose-bound ra-
Sequences are now available for L7 from eight species. To determine the degree of conservation of NBD 1 and NBD 2 the human sequences were aligned with the corresponding L7 sequences from rat, mouse, drosophila, dictyostelium, yeast (Saccharomyces cerevisiae), and plants (Solanum tuberosum, and Arabidopsis thaliana) (Fig. 5). The alignment shows that NBD 2 is highly conserved among all species (Fig. 5A), whereas NBD 1 is only conserved in vertebrate L7 proteins (Fig. 5B).
FIG. 3. Nucleic-acid-binding specificities of L7Dbzip. The binding of L7Dbzip to L7 mRNA was competed with increasing amounts of unlabeled nucleic acids as indicated.
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FIG. 4. The C-terminal NBD of protein L7 binds specifically to 28S rRNA. NBD 1 and NBD 2 of human protein L7, as represented by GST-fused peptides domIII and EP9 (A), respectively, were radiolabeled and bound to 28S rRNA which had been covalently attached to agarose beads. Protein-binding was competed with increasing amounts of unlabeled nucleic acids as indicated, and monitored by SDS-polyacrylamide electrophoresis under reducing conditions (B).
DISCUSSION In this report we demonstrate that human ribosomal protein L7 carries in addition to its N-terminal nucleic acid-binding domain (NBD 1) a C-terminal one (NBD 2) which shows high affinity for 28S rRNA. In a saturation filter binding study an apparent dissociation constant K d 5 20 nM was determined for L7 mRNAbinding of NBD 2, and in RNA-binding competition analyses we have shown that 28S rRNA is an about twentyfold more efficient competitor than L7 mRNA. Based on these data we would predict for 28S rRNAbinding to NBD 2 an apparent dissociation constant in the nanomolar range. NBD 2 also shows high affinity for the highly structured [23, 24] ribohomopolymer poly G. This property of NBD 2 is shared with NBD 1 [8] and NBDs in several unrelated proteins [25–27]. Protein L7 is also capable of binding to 5S rRNA [28]. The affinity of this interaction (K d 5 6 mM) is low compared to binding of 28S rRNA. However one can
not rule out a physiological relevance of this interaction because in the assembled ribosome the affinity of L7 to 5S rRNA may be increased by co-operative effects of neighbouring proteins. In summary, NBD 2 seems to bind with high affinity to one (or more) site(s) on 28S rRNA, and seems to cross-react with sites on some structured RNAs, for instance L7mRNA and poly G. We propose that NBD 2 mediates the association of L7 with the large ribosomal subunit through specific binding to 28S rRNA. This is not meant to exclude that interactions with other ribosomal proteins may also be involved. Our hypothesis is supported by in vitro biotinylation [29] and limited proteolytic degradation [30] studies which show that the N-terminal region of L7 including NBD 1 is solvent exposed, and presumably protrudes from the ribosome, whereas the C-terminal region, although hydrophilic in computer-based structure predictions, is inaccessible for external labeling or a proteolytic attack. NBD 2 thus very likely is buried in the interior of the large ribosomal subunit where it may interact with 28S rRNA. Although NBD 2 is also capable of high affinity-binding to sites on mRNA, it presumably will not come in contact with this potential ligand. For the interaction with mRNAs, the N-terminal part of L7 is in a more suitable position, as it is located on the surface of the ribosome [29], where it may be involved in the regulation of translation through binding of NBD1 with high affinity to cognate sites on specific mRNAs [7, 8, 9] and/or interaction with ribosomal protein S7 on the 40S subunit via the bziplike element [10]. Recently L7 turned out to be a general co-regulator which gears into different steroid hormone receptor controlled signal transduction pathways [11, 12]. The interaction of L7 with nuclear receptors was mapped to the N-terminus which contains the bzip-like element and NBD 1 [12]. It is not known at what stage L7 interferes with nuclear receptor-mediated transcription [11, 12]. Since the protein is also found in the nucleus of human cell lines [13, and unpublished results] and shown to be able to repress binding of VDRRXR heterodimers to a vitamin D response element [12], it is possible that the DNA-binding activity of NBD 1 in addition to the dimerisation property of the bzip-like element confers the regulatory function of L7 in L7/nuclear receptor containing transcription complexes. Searches of protein sequence data bases have not revealed significant similarity of the 50 C-terminal residues of protein L7 to segments of other nucleic-acid binding proteins. Wang et al. have identified in the NBD of nucleolar protein B23.1 a sequence motif (LWQW) that is also present in other nucleic acid binding proteins such as the negative regulator of mitosis in yeast, bacterial transposon TN7 transposition protein T, and bacterial DNA-primases [31]. Interestingly, the most conserved region of L7-NBD 2 contains the
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FIG. 5. Sequences of the C-terminal but not of the N-terminal NBDs are conserved in L7-related proteins from various organisms. (A) Sequence comparison of the C-terminal region of L7-proteins from human (h), rat (r), mouse (m), Drosophila melanogaster (dr), Dictyostelium discoideum (di), Saccharomyces cerevisiae (sc), Solanum tuberosum (st), and Arabidopsis thaliana (at). NBD 2 in human L7 maps to the region between position 198 and the C-terminus. cons 5 consensus sequence. (B) The N-terminal nucleic acid-binding domain (NBD 1, aa 27-48 of human L7, ref. 8) is conserved in L7-proteins from humans, rats and mice (underlined). Sequences are taken from reference 4 and the EMBL sequence data library (Arabidopsis thaliana, Accession Number 3212879; Drosophila melanogaster, Accession Number 417674; Solanum tuberosum, Accession Number Z30162). Yeast sequences are derived from the S. cerevisiae YL8A gene [4]. Known L7 sequences from Schizosaccharomyces pombe are highly conserved when compared with YL8A [4] and have therefore not been included into the alignment here.
sequence LWPF which resembles the conserved LWQW motif of B23.1. In addition, L7 and B23.1 share in their nucleic acid-binding domains two further regions of conserved amino acids with a preponderance of helix-destabilising amino acids (G, P): 214-SPRGG221, and 230-GGDAG-236, respectively, in L7-NBD2 (Fig. 5A) vs 234-TPKGP-241, and 255-GGSLP-262, respectively, in the B23.1 NBD [31]. These conserved regions may represent functionally important elements of a new class of nucleic acid-binding motifs. NBD 2 from L7 and the NBD of B23.1 are also similar in length (50 and 67 aa, respectively). It is not clear whether NBD 1 and NBD 2 are functionally linked. Protein L7, like some other multifunctional ribosomal proteins [32–35], may use distinct domains for unrelated functions. The latter view is supported by (i) the finding that NBD 1 inhibits cellfree translation in the absence of NBD 2 [7], and (ii) by evolutionary considerations. The C-terminal region of human protein L7 carrying NBD 2 is highly conserved among L7 proteins from distantly related organisms, whereas NBD 1 is variable and conserved only in L7 proteins derived from humans and rodents (Fig. 5). One may interpret the conservation of NBD 2 as an indication of its ,,old“ function, for instance mediating
ribosome-association through rRNA-binding, and the function of the less conserved NBD 1 containing a bzip element (i.e., co-regulation of nuclear receptors) apparently may have been acquired during the evolution of vertebrates. Future studies should reveal the physiological RNA (or DNA) binding sites of L7 to elucidate the mechanisms by which NBD 1 and NBD 2 of L7 regulate gene expression at the level of transcription and translation. ACKNOWLEDGMENTS We thank R. Sedlacek for the gift of rRNA, and M. Go¨rlach for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft through Grant C7 in Sonderforschungsbereich 156 to U.K.
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Human Ribosomal Protein L7 Carries Two Nucleic Acid-Binding Domains with Distinct Specificities Anna von Mikecz,* ,† Elli Neu,* Ulrich Krawinkel,* and Peter Hemmerich* ,‡ ,1 *Department of Immunology, University of Konstanz, Konstanz, Germany; †Medical Institute for Environmental Hygiene, Du¨sseldorf, Germany; and ‡Institute for Molecular Biotechnology, Jena, Germany
Received April 12, 1999
Protein L7 is associated with the large subunit of eukaryotic ribosomes that can act as a co-regulator of nuclear receptor-mediated transcription. In this study we show that L7 carries in addition to the known N-terminal nucleic acid-binding domain (NBD 1) a second one (NBD 2) which maps to the 50 C-terminal amino acids of the protein. The amino acid sequence of this region does not contain any of the known nucleic acid binding motifs; thus, NBD 2 may represent a new class of nucleic acid-binding protein motifs. NBD 2 is conserved in all known eukaryotic L7 homologs, whereas NBD 1 is only present in mammalian L7. Binding studies show that NBD 2 is functionally different from NBD 1 in that it binds preferentially to 28S rRNA, suggesting that NBD 2 is involved in the attachment of protein L7 to the large ribosomal subunit. Potential functions of NBD 1 and NBD 2 in translational and nuclear receptor-mediated transcriptional control are discussed. © 1999 Academic Press
Human and rodent protein L7 have been shown to be an integral part of the large subunit of ribosomes [1, and references therein]. The human and mouse L7genes share characteristic structural features with genes of other ribosomal proteins [2, 3]. There is no bacterial homologue of eukaryotic protein L7 [4]. Rodent and human L7 proteins carry in their N-terminal region a motif which is similar to the basic-regionleucine-zipper (bzip) domain of DNA-binding eukaryotic transcription factors [5]. Recombinant L7-peptides containing the bzip-like region bind to RNA and DNA as homodimers, as has been demonstrated by filterbinding, cross-linking analyses, and mass spectrometry studies [5–7]. A 22 amino acid long, alpha-helical 1 To whom correspondence should be addressed at Institute for Molecular Biotechnology, Beutenbergstr. 11, 07745 Jena, Germany. Fax: 149 3641 656225. E-mail: [email protected]. Abbreviations used: His; histidine; GST; glutathione-S-transferase; bzip; basic region leucine zipper; K d, apparent dissociation constant; aa, amino acid(s); NBD, nucleic acid-binding domain.
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arginine- and lysine-rich peptide derived from the bziplike region is sufficient for DNA-binding and specific RNA-binding [8]. In vitro and in vivo studies together with its ribosomal association [1] suggest that L7 is involved in translational regulation of gene expression [7, 9]. The recently reported interaction of L7 with ribosomal protein S7 and the multi-zinc finger protein ZNF7 in vivo may also be relevant for this function [10]. In addition to its role in the translation apparatus L7 has been demonstrated to be a co-regulator of nuclear receptor-mediated transcription. L7 can function as a co-activator that strongly enhances transcription of RU486-occupied human progesterone receptor (hPR) or glucocorticoid receptor (GR), and tamoxifenoccupied human oestrogen receptor (hER) [11]. L7 also co-regulates vitamin D receptor-retinoid X receptor (VDR-RXR) mediated transactivation [12]. Deletion analyses indicated that the N-terminus of L7, which harbours the bzip-like domain, mediates the physical interaction with the vitamin D receptor in vitro and in vivo [12]. Thus, L7 seems to be involved in the control of eukaryotic gene expression at the level of both, transcription and translation. In addition, we have described protein L7 as a major autoantigen in patients with systemic rheumatic diseases. Quantitative and qualitative changes of the anti-L7 autoantibody response appear to correlate with acute disease manifestation [13–17], thus indicating that L7 is involved in as yet unknown pathomechanisms. Ribonucleoprotein particles and ribosomal components are main targets of systemic autoimmune responses [18, 19]. Since the study of the structure and function of autoantigens is believed to provide insights into the cellular and molecular basis of autoimmune processes [18, 19] we further analysed the structurefunction relationship of protein L7. It has been unclear as yet how protein L7 interacts with ribosomal components. In this report we demonstrate that L7 carries in addition to the N-terminal nucleic acid-binding domain (NBD 1) a second one (NBD 2) which resides in the fifty C-terminal amino
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acids. The two NBDs of protein L7 appear to be functionally distinct in that NBD 1 binds 28S rRNA and mRNA with similar affinity, whereas NBD 2 clearly prefers 28S rRNA over all other ligands. In addition, NBD 1 also binds to dsDNA whereas NBD 2 seems to be specific for RNA-binding. MATERIALS AND METHODS Standard procedures. Molecular cloning procedures, analytical and preparative nucleic acid and protein gel electrophoresis, and polymerase chain reactions were performed as described by Ausubel et al. [20]. Construction and expression of L7 fusion proteins. Plasmid pHL7.14 [5] was used as a template to PCR-amplify a DNA-fragment containing the open reading frame of human ribosomal protein L7 with forward primer rat1 (59-CCGGATCCGGCTGGAACCATGGAGGCTG-39, BamHI site underlined) and reverse primer amb4 (59GGGAATTCGCAGGTACTGTTTATTAACCC-39, EcoRI site underlined) lacking the poly-A tail of L7 cDNA. The resulting fragment was cloned into BamHI/EcoRI prepared pBluescript II KS (Stratagene, San Diego, CA, USA) to yield pL7.14A 2. The BamHI/EcoRI fragment and a PstI/EcoRI fragment (coding for aa 87-248 of human protein L7) was further subcloned into expression vector pRSET-B (Invitrogen, San Diego, CA, USA) to obtain pRSET-L7 and pRSET-L7Dbzip. All plasmid inserts were verified by sequencing. His-tagged fusion proteins were expressed from these plasmids in E. coli BL21(DE)3 according to the manufacturers instructions (Invitrogen) and purified by affinity chromatography on Ni 21-NTA beads (Quiagen, Hilden, Germany). The construction and expression of GST-fusion proteins EP6, EP7, EP8, and EP9 has been described previously [14]. Nucleic acids, saturation filter binding assays. The synthesis of S-labeled L7 mRNA, saturation filter binding assays, the synthesis and isolation of unlabeled competitor RNAs and DNAs, and filter binding competition assays have been described in detail in previous reports [5, 7, 8]. Apparent dissociation constants (K d) were graphically determined from the slope of graphs obtained by plotting the number of moles of complexed RNA per mole of total RNA (1/r) versus the reciprocal of the concentration of free protein L7 (1/c) (double reciprocal plot). 35
Solid phase binding competition assay. PolyA 2- and polyA 1fractions of total RNA from peripheral blood mononuclear cells were prepared by affinity chromatography on oligo dT-cellulose. The 28S rRNA in the polyA 2-fraction was subsequently purified by preparative agarose gel electrophoresis, filtration through Spin-X columns (Sigma, Deisenhofen, Germany), and ethanol precipitation. Purified 28S rRNA was then coupled to suspended cyanogen-bromideactivated sepharose (Pharmacia, Uppsala, Sweden) according to the manufacturer’s instructions (10 mg per 100 ml beads). 35S-radiolabeled GST-L7 fragments were obtained by coupled cell free transcription/translation of polymerase chain reaction (PCR) products in a reticulocyte lysate (Promega, Madison, Wisconsin, USA). PCRfragments encoding GST-fused peptide domIII (represents NBD 1, aa 27– 48 of L7) and peptide EP9 (represents NBD 2, aa 199 –248 of L7) were generated using forward primer T3-ATG/GST and reverse primer pGEXrev from plasmids pGEX-L7domIII [8], and pGEX-EP9 [14], respectively. Primer T3-ATG/GST contained the T3 RNApolymerase recognition sequence for transcription, an ATG start codon followed by an in frame sequence complementary to nucleotide position 12 to 29 of Sj26 cDNA [21] and primer pGEXrev is reverse complementary to the 18 nucleotides downstream from the multiple cloning site of pGEX vectors [22]. 50 ml reticulocyte lysate were diluted 1:10 in phosphate buffered saline and GST-fused polypeptides were purified on glutathione-agarose beads. 35S-labeled L7fragments were then incubated for 20 min at room temperature with 10 ml of 28S rRNA-immobilised sepharose beads in 100 ml binding
buffer (10 mM TRIS/HCl, pH 7.0, 100 mM NaCl, 1 mM EDTA, 0.1 mg/ml bovine serum albumin), washed three times, and incubated for another 20 min with increasing concentrations of competitor nucleic acids in 100 ml binding buffer. The beads were then washed again three times in binding buffer, precipitated by centrifugation, resuspended in 10 ml reducing SDS-gel loading buffer, boiled for 2 min, and subjected to SDS-gel electrophoresis. Finally, the gels were soaked in “Amplify” (Amersham, Amersham, UK), dried and bands were visualised by autoradiography.
RESULTS Protein L7 Carries Two Nucleic Acid-Binding Domains The N-terminal region of protein L7, designated L7bzip (see Fig. 1A), carries a nucleic acid-binding domain (NBD 1) which is bzip-like and can bind as a homodimer with an arginine-lysine-rich sequence to dsDNA, and cognate sites on mRNAs [5, 8]. As protein L7 is capable of interfering with the translation from distinct mRNAs in vitro and in vivo [7] we hypothesised that mRNAs of as yet unspecified cellular proteins are the physiological targets of protein L7. In model systems, we demonstrated high affinity binding to L7 mRNA (K d 5 40 nM) and rev-responsive-element (RRE)-RNA of human immunodeficiency virus 1 (K d 5 10 nM) [8]. Upon analysis of saturation filter binding of histidine (His)-tagged complete protein L7 to L7 mRNA we found that it bound to this ligand with an at least twofold higher affinity (K d 5 20 nM) than Histagged or GST-fused L7bzip [7, and data not shown]. These results suggested that protein L7 carries sequences C-terminal to NBD 1 which contribute to the strength of mRNA-binding. Surprisingly, the Histagged C-terminal fragment of protein L7 showed a L7 mRNA-binding curve which was indistinguishable from the one of the complete protein (Fig. 1). This clearly indicated that protein L7 carries C-terminal to NBD 1 a second nucleic acid-binding domain (NBD 2) which is responsible for high affinity binding to L7 mRNA. In contrast to complete protein L7 and L7bzip, there is only weak, but measurable binding of L7Dbzip to double-stranded DNA (data not shown, see also Fig. 3). Mapping of NBD 2 To map NBD 2 within fragment L7Dbzip we performed saturation filter binding analyses with four GST-fused overlapping subfragments and His-L7Dbzip as a control, using radiolabeled L7 mRNA as a ligand (Fig. 2A). Fragment EP9 and L7Dbzip showed identical binding curves, fragment EP8 showed at least 30fold weaker but still significant binding, whereas binding by fragments EP6 and EP7 was hardly above background binding compared to GST alone (Fig. 2B). One can thus locate NBD 2 to EP9, that is to the 50 C-terminal amino acids of protein L7. The 10
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FIG. 1. Protein L7 carries a second NBD. (A) Schematic depiction of human L7 protein and constructs used for filter-binding assays. The positions of the previously characterized bzip-like region and the arginine-rich NBD [8] are indicated. (B) L7 full-length protein (lane-1) and L7Dbzip (lane-2) were expressed as His-tagged fusion proteins, affinity-purified and analyzed in SDS-gels. (C) Filter-binding curves of His-L7 and His-L7Dbzip to radiolabeled L7 mRNA.
N-terminal amino acids of EP9 overlap with the 10 C-terminal ones of EP8 (positions 198-207). These residues may contribute to RNA-binding as the N-terminal part of NBD 2, and could be responsible for the weak binding activity of EP8. Specificity of NBD 2 To functionally discriminate between NBD 1 and NBD 2 we performed competition analyses of RNA
binding. Firstly, we competed binding of radiolabeled L7 mRNA to His-L7Dbzip protein, that is to NBD 2, with various unlabeled nucleic acids on a mass basis (Fig. 3). In this analysis 28S rRNA and poly G compete significantly more efficient than the autologous competitor L7 mRNA. Transfer RNA (tRNA), linearised plasmid pBluescript (dsDNA), and poly dI:dC compete weakly. No competition is observed with synthetic ribohomopolymers poly A, poly U, poly C, and synthetic
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diolabeled L7-polypeptides was monitored by SDS gel electrophoresis (Fig. 4B). One thousand nanograms 28S rRNA per sample are sufficient to compete significantly with the binding of EP9 (NBD 2) to immobilized 28S rRNA, whereas more than 5000 nanograms of L7 mRNA, tRNA, unrelated RNA (ssRNA) or DNA are required to do so. For significant competition with the binding of peptide domIII (NBD 1) to immobilized 28S rRNA, 1000 nanograms of 28S rRNA, L7 mRNA, or DNA per sample are sufficient, and 5000 nanograms tRNA compete weakly. In summary, the two NBDs of protein L7 appear to be functionally distinct in that NBD 1 binds 28S rRNA and L7mRNA with similar affinity, whereas NBD 2 clearly prefers 28S rRNA over L7 mRNA. In addition, NBD 1 also binds to dsDNA whereas NBD 2 seems to be specific for RNA-binding. Conservation of NBD 1 and NBD 2 in L7 Proteins
FIG. 2. NBD 2 maps to the 50 C-terminal amino acids of protein L7. Overlapping fragments of protein L7 were expressed as GSTfusion proteins (schematically shown in A), and their mRNA-binding activity was analyzed by saturation filter binding to radiolabeled L7 mRNA (B).
deoxyribohomopolymers. GST-EP9 exhibited exactly the same profile of RNA-competition as His-L7Dbzip indicating that this function is fully retained in the fifty C-terminal aa of L7 (data not shown). The pattern of competitor efficiencies, namely poly G, 28S rRNA . L7 mRNA . tRNA . dsDNA, defines the relative affinities of a set of natural and synthetic ligands for NBD 2. It is different from the pattern poly G . L7 mRNA, 28S rRNA . dsDNA . tRNA, which we have determined previously by competition of L7 mRNAbinding to NBD 1 [5, 7, 8]. Significantly, calculated on a molar basis, the competitive efficiency of 28S rRNA binding to NBD2 is about twentyfold better than that of L7 mRNA, possibly indicating high affinity binding site(s) of NBD 2 on 28S rRNA. For a direct comparison of specificities, the binding of the 35S-labeled GST-fused polypeptides domIII [8] and EP9, which carry NBD 1 or NBD 2, respectively to 28S rRNA immobilised on agarose beads were competed with unlabeled 28S rRNA, L7 mRNA, tRNA, unrelated RNA (ssRNA, which represents the in vitro transcribed polylinker of plasmid pBluescript), and DNA (linearised pBluescript). The amount of agarose-bound ra-
Sequences are now available for L7 from eight species. To determine the degree of conservation of NBD 1 and NBD 2 the human sequences were aligned with the corresponding L7 sequences from rat, mouse, drosophila, dictyostelium, yeast (Saccharomyces cerevisiae), and plants (Solanum tuberosum, and Arabidopsis thaliana) (Fig. 5). The alignment shows that NBD 2 is highly conserved among all species (Fig. 5A), whereas NBD 1 is only conserved in vertebrate L7 proteins (Fig. 5B).
FIG. 3. Nucleic-acid-binding specificities of L7Dbzip. The binding of L7Dbzip to L7 mRNA was competed with increasing amounts of unlabeled nucleic acids as indicated.
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FIG. 4. The C-terminal NBD of protein L7 binds specifically to 28S rRNA. NBD 1 and NBD 2 of human protein L7, as represented by GST-fused peptides domIII and EP9 (A), respectively, were radiolabeled and bound to 28S rRNA which had been covalently attached to agarose beads. Protein-binding was competed with increasing amounts of unlabeled nucleic acids as indicated, and monitored by SDS-polyacrylamide electrophoresis under reducing conditions (B).
DISCUSSION In this report we demonstrate that human ribosomal protein L7 carries in addition to its N-terminal nucleic acid-binding domain (NBD 1) a C-terminal one (NBD 2) which shows high affinity for 28S rRNA. In a saturation filter binding study an apparent dissociation constant K d 5 20 nM was determined for L7 mRNAbinding of NBD 2, and in RNA-binding competition analyses we have shown that 28S rRNA is an about twentyfold more efficient competitor than L7 mRNA. Based on these data we would predict for 28S rRNAbinding to NBD 2 an apparent dissociation constant in the nanomolar range. NBD 2 also shows high affinity for the highly structured [23, 24] ribohomopolymer poly G. This property of NBD 2 is shared with NBD 1 [8] and NBDs in several unrelated proteins [25–27]. Protein L7 is also capable of binding to 5S rRNA [28]. The affinity of this interaction (K d 5 6 mM) is low compared to binding of 28S rRNA. However one can
not rule out a physiological relevance of this interaction because in the assembled ribosome the affinity of L7 to 5S rRNA may be increased by co-operative effects of neighbouring proteins. In summary, NBD 2 seems to bind with high affinity to one (or more) site(s) on 28S rRNA, and seems to cross-react with sites on some structured RNAs, for instance L7mRNA and poly G. We propose that NBD 2 mediates the association of L7 with the large ribosomal subunit through specific binding to 28S rRNA. This is not meant to exclude that interactions with other ribosomal proteins may also be involved. Our hypothesis is supported by in vitro biotinylation [29] and limited proteolytic degradation [30] studies which show that the N-terminal region of L7 including NBD 1 is solvent exposed, and presumably protrudes from the ribosome, whereas the C-terminal region, although hydrophilic in computer-based structure predictions, is inaccessible for external labeling or a proteolytic attack. NBD 2 thus very likely is buried in the interior of the large ribosomal subunit where it may interact with 28S rRNA. Although NBD 2 is also capable of high affinity-binding to sites on mRNA, it presumably will not come in contact with this potential ligand. For the interaction with mRNAs, the N-terminal part of L7 is in a more suitable position, as it is located on the surface of the ribosome [29], where it may be involved in the regulation of translation through binding of NBD1 with high affinity to cognate sites on specific mRNAs [7, 8, 9] and/or interaction with ribosomal protein S7 on the 40S subunit via the bziplike element [10]. Recently L7 turned out to be a general co-regulator which gears into different steroid hormone receptor controlled signal transduction pathways [11, 12]. The interaction of L7 with nuclear receptors was mapped to the N-terminus which contains the bzip-like element and NBD 1 [12]. It is not known at what stage L7 interferes with nuclear receptor-mediated transcription [11, 12]. Since the protein is also found in the nucleus of human cell lines [13, and unpublished results] and shown to be able to repress binding of VDRRXR heterodimers to a vitamin D response element [12], it is possible that the DNA-binding activity of NBD 1 in addition to the dimerisation property of the bzip-like element confers the regulatory function of L7 in L7/nuclear receptor containing transcription complexes. Searches of protein sequence data bases have not revealed significant similarity of the 50 C-terminal residues of protein L7 to segments of other nucleic-acid binding proteins. Wang et al. have identified in the NBD of nucleolar protein B23.1 a sequence motif (LWQW) that is also present in other nucleic acid binding proteins such as the negative regulator of mitosis in yeast, bacterial transposon TN7 transposition protein T, and bacterial DNA-primases [31]. Interestingly, the most conserved region of L7-NBD 2 contains the
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FIG. 5. Sequences of the C-terminal but not of the N-terminal NBDs are conserved in L7-related proteins from various organisms. (A) Sequence comparison of the C-terminal region of L7-proteins from human (h), rat (r), mouse (m), Drosophila melanogaster (dr), Dictyostelium discoideum (di), Saccharomyces cerevisiae (sc), Solanum tuberosum (st), and Arabidopsis thaliana (at). NBD 2 in human L7 maps to the region between position 198 and the C-terminus. cons 5 consensus sequence. (B) The N-terminal nucleic acid-binding domain (NBD 1, aa 27-48 of human L7, ref. 8) is conserved in L7-proteins from humans, rats and mice (underlined). Sequences are taken from reference 4 and the EMBL sequence data library (Arabidopsis thaliana, Accession Number 3212879; Drosophila melanogaster, Accession Number 417674; Solanum tuberosum, Accession Number Z30162). Yeast sequences are derived from the S. cerevisiae YL8A gene [4]. Known L7 sequences from Schizosaccharomyces pombe are highly conserved when compared with YL8A [4] and have therefore not been included into the alignment here.
sequence LWPF which resembles the conserved LWQW motif of B23.1. In addition, L7 and B23.1 share in their nucleic acid-binding domains two further regions of conserved amino acids with a preponderance of helix-destabilising amino acids (G, P): 214-SPRGG221, and 230-GGDAG-236, respectively, in L7-NBD2 (Fig. 5A) vs 234-TPKGP-241, and 255-GGSLP-262, respectively, in the B23.1 NBD [31]. These conserved regions may represent functionally important elements of a new class of nucleic acid-binding motifs. NBD 2 from L7 and the NBD of B23.1 are also similar in length (50 and 67 aa, respectively). It is not clear whether NBD 1 and NBD 2 are functionally linked. Protein L7, like some other multifunctional ribosomal proteins [32–35], may use distinct domains for unrelated functions. The latter view is supported by (i) the finding that NBD 1 inhibits cellfree translation in the absence of NBD 2 [7], and (ii) by evolutionary considerations. The C-terminal region of human protein L7 carrying NBD 2 is highly conserved among L7 proteins from distantly related organisms, whereas NBD 1 is variable and conserved only in L7 proteins derived from humans and rodents (Fig. 5). One may interpret the conservation of NBD 2 as an indication of its ,,old“ function, for instance mediating
ribosome-association through rRNA-binding, and the function of the less conserved NBD 1 containing a bzip element (i.e., co-regulation of nuclear receptors) apparently may have been acquired during the evolution of vertebrates. Future studies should reveal the physiological RNA (or DNA) binding sites of L7 to elucidate the mechanisms by which NBD 1 and NBD 2 of L7 regulate gene expression at the level of transcription and translation. ACKNOWLEDGMENTS We thank R. Sedlacek for the gift of rRNA, and M. Go¨rlach for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft through Grant C7 in Sonderforschungsbereich 156 to U.K.
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