AU4908599A - Snurpotine 1 human M3G-CAP specific nucleus import receptor protein with new domain structure, the production and use thereof - Google Patents

Description

WO 00/04144 PCT/EP99/04750 Description SNURPORTIN 1, HUMAN M3G-CAP-SPECIFIC NUCLEUS IMPORT RECEPTOR PROTEIN WITH NEW DOMAIN STRUCTURE, AND THE 5 PRODUCTION AND USE THEREOF The present invention relates to a human 5'-2,2,7-terminal trimethylguanosine (hereinbelow: m3G)-cap-specific nucleus import receptor protein obtainable by nucleus extraction, cytosol extraction and 10 expression of recombinant nucleic acids, and its use in particular as medicament or diagnostic reagent. Protein targeting within the cell is of particular importance since there is the prospect of promising diagnostic and pharmaceutical applications. 15 Proteins originating from de novo biosynthesis contain signals which determine their final localization in the cell. This is also the case with the so-called protein import pathway from the cytosol into the nucleus, which pathway uses a nuclear pore complex (NPC) and is generally mediated by 20 a saturable transport receptor which specifically recognizes a nuclear localization signal (NLS) (G6rlich and Mattaj (1996), Science, 107, 1513 in particular Table 1; Nigg (1997), Nature, 386; 779; Ohno et al. (1998), Cell, 92, 327). 25 The so-called basic NLSs are in most cases composed of one amino acid or a pool of several basic amino acids (review in Dingwall and Laskey (1991), Trends Biochem. Sci., 16, 478). The proteins which have such a basic NLS are hereinbelow termed karyophilic proteins. 30 Gbrlich describes the isolation of the transport receptor, so-called importin, composed of the complex heterodimeric subunits a (60 kD) and P (90 kD). Importin a contains an N-terminal motive, the importin P binding (hereinbelow IBB) domain, which mediates complexing with importin 0, and a C-terminal domain, which generates the NLS binding site and is 35 composed of eight so-called "armadillo" repeats (G6rlich et al. (1996), EMBO J., 15, 1810; Moroianu et al. (1996), Proc. Nati. Acad. Sci. USA, 92, 2008; Weis et al. (1996), EMBO J., 15, 1818). Importin 0 causes the NLS importin complex to be bound to the NPC (Chi et al. (1995), J. Cell Biol., 130, 265; G6rlich et al. (1995), Curr. Biol. 5, 383 and (1995) Nature, 377, 2 246; Imamoto et al. (1995), FEBS Left., 368, 415; Moroianu et al. (1995), Nati. Acad. Sci. USA., 92, 2008). These are also termed karyopherin a/P (Moroianu et al. 1995 supra; Radu et al. (1995). Proc. Nati. Acad. Sci. USA, 92,1769). 5 Translocation of the complex through the pore of the nucleus requires additional energy-supplying transport factors such as GTP and Ran (Melchior et al, (1993) J. Cell. Biol., 123, 1649; Moore and Blobel (1993), Nature, 365, 661) and plO/NTF2 (Moore and Blobel, (1994), Proc. Nati. 10 Acad. Sci. USA, 91, 10212; Paschal and Gerace (1995), J. Cell. Biol., 129, 925). Present-day studies on the transport signals of hnRNP Al and K have identified a novel protein import pathway, which differs from the known 15 basic NLS recognition (Pollard et al. (1996), Cell, 86, 985; Michael et al. (1997), EMBO J., 16, 3587). Here, the import of hnRNP Al into the nucleus depends on a transport signal 38 amino acids in length, so-called M9, whose sequence shows no similarity with the basic NLS (Siomi and Dreyfuss (1995), J. Cell. Biol., 129, 551; Weighart et al. (1995), J. Cell. Sci., 20 108, 545; Michael et al. (1995), Cell, 83, 415). M9 is recognized directly by transportin (Pollard et al. 1996, Cell, 86, 985; Nakielny et al., Exp. Cell. Res., 1996, 261; Fridell et al., J. Cell Sci. 1997, 1325). In contrast to importin P, transportin is functional without an a subunit, while binding to the NPC and the ran-dependent translocation of the hnRNP Al/transportin 25 complex into the nucleus takes place analogously to importin P (Nakielny et al. (1996), Exp. Cell. Res., 229, 261; Izaurralde et al. (1997), J. Cell Biol., 137, 27). A homolog to transportin, Kap104p, which imports a specific group of mRNA-binding proteins, has been described in yeast (Aitchinson et al. (1996) Science, 274, 624). 30 Two other proteins which are similar to importin P, Kapl23pNrb4p and Psel p, are suspected for the import of ribosomal proteins into the nucleus in yeast (Rout et al. (1997), Cell, 89, 715; Schlenstedt et al. (1997), EMBO J., 16, 6237). These novel importin-a-independent transport receptors all 35 belong to the large family of the importin-p-like transport factors (Fornerod et al. (1997), EMBO J., 16; 807, G6rlich et al. (1997) J: Cell. Biol., 138, 65). However, the import mechanism of the nucleus-specific spliceosomal U snRNP is largely unknown. Each snRNP particle is composed of one (Ul, 3 U2 and U5) or two (U4/U6) snRNA molecules and of a joint set of eight basal proteins (B, B', D1, D2, D3, E, F and G, so-called Sm proteins), which are bound to each of the m3G-cap-containing snRNAs U1, U2, U4 and U5 and which differ with regard to the respective snRNA in specific, 5 other, associated proteins (Will and LOhrmann (1997), Curr. Opin. Cell. Biol., 9, 320). With the exception of the U6 snRNP, which does not leave the nucleus (Vankan et al. (1990), EMBO J., 9, 1075), biogenesis of the snRNPs 10 depends on a bidirectional transport of the snRNA through the nuclear membrane. The snRNAs (U1, U2, U4 and U5) are synthesized specifically with a 5'-terminal 7-monomethyl-guanosine (m G-) cap structure, while the Sm proteins originate in the cytoplasm and do not migrate into the nucleus without binding to U snRNA. Rather, newly synthesized U snRNAs are 15 exported transiently to the cytoplasm, where they bind the Sm proteins at the specific snRNA Sm binding site to form a ribonucleoprotein complex (so-called Sm Core as described by Mattaj and De Robertis (1985), Cell, 40, 111; Raker et al. (1996), EMBO J., 15, 2256). The stable association of all Sm proteins is essential for the hypermethylation of the m 7 G cap to the 20 m 3 G cap (Mattaj (1986) Cell, 46, 905; Plessel et al. (1994), Mol. Cell. Biol., 14, 4160). After these processes, and after processing of the 3' end, the mature snRNP particle is transported back into the nucleus in a receptor and energy-dependent fashion (Neuman de Vegvar and Dahlberg (1995) Mol. Cell. Biol., 10, 3365). 25 The NLS of the U1 snRNP in Xenopus laevis oocytes, which is also complex in nature, has been described. Firstly by the m 3 G cap (Fischer and LOhrmann (1990), Science, 249, 786; Hamm et al. (1990), Cell, 62, 569), secondly by a portion in the Sm basal domain, which portion is not 30 characterized in greater detail (Sm Core NLS; Fischer et al. (1993) EMBO J. 12, 573). Not all spliceosomal snRNAs require m 3 G cap to the same extent for transport into the nucleus in oocytes. While U1 and U2 snRNA require m 3 G cap for transport into the nucleus at any rate, U4 and U5 snRNAs are also capable of reaching the nucleus in the form of ApppG cap 35 derivatives, even though the efficiency is reduced considerably hereby (Fischer et al. (1991), J. Cell. Biol., 113, 705; Michaud and Golgfarb (1992) J. Cell. Biol., 116, 851). Although m 3 G cap is not essential for the transport into the nucleus of the snRNAs U4 and U5, it accelerates the transport thereof considerably. This demonstrates that m 3 G cap has a signal function 4 for the import of all U snRNAs (Fischer et al., J. Cell. Biol., 1994, 971; Marshallsay and Luhrmann, 1994, Nucleic Acids Res, 13, 222). The object of the invention is therefore to provide a polypeptide which 5 interacts specifically with m 3 G cap and which is used as m3G-cap-specific nucleus import protein receptor in vivo and in vitro. The object is achieved by providing a 45 kD protein of cytoplasmic extracts of HeLa cells with high specificity for m 3 G cap structures. 10 Nucleus import receptor protein means, for the purposes of the present invention, that the polypeptide acts firstly as receptor and secondly mediates the transport of received molecules from the cytosol (cytoplasm) into the nucleus. 15 m3G-cap-specific nucleus import protein receptor means, for the purposes of the present invention, that the above-defined nucleus import receptor specifically receives, i.e. recognizes in the widest sense, molecules with m 3 G cap structures and mediates their transport into the nucleus, both 20 in vitro and in vivo. m3G-cap-containing molecules means, for the purposes of the present invention, that these are accessible to the m3G-cap-specific nucleus import protein receptor according to the invention. Fusion molecules of m 3 G cap 25 and protein or other biologically relevant molecules are therefore also feasible. In vivo means, for the purposes of the present invention, a conversion, i.e. a reaction, which proceeds within a live organism. 30 In vitro means, for the purposes of the present invention, a conversion, i.e. a reaction, which proceeds outside a live organism. "Cap" for the purposes of the present invention has the meaning which is 35 known to the skilled worker in the field of U snRNA and snRNP studies. To execute the invention, identification of a potential m3G-cap-binding protein is carried out with the aid of a UV-crosslinking assay and the synthetic substrate (m3G)pppAmpUmpA oligonucleotide (m3G-cap-oligo) 5 as substrate. Following UV irradiation of cytosolic S100 extracts which contain the m3G-cap-oligo radiolabeled with [ 3P]pCp at the 3' end, the proteins are analyzed by SDA-polyacrylamide gel electrophoresis (PAGE) and autoradiography the crosslinked radiolabeled proteins. Figures 1A and 5 1 B show that a radiolabeled main band with an apparent molecular weight of 45 kD (arrow in Figures 1 A and 1 B) and three less intensively labeled proteins of molecular weights 25, 35 and 150 kD are detected reproducibly. The following m3G-cap specificity for the polypeptide according to the 10 invention, preferably 45 kD protein, is essential to the invention and recognized with the aid of competition studies using various unlabeled cap structures. While a 10 000-fold excess of m Gppp-G or ApppG cap dinucleotide only has very little effect on the efficacy of the UV crosslink with the radiolabeled m3G-cap-oligo to the 45 kD protein (Figure 1A, tracks 15 2-4 and 5-7) a 10- to 100-fold excess of unlabeled m3G-cap-oligo suffices to completely prevent binding to the 45 kD protein (Figure 1A, tracks 9-11). In contrast, significant inhibition of the crosslinking by-products (with the exception of 150 kD protein), was only observed with a 1000-fold excess of the unlabeled m3G-cap-oligo. In addition, a synthetic m3GpppG cap 20 dinucleotide inhibited binding to the 45 kD protein less effectively by one order of magnitude than the unlabeled m3G-cap-oligo (Figure 3A, compare tracks 8-11 and 12-15). The polypeptide of the object, preferably a 45 kD protein, binds not only to 25 isolated m 3 G cap structures and to native m 3 G caps in the U1 snRNA, but also to native U1 snRNP particles. This was demonstrated by inhibiting the UV crosslinking of the m3G-cap-oligos to the 45 kD protein by U1 snRNA and U1 snRNP (Figure 1B, tracks 2-5 and 11-14). The interaction of the 45 kD protein with U1 snRNA or U1 snRNP depends stringently on the 30 presence of the 5'-terminal m 3 G cap structure. U1 snRNA and U1 snRNP preparations whose 5'-terminal ends had been removed with the aid of oligonucleotide-driven Rnase H hydrolysis were not capable of inhibiting the UV crosslinking of the m3G-cap-oligo to the 45 kD protein (Figure 1 B, tracks 6-9 and 15-18). Equal concentrations of m 3 G cap oligo, U1 snRNA 35 or U1 snRNP suffice to completely inhibit crosslinking of the 45 kD protein with the radiolabeled m3G-cap-oligo (compare Figures 1A and 1B). This - -experiment demonstrates that neither additional RNA sequences nor Sm 6 core proteins were capable of increasing the affinity of the 45 kD protein for the 5'-terminal m 3 G cap structure of U1 snRNA/snRNP. The present invention therefore relates to the polypeptide of the object as 5 such with an amino acid sequence as shown in SEQ ID No. 1 or a functional variant thereof, and portions thereof with at least six amino acids, preferably with at least twelve amino acids, in particular with at least 65 amino acids and especially with 360 amino acids (hereinbelow termed "polypeptide according to the invention" or "snurportin 1"). 10 For example, a polypeptide approximately 6-12, preferably approximately 8, amino acids in length can contain an epitope which, after coupling to a support, serves for the production of specific poly- or monoclonal antibodies. Polypeptides at least approximately 65 amino acids in length can also serve directly, without support, for the production of poly- or 15 monoclonal antibodies. The term "functional variant" is to be understood as meaning, for the purposes of the present invention, polypeptides which are functionally related to the peptide according to the invention, i.e. exhibit an m3G-cap 20 specific nucleus import receptor activity. Variants are also to be understood as meaning allelic variants or polypeptides derived from other human cells or tissue. For the purposes of the present invention, this also applies to polypeptides originating from different human individuals. 25 In the wider sense, this is also understood as meaning polypeptides which have a sequence homology, in particular a sequence identity, of approx. 70%, preferably approx. 80%, in particular approx. 90%, especially approx. 95%, to the polypeptide with the amino acid sequence in accordance with SEQ ID No. 1. Furthermore, this also includes deletion of the polypeptide in 30 the region of approx. 1-65, preferably of approx. 1-30, in particular of approx. 1-15, especially of approx. 1-6, amino acids. For example, the first amino acid methionin may be absent without this substantially altering the function of the polypeptide. In addition, this also includes fusion proteins which contain the above-described polypeptides according to the invention, 35 where the fusion proteins themselves already have the function of a human m3G-cap-specific nucleus import receptor or where they can acquire the specific function only after elimination of the fusion portion. These especially include fusion proteins with a content of, in particular non human, sequences of approx. 1-150, preferably approx. 1-100, in particular 7 approx. 1-65, especially approx. 1-30, amino acids. Examples of non human peptide sequences are prokaryotic peptide sequences, for example the E.coli galactosidase or a so-called histidin label, for example a Met-Ala His6 label. A fusion protein with a so-called histidin label is particularly 5 advantageously suitable for the purification of the expressed protein over columns containing metal ions, for example over an Ni -NTA column. "NTA" stands for the chelating agent "nitrilotriacetic acid" (Qiagen GmbH, Hilden). 10 The portions of the polypeptide according to the invention represent for example epitopes which can be recognized specifically by antibodies. This cDNA, which encodes the polypeptide according to the invention, codes for 360 amino acids (AAs) with the predicted molecular weight of 15 45 kD (Fig. 1A). A database search with human snurportin 1 surprisingly revealed a high homology in the N-terminal region (AA 27 to 65, Fig. 6B to the IBB domain of importin a (31% identity, 62% similarity to hSRP1, similar agreements were observed with hRchl, xlmpa and ySRP1, Fig. 6B). In addition, AA regions in the IBB domain of snurportin 1 are 20 highly homologous to IBB domain sequences of other members of the importin family (G6rlich et al., EMBO J., 1996, 1810; Weis et al., 1996, supra; indicated by black dots in Fig. 6B). In contrast to the extensive N-terminal IBB domain, the C-terminal portion of snurportin 1 differs structurally from importin a (for example less than 10% sequence identity 25 with the C terminus of hSRP1). In particular, no significant sequence homology could be detected between snurportin 1 and the arm repeat domain of importin a (Fig. 6B). In particular the abovementioned parts of the polypeptide can also be 30 synthesized with the aid of traditional peptide synthesis (Merrifield technique). They are suitable, in particular, for obtaining antisera, with the aid of which suitable gene expression libraries can be screened in order to arrive at further, functional variants of the polypeptide according to the invention. 35 In an especially preferred embodiment, the polypeptide according to the invention is a recombinant protein. This is to be understood as meaning, for the purposes of the present invention, a protein which has been produced by introducing a DNA sequence which encodes the protein into a host cell 8 and expressing it therein. The protein can then subsequently be obtained from the host cell and/or the culture medium. The host cell is preferably a bacterium (for example E.coli strains such as DH5, HB101 or 121), yeasts (for example Saccharomyces cerevisiae or Pichia pastoris), fungi (for 5 example Aspergillus spec.), mammalian cell lines (for example 293, Vero, HeLa, Cos or BHK cells), insect cell lines or a protist (microalgae or protozoa, for example of the genus Tetrahymena such as, for example, defined in Schlegel "Allgemeine Mikrobiologie" [General Microbiology] (Georg Thieme Verlag, 1985, 1-2). It is especially preferred for snurportin to 10 be secreted by the host cell. Such host cells for the production of a recombinant snurportin can be generated by methods known to the skilled worker. A survey over various expression systems is found, for example, in 15 Methods in Enzymology 153 (1987), 385-516 and in Bitter et al. (Methods in Enzymology 153 (1987), 516-544). Expression vectors are described extensively in the literature. As a rule, they comprise, besides a selection marker gene and a replication origin which ensures replication in the selected host, a bacterial or viral promoter and in most cases a 20 transcription termination signal. Between promoter and termination signal there are at least one restriction cleavage site or a polylinker which allow insertion of a coding DNA sequence. The DNA sequence which naturally controls transcription of the gene in question may be used as promoter sequence as long as it is active in the selected host organism. However, 25 this sequence may also be exchanged for other promoter sequences. Not only promoters which cause constitutive expression of the gene may be used, but also inducible promoters, which permit targeted regulation of the expression of the subsequent gene. Bacterial and viral promoter sequences with these properties are described in detail in the literature. 30 Regulatory sequences for expression in microorganisms (for example E.coli, S. cerevisiae) are sufficiently described in the literature. Promoters which allow particularly strong expression of the subsequent gene are, for example, the T7 promoter (Studier et al., Methods in Enzymology (1990), 185, 60-89), lacuv5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez and 35 Chamberlin (Eds), Promotors, Structures and Function; Praeger, New York, (1982), 462-481; DeBoer et al., Proc. NatI. Acad. Sci. USA (1983), 21-25), 1p1, rac (Boros et al., Gene (1986), 42, 97-100).

9 The transformation of the host cell with the DNA encoding the polypeptide according to the invention can be carried out, as a rule, by standard methods, for example such as described in Sambrook et al. (Molecular Cloning: A Laboratory Course Manual, 2nd edition (1989), Cold Spring 5 Harbor Press, New York). The host cell is grown in nutrient media which meet the requirements of the host cell used in each case, in particular taking into consideration pH, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements and the like. The expression vectors may be, for example, prokaryotic or eukaryotic 10 expression vectors. Examples of prokaryotic expression vectors are, for expression in E.coli, for example the T7 expression vector pGM10 (Martin, 1996), which encodes an N-terminal Met-Ala-His6 label which allows advantageous purification of the expressed protein over an Ni2+-NTA column. Suitable eukaryotic expression vectors for expression in 15 Saccharomyces cerevisiae are, for example, the vectors p426Met25 or p426GAL1 (Mumberg et al., Nucl. Acids Res., 1994, (22), 5767), for expression in insect cells, for example, baculovirus vectors such as disclosed in EP-B1-0127839 or EP-B1-0549721, and for expression in mammalian cells, for example, SV40 vectors, which are universally 20 available. In general, the expression vectors also contain regulatory sequences which are suitable for the host cell such as, for example, the trp promoter for expression in E.coli (see, for example, EP-B1-0154133), the ADH-2 25 promoter for expression in yeasts (Russel et al. (1983), J. Biol. Chem. 258, 2674), the baculovirus polyhedrin promoter for expression in insect cells (see, for example, EP-B1-0127839) or the early SV40 promoter or LTR promoters, for example of MMTV (Mouse Mammary Tumour Virus; Lee et al. (1981) Nature, 214, 228). 30 Purification of the enzyme produced by the host cells can be effected by conventional purification methods such as precipitation, ion-exchange chromatography, affinity chromatography, gel filtration, HPLC reversed phase chromatography and the like. 35 Modification of the DNA expressed in the host cells and encoding the polypeptide according to the invention allows production, in the host cell, of a polypeptide which can be isolated from the culture medium more readily owing to certain properties. Thus, it is possible to express the protein to be 10 expressed in the form of a fusion protein with a further polypeptide sequence whose specific binding properties allow isolation of the fusion protein by affinity chromatography (for example Hopp et al. (1988), Bio/Technology, 6, 1204-1210; Sassenfeld (1990), Trends Biotechnol. 8, 5 88-93). The present invention therefore furthermore relates to a nucleic acid encoding an m3G-cap-specific nucleus import receptor protein with a nucleic acid sequence in accordance with Figure 2 or a functional variant 10 thereof, and portions thereof with at least 8 nucleotides, preferably with at least 15 or 20 nucleotides, in particular with at least 100 nucleotides, especially with at least 300 nucleotides (termed "nucleic acid according to the invention" hereinbelow). 15 The complete nucleic acid encodes a protein with 360 amino acids and a molecular mass of 45 kD. Expression of the nucleic acid in E.coli leads to a protein which has similar enzymatic activities to those of a human m 3

G

cap-specific nucleus import receptor. Further experiments in accordance with the present invention confirm that 20 the nucleic acid is one which encodes a human snurportin 1. In a preferred embodiment, the nucleic acid according to the invention is a DNA or RNA, preferably a double-stranded DNA, and in particular a DNA with a nucleic acid sequence in accordance with SEQ ID No. 2. 25 The term "functional variant" is to be understood as meaning in accordance with the present invention a nucleic acid which is functionally related to the human m3G-cap-specific nucleus import receptor and is, in particular, of human origin. Examples of related nucleic acids are, for example, nucleic acids from different human cells or tissues or allelic variants. Also, the 30 present invention covers variants of nucleic acids which can originate from different human individuals. In the wider sense, the term "variants" is to be understood as meaning in accordance with the present invention nucleic acids which have a 35 homology, in particular a sequence identity, of approximately 60%, preferably of approximately 75%, in particular of approximately 90% and especially of approximately 95%.

11 The portions of the nucleic acid according to the invention can be used, for example, for generating individual epitopes, as probes for identifying further functional variants or as antisense nucleic acids. For example, a nucleic acid of at least approximately 8 nucleotides is suitable as antisense nucleic 5 acid, a nucleic acid of at least approx. 15 nucleotides as primer in the PCR method, a nucleic acid of at least approximately 20 nucleotides for identifying further variants, and a nucleic acid of at least approx. 100 nucleotides as probe. 10 In a further preferred embodiment, the nucleic acid according to the invention comprises one or more noncoding sequences. For example, the noncoding sequences are intron sequences or regulatory sequences such as promoter or enhancer sequences for the controlled expression of the m3G-cap-specific nucleus import receptor. 15 In a further embodiment, the nucleic acid according to the invention is therefore present in a vector, preferably in an expression vector or a gene therapeutically active vector. 20 Examples of gene-therapeutically active vectors are viral vectors, preferably adenovirus vectors, in particular replication-deficient adenovirus vectors, or adeno-associated viral vectors, for example an adeno associated viral vector which is exclusively composed of two inserted terminal repeats (ITRs). 25 Suitable adenovirus vectors are described, for example, in McGrory, W.J. et al. (1988) Virol. 163, 614; Gluzman, Y. et al. (1982) in "Eukaryotic Viral Vectors" (Gluzman, Y. ed.) 187, Cold Spring Harbor Press, Cold Spring Harbor, New York; Chroboczek, J. et al. (1992) Virol. 186, 280; Karlsson, S. et al. (1986) EMBO J., 5, 2377 or W095/00655. 30 Suitable adeno-associated viral vectors are described, for example, in Muzyczka, N. (1992) Curr. Top. Microbiol. Immunol. 158, 97: W095/23867; Samulski, R.J. (1989) J. Virol, 63, 3822; W095/23867; Chiorini, J.A. et al. (1995) Human Gene Therapy 6, 1531 or Kotin, R.M. (1994) Human Gene 35 Therapy 5, 793. Gene-therapeutically active vectors can also be obtained by complexing the nucleic acid according to the invention with liposomes. Lipid mixtures which are suitable for this purpose are those described by FeIgner, P.L. et 12 al. (1987) Proc. Nati. Acad. Sci., USA 84, 7413; Behr, J.P. et al. (1989) Proc. Nati. Acad. Sci. USA 86, 6982; FeIgner, J.H. et al. (1994) J. Biol. Chem. 269, 2550 or Gao, X. & Huang, L. (1991) Biochim. Biophys. Acta 1189, 195. When preparing the liposomes, the DNA is bound ionically at 5 the surface of the liposomes, namely in such a ratio that a positive net charge remains and the DNA is complexed fully by the liposomes. For example, the nucleic acid according to the invention can be synthesized chemically with the sequence disclosed in SEQ ID No. 2 or 10 with the peptide sequence disclosed in SEQ ID No. 1 taking into consideration the genetic code, for example using the phosphotriester method (see, for example, Uhlman, E. & Peyman, A. (1990) Chemical Reviews, 90, 543, No. 4). Another possibility of obtaining the nucleic acid according to the invention is to isolate it from a suitable genomic library, for 15 example from a human library, using a suitable probe (see, for example, Sambrook, J. et al. (1989) Molecular Cloning. A laboratory manual. 2nd Edition, Cold Spring Harbor, New York). Suitable probes are, for example, single-stranded DNA fragments approximately 100 to 1000 nucleotides in length, preferably approximately 200 to 500 nucleotides in length, in 20 particular approximately 300 to 400 nucleotides in length, whose sequence can be deduced from the nucleic acid sequence as shown in SEQ ID No. 2. The present invention furthermore also relates to antibodies which react specifically with the polypeptide according to the invention, the 25 abovementioned portions of the polypeptide either being immunogenic themselves or capable of being made immunogenic, or increased in their immunogenicity, by coupling to suitable supports such as, for example, bovine serine albumin. 30 The antibodies are either polyclonal or monoclonal. Their preparation, which is also a subject-matter of the present invention, is carried out for example by generally known methods by immunizing a mammal, for example a rabbit, with the polypeptide according to the invention or the abovementioned portions thereof, if appropriate in the presence of, for 35 example, Freund's adjuvant and/or aluminum hydroxide gels (see, for example, Diamond, B.A. et al. (1981) The New England Journal of Medicine, 1344). The polyclonal antibodies which have formed in the animal owing to an immunological reaction can subsequently be isolated readily from the blood and purified, for example, by column 13 chromatography, using generally known methods. The antibodies are preferably subjected to affinity purification, in which for example the C-terminal DAN fragment has been coupled to an NHS-activated HiTrap column. 5 Monoclonal antibodies can be produced for example by the known method of Winter & Milstein (Winter, G. & Milstein, C. (1991) Nature, 349, 293). Also, the present invention furthermore relates to a pharmaceutical 10 comprising a nucleic acid according to the invention or a polypeptide according to the invention and, if appropriate, suitable additives or adjuvants, and to a method of producing a pharmaceutical for the treatment of cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer's disease, allergies, in particular 15 neurodermitis, type I allergies or type IV allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and/or diabetes and/or for controlling cell metabolism, in particular in immunosuppression, especially in the case of transplantations, in which method a nucleic acid according to the invention, for example a so-called 20 antisense nucleic acid, or a polypeptide according to the invention is formulated together with pharmaceutically acceptable additives and/or adjuvants. Especially suitable for use in gene therapy in humans is a pharmaceutical 25 comprising the nucleic acid according to the invention in naked form or in the form of one of the above-described gene-therapeutically active vectors or in the form of a complex with liposomes. Examples of suitable additives and/or adjuvants are a physiological saline, 30 stabilizers, proteinase inhibitors, nuclease inhibitors and the like. The present invention furthermore also relates to a diagnostic comprising a nucleic acid according to the invention, a polypeptide according to the invention or antibodies according to the invention and, if appropriate, 35 suitable additives and/or adjuvants and to a method of producing a diagnostic for diagnosing cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer's disease, allergies, in particular neurodermitis, type I allergies or type IV allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and/or

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14 diabetes and/or for analyzing cell metabolism, in particular of the immune state, especially in the case of transplantations, in which method a nucleic acid according to the invention, a polypeptide according to the invention or an antibody according to the invention are combined with suitable additives 5 and/or adjuvants. For example, the present invention allows a diagnostic to be produced with the nucleic acid according to the invention, based on the polymerase chain reaction (PCR diagnostics, for example in accordance with EP-0200362) or 10 a northern blot. These tests are based on the specific hybridization of the nucleic acid according to the invention with the complementary counter strand, conventionally of the corresponding mRNA. The nucleic acid according to the invention can also be modified for this purpose, such as described, for example, in EP0063879. Preferably, a DNA fragment 15 according to the invention is labeled, for example radiolabeled using a-P -dATP or nonradio labeled using biotin, by generally known methods using suitable reagents and incubated with isolated RNA which has preferably been bound to suitable membranes, for example made of cellulose or nylon. In addition, it is advantageous for the isolated RNA to be 20 separated by size prior to hybridization and binding to a membrane, for example by means of agarose gel electrophoresis. Thus, with equal quantities of test RNA from each tissue sample, the mRNA quantity labeled specifically by the probe can be identified. 25 A further diagnostic comprises the polypeptide according to the invention or the immunogenic portions thereof which have been described above in greater detail. The polypeptide or portions thereof which are preferably bound to a solid phase, for example made of nitrocellulose or nylon, can be contacted in vitro for example with the body fluid to be tested, such as 30 blood, in order to react for example with autoimmune antibodies. The antibody/peptide complex can subsequently be detected for example with labeled anti-human-IgG or anti-human-IgM antibodies. The label is for example an enzyme, such as peroxidase, which catalyzes a color reaction. The presence and quantity of the autoimmune antibodies present can thus 35 be detected in a simple and rapid manner by the color reaction. Another diagnostic comprises the antibodies according to the invention themselves. Using these antibodies it is possible to examine for example a human tissue sample in a simple and rapid manner for the presence of the 15 polypeptide in question. In this case, the antibodies according to the invention are labeled, for example with an enzyme as already described above. The specific antibody/peptide complex can thus be detected in a simple fashion and equally rapidly by an enzymatic color reaction. 5 Another subject-matter of the present invention also relates to an assay for identifying functional interactors, such as, for example, inhibitors or stimulators, comprising a nucleic acid according to the invention, a polypeptide according to the invention or the antibodies according to the 10 invention and, if appropriate, suitable additives and/or adjuvants. A suitable assay for identifying functional interactors is, for example, the so called "two-hybrid system" (Fields, S. & Sternglanz, R. (1994) Trends in Genetics, 10, 286). In this assay, a cell, for example a yeast cell, is 15 transformed or transfected with one or more expression vectors which express a fusion protein which contains the polypeptide according to the invention and a DNA binding domain of a known protein, for example Gal4 or LexA from E. coli, and/or which express a fusion protein which contains an unknown polypeptide and a transcription activation domain, for example 20 of Gal4, herpesvirus VP16 or B42. In addition, the cell comprises a reporter gene, for example the E. coli LacZ gene, green fluorescence protein, or the yeast amino acid biosynthesis genes His3 or Leu2, which is under the control of regulatory sequences such as, for example, the lexA promoter/operator or of a so-called yeast upstream activation sequence 25 (UAS). The unknown polypeptide is encoded, for example by a DNA fragment which is derived from a genomic library, for example a human genomic library. Usually, a cDNA genomic library is immediately prepared in yeast with the aid of the expression vectors described, so that the assay can be carried out immediately thereafter. 30 For example, the nucleic acid according to the invention is cloned in as a functional unit with the nucleic acid encoding the lexA DNA binding domain, in a yeast expression vector, so that a fusion protein of the polypeptide according to the invention and the LexA DNA binding domain is expressed 35 in the transformed yeast. In another yeast expression vector, cDNA fragments from a cDNA genomic library are cloned as a functional unit with the nucleic acid encoding the Gal4 transcription activation domain so that a fusion protein of an unknown polypeptide and the Gal4 transcription activation domain is expressed in the transformed yeast. The yeast, which 16 is transformed with the two expression vectors and which is, for example, Leu2-, additionally comprises a nucleic acid which encodes Leu2 and which is under the control of the LexA promoter/operator. In the event of functional interaction between the polypeptide according to the invention 5 and the unknown polypeptide, the Gal4 transcription activation domain binds to the LexA promoter/operator via the LexA DNA binding domain, the LexA promoter/operator thus being activated and the Leu2 gene being expressed. As a consequence, the Leu2 yeast is capable of growth on minimal medium lacking leucin. 10 When using the LacZ or green fluorescence protein reporter gene in place of an amino acid biosynthesis gene, transcriptional activation can be detected by the formation of colonies with blue or green fluorescence. However, the blue or fluorescence coloration can also be readily quantified 15 in a spectrophotometer, for example at 585 nM in the case of blue coloration. In this manner, expression genomic libraries can be screened in a simple and rapid fashion for polypeptides which interact with the polypeptide 20 according to the invention. The novel polypeptides which have been found can subsequently be isolated and characterized further. A further possible use of the two-hybrid system is the control of the interaction between the polypeptide according to the invention and a known 25 or unknown polypeptide by other substances such as, for example, chemical compounds. In this manner, novel valuable active ingredients which can be synthesized chemically and employed as therapeutics can be found readily. The present invention is therefore not only intended for a method of finding polypeptide-like interactors, but also extends to a method 30 of finding substances which can interact with the above-described protein/protein complex. Such peptide-like or else chemical interactors are therefore termed, for the purposes of the present invention, functional interactors, and these may have an inhibitory or a stimulatory action. 35 In the following text, experiments and figures will be explained which characterize the polypeptide according to the invention, snurportin 1, in greater detail (see also Examples).

17 Purification of the polypeptide according to the invention, a 45 kD m3G-cap-binding protein, termed snurportin 1, and detection of its activity: To purify the protein, a cytosolic S100 extract from HeLa cells was first 5 purified over a CM-Sepharose column, and the eluate, which contained snurportin 1, was separated by means of Q-Sepharose chromatography. The fractions containing snurportin 1 (assayed using the UV crosslinking assay) were subsequently applied to an M 3 G cap affinity column. To this end, biotinylated m3G-cap-oligo (m3GpppAmpUmpA-/CH 2 )6-biotin) was 10 bound to a streptavidin agarose column. The bound proteins were eluted from the column using increasing NaCl concentrations and analyzed by means of SDS-PAGE. A pure protein, the polypeptide according to the invention, with an apparent molecular weight of 45 kD was eluted from the column at a salt concentration of 0.6 to 1 M NaCl (Figure 2A, tracks 9-11). 15 The purified 45 kD protein was crosslinked efficiently with radiolabeled m3G-cap-oligo with the aid of UV light (Figure 2B). These experiments show that the polypeptide according to the invention, which had been purified from HeLa S100 extracts, is necessary and sufficient to form the 45 kD UV crosslink. This means that the 45 kD protein bears the m 3 G cap 20 binding activity. snurportin 1 contains an IBB domain, but not the known armadillo repeats: In order to clone the protein, peptide sequences of the purified protein were 25 generated by microsequencing. All five peptide sequences generated were found in GenBank in an EST (expressed sequence tag) (Figure 6A). This snurportin 1 encoding cDNA encodes 360 amino acids (AAs) with the predicted molecular weight of 45 kD (Figure 1A). A database search with human snurportin 1 surprisingly revealed high homology in the N-terminal 30 region (AA 27 to 65, Figure 6B) with the IBB-domain of importin a (31% identity, 62% similarity with hSRP1, similar agreements were observed with hRchl, xlmpaxand ySRP1, Figure 6B). In addition, AA regions in the IBB domain of snurportin 1 are highly homologous to IBB domain sequences of other members of the importin a family (G6rlich et al., 1996; supra; Weis et 35 al., 1996; supra; marked by black dots in Figure 6B). In contrast to the extensive N-terminal IBB domain, the C-terminal portion of snurportin 1 differs structurally from importin a (for example less than 10% sequence identity with the C-terminus of hSRP1). In particular, no significant sequence homology was detected between snurportin 1 and the "armadillo" 18 repeat domain of importin a (Figure 6B). This means that no evolutionary conservation exists of the C-terminal regions of these two proteins. Surprisingly, snurportin 1 shows a total sequence homology with various 5 ORFs (open reading frames) of mouse ESTs (for example AA571557, over 90% identity), a Drosophila EST (AA541081, over 40% identity) and with a C. elegans protein of unknown function (ACC AF024493). The homology between snurportin 1 and the C. elegans protein is not limited to the N-terminal IBB domain (43% identity, 59% similarity; see Figure 6A). It 10 therefore emerges that the protein constitutes the functional homolog to snurportin 1. Identification of the C. elegans homolog shows that snurportin 1 was conserved in terms of evolution and therefore has an essential specific function as nucleus import receptor protein. 15 snurportin 1 binds importin P in vitro in an IBB-domain-dependent fashion: The presence of an IBB domain at the N-terminus of snurportin 1 causes mediation of the transport of m3G-cap-containing molecules into the nucleus. This is detected by an in-vitro binding of the polypeptide according 20 to the invention to importin p. Histidine fusion proteins of total snurportin or N-terminal deletion mutants of the polypeptide according to the invention (Al-65 snurportin 1 causes a lacking IBB domain as shown in Fig. 3B) and also total hSRP1a and 25 Xenopus importin a were incubated with in-vitro-translated 35S-labeled importin P. The protein complexes were subsequently precipitated with Ni-NTA-agarose beads, and importin P binding was analyzed by means of SDS-PAGE and subsequent autoradiography. Importin 1 coprecipitated with total snurportin 1, hSRP1ax and importin a, but not with A1-65 30 snurportin 1 (Figure 3A, tracks 1-4). It has been proven that snurportin 1 binds importin P in vitro, and the N-terminal IBB domain is necessary for this binding. The C-terminal domain of snurportin 1 has an m3G-cap-binding activity: 35 Importin a requires its C-terminal domain for binding the NLS of karyophilic proteins (Cortes et al. (1994), Proc. Nati. Acad. Sci. USA, 91, 7633). To test whether the C-terminal domain of snurportin 1 is involved analogously in the binding of the m3G-cap-NLS of snRNPs, crosslinking studies were 19 carried out on the m3G-cap-oligo and deletion mutants of snurportin 1. To this end, purified recombinant snurportin 1 lacking the N-terminal amino acids 1-65 (inclusive of the IBB domain) were crosslinked as efficiently with the radiolabeled m3G-cap-oligo as the recombinant total snurportin 1 5 (Figure 3B, compare tracks 6 and 7). Deletion of the C-terminal 32 AAs did not prevent crosslinking. Deletion of additional 120 AAs starting from the C-terminal end, in contrast, fully prevented binding to the m3G-cap-oligo. It can therefore be assumed that the middle portion of the C-terminal region of snurportin 1 is responsible for binding to the m 3 G cap. 10 In Xenopus oocytes, snurportin 1 stimulates the import of U snRNP into the nucleus in an IBB-domain-dependent fashion: The role of snurportin 1 in the transport of U snRNPs into the nucleus was 15 studied by microinjection into Xenopus laevis oocytes. First, m3G-cap modified HeLa Ul and U5 snRNA together with ApppG-cap-modified U6 snRNA were microinjected into the cytoplasm of oocytes. After one hour, the oocytes received a second injection with purified snurportin 1 or buffer. Transport into the nucleus was studied 3, 5 and 8 hours later (Figure 5). U6 20 snRNA was injected as control, since studies had shown that this RNA is transported into the nucleus via the karyophilic proteins (cf. Michaud and Goldfarb (1991), J. Cell. Biol., 112, 215 and (1992), J. Cell. Biol. 116, 851). Exogenous addition of snurportin 1 stimulated the import of Ul and U5 snRNA into the nucleus significantly by approx. 50-70%, while no effect 25 was observed on the ApppG-cap-modified U6 snRNA (Figure 7A, compare tracks 4-12, top, with tracks 13-21, middle, and Figure 7B for quantification). The same nucleus import stimulation was observed with snurportin 1 which had been isolated from HeLa cells by affinity purification and added exogenously. In summary, these results demonstrate that 30 snurportin 1 is a novel snRNP-specific nucleus import factor. Importin a requires an intact IBB domain for its function (G6rlich et al., 1996; supra, Weis et al., 1996; supra). To study the role of the IBB domain of snurportin 1 in the transport of snRNPs into the nucleus, the N-terminal 35 deletion mutant of snurportin 1 (A1-65 snurportin 1) together with m3G-cap modified Ul and U5 snRNAs was also microinjected into oocytes. While this mutant lacks the IBB domain, it still retains all of the m 3 G cap binding ability (see Figure 3B, track 7). A1-65 snurportin 1 was not only capable of accelerating the import of snRNPs into the nucleus, but even had a strong 20 inhibitory effect on the import of the m3G-cap-modified U1 and U5 snRNAs (Fig. 7A, tracks 22-30). The unhindered transport of ApppG-cap-modified U6 snRNA (Figure 4A, tracks 22-30) excludes an unspecific effect of Al-65 snurportin 1 on nucleus import. This shows that the U1 and U5 snRNA 5 import can be attributed to competition between A1-65 snurportin 1 and the endogenous Xenopus laevis snurportin 1 for the m 3 G cap. These results are the reason for the essential role of the IBB domain for the function of snurportin 1. Inhibition by the snurportin 1 A1-65 deletion mutant of the import of U1 and U5 snRNAs into the nucleus at the same time 10 emphasizes the crucial role of snurportin 1 in the m3G-cap-dependent nucleus import in Xenopus oocytes. snurportin 1 greatly accelerates the in vitro nucleus import of U1 snRNPs into cells which have been permeabilized with digitonin: 15 For the purposes of "nucleus import assays" (see Example, "in-vitro transport system" following Marshallsay and LOhrmann (1994), EMBO J., 13, 222), purified HeLa U1 snRNP were injected into Xenopus oocytes. The material used as specific control for import into the nucleus was A5'U1 20 snRNP, where 10 nucleotides had been removed from the 5' terminus including the m 3 G cap by DNA-oligonucleotide-induced Rnase-H-mediated hydrolysis. The protein moieties of both forms of U1 snRNPs were labeled by modification with the fluorescence dye Cy3 (Ul snRNP* and: A5'U1 snRNP* hereinbelow). SDS-PAGE and glycerol gradient centrifugation 25 confirm that the integrity of the snRNP particles have not been affected by the tagging method and that the level of tagging is comparable in both forms. As shown in Figure 5, intact U1 snRNP* is transported into the nucleus 30 much more efficiently in the presence of cytosolic HeLa S100 extract than A1-65 U1 snRNP*. In both cases, the transport was energy- (Figures 5C and D) and temperature-dependent. These results agree with the assumption that the endogenous snurportin 1 in HeLa cell cytosol should contribute significantly to the import of U1 snRNPs into the nucleus. 35 Competition studies on unlabeled U1 snRNPs or m3GpppG cap dinucleotide were carried out for this purpose. In the presence of 100-fold excess of unlabeled A5'U1 snRNP, the import of intact U1 snRNP* into the nucleus was reduced by approx. 35-40% (compare 8E and 8A), while the import of A5'U1 snRNP* was completely inhibited (compare 8F and 8B).

21 This means that exogenous A5'U1 snRNP particles titrated an snRNP import receptor which exists in the HeLa cell cytosol in limited quantities and differs from snurportin 1 (possibly the Sm-Core NLS binding receptor). 5 Since a significant proportion of the intact U1 snRNPs continued to be imported in the presence of competitive A5'U1 snRNP, the import of these particles is mainly m3G-cap-dependent. The fact that the import of U1 snRNP* into the nucleus by an excess of intact unlabeled U1 snRNP (G in Fig. 5) or by the simultaneous addition of A5'U1 snRNP and synthetic 10 m3GpppG cap dinucleotide was inhibited by approx. 90% (H in Fig. 5) tallies with this experiment. To prove directly that snurportin 1 is responsible for nucleus transport in somatic cells, in-vitro import studies were carried out in the presence of 15 recombinant snurportin 1. Addition of snurportin 1 to cytosolic S100 extract led to a significant rise in the accumulation of U1 snRNP* in the nucleus (up to 180% in the presence of 100 pM exogenous snurportin 1; compare I and A in Fig. 5). This stimulation depends strictly on an m 3 G cap since the uptake into the nucleus of A5'U1 snRNP* is not accelerated (K in Fig. 5). In 20 addition, preincubation of snurportin 1 with an excess of m3GpppG cap dinucleotide prevented the stimulation of the U1 snRNP import by snurportin 1. Finally, and in agreement with the results of the studies on Xenopus oocytes, stimulation of the nucleus import by exogenous snurportin 1 requires the presence of its N-terminal IBB domain in 25 snurportin 1. Addition of 100 pM of A1-65 snurportin 1 to cytosolic S100 extract from HeLa cells did not accelerate, but rather inhibited, U1 snRNP import by approx. 30-40% (compare L and A in Fig. 5). As expected, Al -65 snurportin 1 did not inhibit the import of A5'U1 snRNP* (compare M and K, and M and B, in Fig. 5). In summary, these data confirm that, in HeLa cells, 30 at least two different import receptors mediate the nucleus import of U1 snRNP. They are snurportin 1 and probably the Sm-Core-NLS receptor. Moreover, snurportin 1 contributes significantly to the accumulation of U1 snRNP in the nucleus of somatic cells. 35 The polypeptide according to the invention can be used in all cells, but preferably in mammalian cells and human cells. In principle, it can be embodied for all molecules containing an m 3 G cap structure.

22 In a particular embodiment, the polypeptide according to the invention is deleted specifically at the above-described IBB domain. To this end, the amino acids 1-65 are deleted at the polypeptide according to the invention, termed hereinbelow A 1-65 snurportin 1. 5 This A 1-65 snurportin 1 can preferably be overexpressed in the target cell, preferably mammalian cells and human cells, by methods known to the skilled worker using an above-described gene-therapeutic vector. This is done to prevent the nucleus import of m3G-cap-containing molecules. 10 The deletion polypeptide according to the invention is preferably produced as recombinant protein using known recombinant methods, preferably from the nucleic acid in accordance with SEQ ID No. 3. 15 Accordingly, another subject-matter of the invention is a nucleic acid or a functional variant (as defined for snurportin 1) in accordance with SEQ ID No. 3 obtainable from the nucleic acid in accordance with SEQ ID No. 2 encoding A 1-65 snurportin 1. SEQ ID No. 3 shows the necessary sequence with the introduction of a start codon ATG by known recombinant 20 methods, by which it is expressed in accordance with the teaching on the above-described nucleic acid (SEQ ID No. 2) and is accessible to the other applications described, in particular as pharmaceutical and diagnostic. Especially preferred is the use of the nucleic acid in accordance with SEQ ID No. 3 for the purposes of a gene-therapeutic vector for the desired 25 overexpression of A 1-65 snurportin 1 in cells. Figure 4 shows the amino acid sequence of A 1-65 snurportin 1, which can be obtained as expression product by the methods which have already been described. 30 Another subject-matter of the invention is the possibility of recognizing, identifying, quantifying, isolating and purifying m3G-cap-containing molecules in the presence of snurportin 1 or A 1-65 snurportin 1 in in-vitro experiments. 35 The examples which follow are intended to illustrate the invention in greater detail without limiting it to the products and embodiments described in the examples.

23 Examples Material and methods 5 All enzymes for DNA manipulations were purchased from New England Biolabs. RNA polymerase and Rnasin were obtained from Promega. Pfu polymerase was obtained from Stratagene and RNaseH from Boehrimnger Mannheim. The cap homologs ApppG and m7GpppG were purchased from Pharmacia. M3GpppG was synthesized and purified as described (Iwase et 10 al. (1989), Nucleic Acids Res., 17, 8979). Radiolabeled triphosphate nucleotides and [ 32P]pCp were obtained from Amersham. Sequencing was carried out with an automatic sequencer using Taq polymerase and double stranded templates (PRISM Ready Reaction DyeDeoxy Terminator cycle sequencing kit) from Applied Biosystems. 15 Example 1: Preparation of snRNPs and snRNAs The preparation of nuclear extracts from HeLa cells (Computer Cell Culture, Mons, Belgium) is described by Dignam (Dignam et al. (1983), 20 nucleic Acids Res., 11, 1475). Native Ul and U5 snRNPs were obtained by affinity chromatography with the monoclonal antibody (mAb) H20, which had been bound covalently to CnBr-activated Sepharose 4B (Bochnig et al. (1987), Eur. J. Biochem, 168, 461) and by chromatography on MonoQ (Bach et al. (1987), Methods Enzymol., 181, 232). For competition studies 25 and Rnase H digests, Ul snRNPs were concentrated to 12 pg/ml by centrifugation at 160 000 x g (2.5 h, 4 0 C). To remove the 5' ends from Ul snRNA, Ul snRNPs (60 pg) were incubated with 10 U RNase H and a DNA oligonucleotide (5- CAGGTAAGTAT-3'; 1.4 pg/pl) in a volume of 50 pl (Lamond and Sproat (1994), RNA processing: A Practical Approach, IRL 30 Press, Oxford UK, 103 et seq.). Remaining m 3 G capped Ul snRNPs were removed from the reaction by immunoprecipitation with 25 pl H20-mAb on Sepharose beads coupled in an end volume of 100 pl of PBS (pH 8). After incubation for two hours at 4 0 C, the samples were spun briefly, and the A5'U1 snRNPs were obtained in the supernatant and concentrated to a 35 concentration of 30 pg/ml using an Amicon ultrafiltration unit (Micrcon-100 concentrator). Purity and intactness of the particles was checked by SDS PAGE and sedimentation analysis on a glycerol gradient (5-20%, PBS, pH 8) in a Beckmann TLS-55 rotor (Marshallsay and LOhrmann, 1994, 24 supra). U1, A5'U1 and U5 snRNA were isolated as described by Sumpter (Sumpter et al. (1992), Mol. Biol. Rep. 16, 229). Example 2: UV crosslinking studies 5 An m 3 G cap oligonucleotide (m3GpppAmpUmpA), which is identical with the 5' end of the HeLa U1 snRNA, was synthesized as described by Sekine (Sekine et al. (1 994) Nucleic Acids Symp. Ser., 31, 61; Sekine et al. (1996), J. Org. Chem., 61, 4412). Preparative [ P]pCp labeling of the m 3 G cap 10 oligos (5 pg) was carried out as described by Fischer (Fischer et al. (1993), EMBO J., 12, 573), with the exception that 250 pCi [ P]pCp were used. Following phenol extraction and ethanol precipitation, the radiolabeled m 3 G cap oligos were purified with 7.5 M urea on a 20% polyacrylamide gel. To identify the m3G-cap-binding proteins in cytosolic HeLa cell extracts by 15 means of UV crosslinking, a pM [ 32P]pCp 3' end-labeled m 3 G cap oligos (2.5 x 106 cpm/pM) was incubated for 10 min on ice, either with 25 pg of S100 cytosolic extract or 1.5 pg of purified HeLa or recombinant snurportin 1 in a volume of 10 pl. The reaction was subsequently irradiated for 5 min at 254 nm with a Sylvania G8T5 UV lamp at a distance of 2 cm. 20 The crosslinked proteins were separated by SDS-PAGE and visualized by autoradiography. Example 3: Purification of the 45 kD m3G-cap-binding protein 25 HeLa S100 extract prepared as described by Dignam (Dignam et al. (1983), Nucleic Acids Res., 11, 1475) was pre-purified on a 240 ml CM Sepharose FF column (Pharmacia). To this end, 960 ml of the extract (approx. 3.5 mg/mI protein) were separated over a column (volume 240 ml) equilibrated in buffer D (25 mM HEPES-KOH, 100 mM NaCl, 2.5 mM 30 MgCI 2 , 0.25 mM EDTA, 8.7% glycerol, 2 mM DTT, 1 mM PMSF, 0.1 mM benzamidine, 10 pg/ml bacitracin, pH 7.9). The eluate, which contained the 45 kD m3G-cap-binding protein, was applied directly onto a Q-Sepharose FF column (volume 240 ml, Pharmacia) equilibrated in buffer D. Then, the column was washed with 2 I of buffer D, and the bound proteins were 35 eluted with a linear NaCl gradient (100-750 mM in buffer D) over 900 ml. Aliquots (0.5 ml) were dialyzed for 4 hours at 4*C against buffer D and analyzed for an m3G-cap-binding activity by means of the UV crosslinking assay. The activity eluted mostly between 170 and 280 mM NaCl. These 25 fractions were combined (210 ml, 627 mg protein), diluted to 100 mM NaCl in buffer D (approx. 1.6 mg/ml) and applied to an m3G-cap-affinity column (preparation of the column, see next section). The column matrix was washed with 10 volumes of buffer D and eluted from the column using a 5 salt concentration which was increased stepwise (in each case after 2 ml 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 1 and 1.5 M NaCl in buffer D). A 0.5 ml aliquot of each fraction was dialyzed against buffer D and concentrated to a concentration of 30 pg/ml protein by means of an Amicon ultrafiltration unit. Then, the m3G-cap-binding activity was detected by means of the UV 10 crosslinking assay. The total yield amounted to 0.36 mg of protein, which corresponded to 0.01% of the starting protein quantity. Example 4: Preparation of the m 3 G cap affinity column 15 To carry out the affinity purification of the m3G-cap-binding protein, a biotinylated m3G-cap-oligo (m3GpppAmpUmpAp-(CH2)6-biotin) was synthesized chemically. Coupling of the biotinylated m3G-cap-oligo was carried out by the method of Lamond and Sproat (1994, supra). 50 nM of the biotinylated m3G-cap-oligo was bound to 1 ml of blocked streptavidin 20 agarose for 18 h at 4 0 C in an equal volume of binding buffer (25 mM Hepes-KOH, 500 mM KCL, 1 mM EDTA, 1 mM DTT, 10% glycerol, pH 7.9). The beads were washed with 5 volumes of buffer D prior to use. Example 5: Microsequencing, cDNA cloning and expression of snurportin 1 25 Purified snurportin 1 was digested with Lys-C, the peptides were separated by means of HPLC, and the AA sequence of various eluted fractions was determined by means of an ABI 477A protein sequencer. The following protein sequences, which [lacuna] with three overlapping ESTs of the 30 ATCC (GenBank Accession Numbers: H43467, H08432, R14245) were characterized: (a) KYSSLEQSERRRRLLELQK, (b) KRLDYVNHARRLAEDD, (c) KRLAIVASRGSTSAYTK, (d) KLPEEEGLGEK (e) KLTHK. As was found by DNA sequencing, clone R14245 contained a 1.6 kb fragment with an ORF encoding all five snurportin 1 peptide 35 sequences. To express his-tag snurportin 1 and N-terminal deletion mutants (A1-65 snurportin 1), either the complete snurportin 1 sequence or the sequence encoding AA 66-360 was amplified by means of PCR from a pBluescript plasmid containing all of the snurportin 1 cDNA (pBS/spnl) and reckoned into the Ncol/BamHl restriction cleavage sites of plasmid pET28b 26 (Novagen). The resulting plasmids (pET28b/spnl and pET28b/A1-65spnl) were transformed into the E. coli bacterial strain BL21[LysS]. The transformed strains were incubated up to an OD 600 of 0.8, and protein expression was induced for 4 hours at 30*C using isopropyl-3-D 5 thiogalactopyranoside. For lysis, the cells of a 2-1 culture were sonicated for 1 min on ice in resuspension buffer D (25 mM HEPES-KOH, 100 mM NaCl, 2.5 mM MgCI 2 , 1 mM PMSF, 0.1 mM benzamidine, 10pg/ml bacitracin, 20 pg/ml leupeptin, 5 mM imidazole, 10 mM 0 mercaptoethanol, pH 7.9). After the solution had been clarified by centrifugation for 45 mins at 10 25 000 g and for 120 min at 100 000 x g, the supernatant was applied to a nickel-nitrilo-acetic acid (Ni-NTA)-agarose column (Qiagen). The bound proteins were eluted with resuspension buffer comprising 200 mM imidazole and 8.7% of glycerol. For further purification, the proteins were dialyzed for 2 hours at 40C against buffer D and subjected to m3G cap 15 affinity chromatography as described above. The following primers were used for the PCR amplification: (i) pET28b/spnl-for [5' GGGCCATGGAAGAGTTGAGTCAGGCCCTG-3']; (ii) pET28b/A-1-65 spnl-for [GGGCCATGGCTGAAGATGACTGGACAGGGATG-3'], (iii) pET28b/spnl-rev and pET28b/A1 -65/spnl -rev 20 [TTTGGATCCGCATTCTCCATGAGGCATCCAGGGTG-3'-]. All constructs which had been generated by PCR were confirmed by sequencing. Expression and purification of hSRP1a and Xenopus importin a have already been described (Weis et al., 1995; G6rlich et al., 1994). 25 Example 6: In-vitro translation and protein binding assays. Importin 0 was produced with the aid of rabbit reticulocyte lysate by in-vitro translation of the plasmid pKW275 (Weis et al., 1996, supra) using the TnT kit (Promega) following the manufacturer's instructions. Binding of 30 snurportin 1, hSRP1a or importin a to importin P by means of Ni-NTA beads was carried out exactly as described by Weis (Weis et al. (1996), supra). A) Labeling RNA and U snRNPs: Procedure 35 The [ 32P]pCp labeling of gel-purified HeLa U1 and U5 snRNAs was carried out as described by Fischer (Fischer et al. (1993), EMBO, J., 12, 573). The in-vitro transcription of the [ P]pCp-labeled ApppG U6 snRNA was carried out as described by Fischer (Fischer et al. (1991), J. Cell. Biol. 113, 705).

27 For in-vitro nucleus import assays, the isolated U1 snRNPs or A5'-U1 snRNPs were carried out with the monofunctional reactive fluorescence dye Cy3 following the manufacturer's instructions (Amersham). The 5 unbound stain molecules were removed by ultrafiltration in an Amicon unit followed by dialysis with PBS (pH 8) until stain molecules were no longer detectable in the fractions which passed through. Sedimentation analysis of the fluorescence-labeled snRNPs was carried out as described above. 10 B) Injections into the oocytes Microinjections were carried out as described by Fischer (Fischer et al. (1993), supra), with the exception that OR-2 buffer was used in place of MBS buffer. After incubation at 18*C for the stated period, the oocytes were 15 transferred into J buffer (70 mM NH 4 CI, 7 mM MgCI 2 , 0.1 mM EDTA, 2.5 mM DTT, 20 mM Tris-HCI, 10% glycerol, pH 7.5) and separated manually. The RNA was purified and analyzed as described (Fischer et al., 1993, supra). The gels were quantified by means of a Molecular Dynamics (Sunnyvale, CA) phosphorimaging system using Image Quant Software 20 Version 3.0. Example 7: Nucleus import assays Nucleus import reactions were carried out on HeLa cells which had grown 25 on glass coverslips at 370C, 5% C02, in Dulbecco's modified Eagle medium (Gibco-BRL) supplemented with 10% fetal calf serum and penicillin/streptomycin (Gibco-BRL) to 50-70% confluence. Following permeabilization with digitonin (Adams et al. (1990), J. Cell Biol., 111, 807) the cells were washed with ice-cold import buffer (25 mM Hepes pH 7.9, 30 100 mM NaCl, 2.5 mM MgCI 2 , 0.25 mM EDTA) and incubated with 25 pl of import buffer comprising 0.2 mg/ml tRNA, 1 mM ATP, 1 mM creatine phosphate, 20 U/mI creatine phosphokinase (Sigma), 4 pg/ml U1 snRNP* or A5'-U1 snRNP* (labeled as described above) and 101pl of HeLa S100 cytosolic extract (5 mg/ml protein). Additional reagents were added as 35 described in the keys of the figures. The import mix was ATP-depleted by not adding ATP, creatine phosphate and creatine phosphate kinase and preincubating for 30 min at 250C in the presence of 20 U/ml Apyrase (Sigma). Incubation of the import reactions was carried out for 30 min at 25 0 C and the reaction was stopped as described by Marshallsay and 28 Luhrmann (1994, supra). After covering the samples with Fluoprep (bioMerieux), they were analyzed with the aid of 50 x lens of a Leica DM/IRB inverted fluorescence microscope and stored with the aid of a CCD camera in the form of digitalized images. The images were processed with 5 the software Adobe Photoshop Version 3.0 and quantified with the aid of the NIH Image Software Version 1.6. For each sample, the average fluorescence of approximately 100 random nuclei from at least three independent assays was calculated and averaged.

Claims (23)

1. A polypeptide with an amino acid sequence as shown in SEQ ID No. 1 or a functional variant thereof and portions thereof -vith at least 5 6 amino acids.

2. The polypeptide as claimed in claim 1, which is an m3G-cap-specific nucleus import receptor protein. 10

3. The polypeptide as claimed in claims 1 and 2 obtainable from cytoplasmatic extracts of HeLa cells.

4. The polypeptide as claimed in any of claims 1 to 3, which comprises an m3G-cap-specific domain. 15

5. An antibody against the polypeptide as claimed in any of claims 1 to 4.

6. A nucleic acid encoding a polypeptide as claimed in any of claims 1 20 to 4 or a functional variant thereof and portions thereof with at least 8 nucleotides, SEQ ID No. 2 being part of the claim.

7. A nucleic acid as shown in SEQ ID No. 3 obtainable from a nucleic acid as claimed in claim 6, encoding a deletion polypeptide as 25 shown in SEQ ID No. 4, SEQ ID No. 3 being part of the claim.

8. The nucleic acid as claimed in claims 6 and 7, which is a DNA or RNA, preferably a double-stranded DNA. 30

9. The nucleic acid as claimed in any of claims 6 to 8, which comprises one or more noncoding sequences.

10. The nucleic acid as claimed in any of claims 6 to 9, which is present in a vector, preferably in an expression vector or a gene 35 therapeutically active vector.

11. The polypeptide with an amino acid sequence as shown in SEQ ID No. 4 obtainable from a nucleic acid as claimed in claim 7, which does not contain an IBB domain. 30

12. A method of generating a nucleic acid as claimed in any of claims 6 to 9, wherein the nucleic acid is synthesized chemically or isolated from a genomic library with the aid of a probe. 5

13. A method of producing an antibody as claimed in claim 5, wherein a mammal is immunized with a polypeptide as claimed in claim 1 or 9 and, if appropriate, the antibodies formed are isolated. 10

14. A pharmaceutical comprising a nucleic acid as claimed in any of claims 6 to 9 and/or a polypeptide as claimed in any of claims 1 to 5 or 11 and, if appropriate, pharmaceutically acceptable additives and/or adjuvants.

15 15. A method of producing a pharmaceutical for the treatment of cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer's disease, allergies, in particular neurodermitis, type I allergies or type IV allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and/or diabetes 20 and/or for controlling cell metabolism, in particular in immunosuppression, especially in the case of transplantations, which comprises formulating a nucleic acid as claimed in any of claims 6 to 9 or a polypeptide as claimed in claim 1 or 11 or antibody as claimed in claim 5 with a pharmaceutically acceptable additive 25 and/or adjuvant.

16. A diagnostic comprising a nucleic acid as claimed in any of claims 6 to 9 or a polypeptide as claimed in claim 1 or 11 or antibody as claimed in claim 5 and, if appropriate, suitable additives and/or 30 adjuvants.

17. A method of producing a diagnostic for diagnosing cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer's disease, allergies, in particular neurodermitis, 35 type I allergies or type IV allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and/or diabetes and/or for analyzing cell metabolism, in particular of the immune state, especially in the case of transplantations, which comprises combining a nucleic acid as claimed in any of claims 6 to 9 or a 31 polypeptide as claimed in claim 1 or 11 or antibody as claimed in claim 5 [lacuna] pharmaceutically acceptable carriers.

18. An assay for identifying functional interactors comprising a nucleic 5 acid as claimed in any of claims 6 to 9 or a polypeptide as claimed in claim 1 or 11 or antibody as claimed in claim 5 and, if appropriate, suitable additives and/or adjuvants.

19. The use of a nucleic acid as claimed in any of claims 6 to 9 or of a 10 polypeptide as claimed in claim 1 or 11 for identifying functional interactors.

20. The use of a nucleic acid as claimed in any of claims 6 to 9 for finding variants of a human m3G-cap-specific nucleus import 15 receptor, which comprises screening a genomic library with the abovementioned nucleic acid and isolating the variant which has been found.

21. The use of a polypeptide as claimed in any of claims 1 to 4 as m 3 G 20 cap-specific nucleus import receptor and for transporting m3G-cap containing molecules into the nucleus.

22. The use of a polypeptide as claimed in any of claims 1 to 4 or 11 for recognizing and/or identifying m3G-cap-containing molecules. 25

23. The use of a polypeptide as claimed in any of claims 1 to 4 or 11 for purifying and quantifying m3G-cap-containing molecules.