WO2000075309A1

WO2000075309A1 - Spliceosome protein and its use

Info

Publication number: WO2000075309A1
Application number: PCT/EP2000/003949
Authority: WO
Inventors: Bettina Bauer; Reinhard Lührmann; Cindy Will
Original assignee: Aventis Research & Technologies Gmbh & Co Kg
Priority date: 1999-06-04
Filing date: 2000-05-03
Publication date: 2000-12-14
Also published as: IL146727A0; EP1190049A1; NO20015906L; NO20015906D0; AU4915900A; DE19925668A1; JP2003501085A; PL352803A1; CA2376055A1

Abstract

The invention relates to a spliceosome protein that is associated with the U11/U12 snRNP complex of the U12-dependent spliceosome and that is specific of said spliceosome. Said protein and the DNA sequence encoding it are useful for the diagnostics of certain autoimmune diseases and of diseases that are caused by defects in the splicing apparatus.

Description

Spliceosome protein and its use

description

The present invention relates to a spliceosome protein which is associated with the U11 / U12 snRNP complex of the AT-AC spliceosome and is specific for this spliceosome. The invention further relates to the use of this protein and the DNA sequences coding therefor in the diagnosis of autoimmune diseases and diseases which are based on defects in the splicer.

In patients suffering from Grave ^'s disease, it has been shown that incorrect splicing results in a crucial enzyme (thyroperoxidase) in an inactive form (Zanelli, E. (1990) Biochem. Biophys. Res. Comm., 170, 725). Studies for the disease spinal muscular atrophy indicate that a defective gene product of the SMN gene (survival of motor neurons) leads to a considerable disruption of the formation of the snRNPs. By inhibiting the splicing apparatus of the muscle neurons, there is paralysis of the nerve cells and a breakdown of the muscle tissue (Fischer, U. et al., (1997), Cell, 90: 1023-9; Liu, Q. et al. ( 1997), Cell, 90: 1013-21; Lefebvre, S. et al, (1997) Nat. Geriet., 16, 265). In the metastasis of cancer cells, certain alternative splice variants of the membrane-bound molecule CD44 seem to play a decisive role. The CD44 gene contains several exons, of which 10 adjacent exons in a different arrangement are spliced from the pre-mRNA during mRNA generation. In rat carcinoma cells, it has been demonstrated that metastatic variants carry exons 4 to 7 or 6 to 7. With the help of antibodies against the part of the protein encoded by exon 6, the metastasis could be effectively suppressed (Sherman, L, et al., (1996) Curr. Top. Microbiol. Immunol. 213: 249-269).

Incorrect splicing can lead to pronounced phenotypes of the affected

Organism. It is known that a point mutation in an intron of the ß-globin can lead to a ß ⁺ thalemia. The point mutation creates an incorrect splice location, which leads to a changed reading frame and an early one Termination of the peptide chain leads (Weatherall, D. & Clegg, JB (1982) Cell, 29, 7; Fukumaki, Y. et al. (1982) Cell, 28, 585). In Arabidopsis thaiiana mutants z. B. a point mutation at the 5 ^' splice of the phytochrome B gene to an incorrect expression of the gene. This change does not remove an intron that contains a stop codon in its sequence. The development of the plants is disturbed because the gene is involved in phytomorphogenesis (Bradley, JM et al. (1995) Plant Mol. Biol, 27, 1133).

Numerous eukaryotic protein or RNA-coding genes are composed like a mosaic, with individual ones for sub-areas of the

Alternate gene product coding sequences (exons) and intervening sequences (introns) not coding for the gene product. The primary transcripts of such mosaic genes therefore contain both the sequences of the coding exons and of the non-coding introns in their RNA chain and still have to be processed for correct gene expression.

One of the essential processing steps is splicing, which takes place in the cell nucleus. In the case of the expression of protein-coding genes, intron-RNA sequences are cut out from longer-chain primary transcripts, the so-called pre-mRNA, with the formation of mature mRNA, and exon-RNA sequences are linked to one another. The splicing process is catalyzed by a so-called spliceosome, a large ribonucleoprotein complex, which is gradually made up of several small nuclear ribonuclein protein particles (small nucleic RNPs, snRNPs), which in turn consist of uridine-rich RNAs (small nucleic RNAs, snRNAs) and specifically consist of these binding proteins, and are composed of proteins which are not firmly bound to these snRNPs, the so-called πicht-snRNP splice factors. Splicing is generally a two-step mechanism, with a transesterification step involved in each step. In the first step, a free 5'-exon and a so-called lariat structure of the intron are generated after binding the spliceosome to the 5'-splice point and the so-called branching point in the intron, the intron still being connected to the 3'-exon . In the second step, the two exons are ligated and the intron released. The majority of introns of pre-mRNA in metazoa have invariable GU and AG dinucleotides at the ends. These introns are cut out by the so-called independent spliceosome (or major spliceosome), which recognizes these dinucleotides. The spliceosome contains the U1, U2, U5 and U4 / U6 snRNPs, which are composed of one (U1, U2, U5) or two (U4 / U6) RNAs and numerous proteins, for example proteins of the Sm class. The splice and branching point of the so-called U2-dependent introns is recognized by the spliceosome by means of interactions in which both RNA and proteins are involved. The duplex formation between the U1 snRNA and the 5 'splice site is caused by various polypeptides, such as the 70K and the C protein of the

U1 snRNPs and proteins of the family of Ser and Arg-rich SR proteins, relieved. The base pairing between U2-snRNA and the branching point likewise requires numerous U2-snRNP proteins, in particular the subunits of the heteromeric splice factors SF3a and SF3b [R. Reed, Curr. Opin. Genet. Dev. 6, 215 (1996); C.L Will and R. Lührmann, Curr. Opin. Cell Biol. 9,

320: 1997; A. Kramer, Annu. Rev. Biochem. 65: 367 (1996)]

More recently, an alternative spliceosome, the so-called AT-AC spliceosome, has been identified, which is composed of the U11, U12, U5 and U4atac / U6atac snRNPs and spliced a rare class of pre-mRNA introns that attach to theirs

Termini have AU and AC dinucleotides or GT and AG dinucleotides [C.B. Bürge et al. In: The RNA World II, R.F. Gesteland and J.F. Atkins, ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1999, p. 525]. These independent introns, which are therefore called, are found only with a low frequency in comparison with the U2-dependent introns and contain highly conserved sequence elements at the 5 'splice point and the branching point, which differ from the weakly conserved sequences of the U2-dependent pre-mRNA introns [CB Bürge et al. In: The RNA World II, R.F. Gesteland and J.F. Atkins, ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1999, p. 525; S.L. Hall and R.A. Padgett, J. Mol. Biol. 239: 357 (1994); P.A. Sharp and C.B. Burge, Cell 91: 875

(1997)]. During assembly of the U12-dependent spliceosome, the U11-snRNP binds to the 5'-splice site by base pairing and the U12-snRNP to the branching site [SL Hall and RAPadgett, Science 271: 1716 (1996); W.-Y. Tarn and JA Steife, Cell 84: 801 (1996); W.-Y. Tarn and JA Steitz, Proc. Natl. Acad. Be. USA 94: 6030 (1997); I Kolossova and RA Padgett, RNA 3: 227 (1997)]. The mature spliceosome is then formed by association of the U5 and U4atac / U6atac snRNPs [W.-Y. Tarn and JA Steitz, Science 273: 1824 (1996)]. The U11 and U12 snRNPs lie in core extracts as highly stable 18S

U11 / U12 complexes before [K.M. Wassarman and J.A. Steitz, Mol. Cell Biol. 8, 1276 (1992)], and more recent in vitro binding studies suggest that U11 and U12 interact with the pre-mRNA as a pre-formed complex [M. Frilander and J.A. Seitz, Genes & Dev. 13: 851 (1999)]. These observations, together with the fact that U12-type introns are not essential

Pyrimidine tract of the 3 'splice of the main class introns, suspect that there are differences between the mechanisms of splice detection in the different spliceosomes.

The identification and characterization of the proteins characteristic of the U12-dependent spliceosome could therefore not only provide information about the exact mechanism of the splicing processes taking place in this spliceosome, but at the same time enable diagnosis and therapy of diseases which are due to disorders in the splice mechanism of this spliceosome. The provision of specific proteins also enables them to be found and

Development of potential splice modulators that can also be used to advantage in the treatment of diseases caused by disorders of the splicing process.

The object of the present invention was therefore to provide one for the U12

To provide spliceosome characteristic protein.

This problem was solved by a spliceosome protein that is associated with the 18S U11 / U12 snRNP complex of the U12-dependent spliceosome and is specific for this spliceosome.

The subject of the present invention is therefore a spliceosome protein with the function of the 35kD protein associated with the U11 / U12 snRNP complex of the U12-dependent spliceosome, which by a nucleic acid (hereinafter nucleic acid according to the invention) which is selected from the group consisting of

a) DNA sequences that contain a protein with the amino acid sequence according to SEQ ID No. 18 encode;

b) DNA sequences which hybridize with the sequences complementary to the sequences under a) and are capable of coding a protein with the function of the 35 kD protein of the U11 / U12 snRNP complex; and

c) DNA sequences which are degenerate in their genetic code with respect to the sequences mentioned under a) or b);

“Spliceosome protein with the function of the 35 kD protein associated with the U11 / U12 snRNP complex of the U12-dependent spliceosome (hereinafter the spliceosome protein according to the invention)” is to be understood here to mean any polypeptide which essentially has the properties of the naturally occurring, preferably human, Has 35kD protein of the U11 / U12 snRNP complex, i.e. in the spliceosome complex, it ensures the correct function of the spliceosome mentioned.

The term hybridization here means hybridization under customary hybridization conditions, in particular under stringent hybridization conditions as are known to the person skilled in the art [cf. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NN. (1989)].

The spliceosome protein preferably has the amino acid sequence according to SEQ ID Νo. 18. However, the protein can also contain one or more amino acid deletions,

Amino acid exchanges or amino acid additions or insertions, as long as the function of the protein is not significantly affected. For example, the spliceosome protein can also contain foreign protein sequences (eg as a fusion protein).

The preferred spliceosome protein according to the invention with a length of 246 amino acids has an apparent molecular weight, determined by SDS-Page, of approximately 35kD and a calculated molecular weight of 29kD and an isoelectric point of 9.88.

Another subject are the DNA sequences coding for this protein according to claim 1, sequences complementary to these sequences, and

Fragments of it.

These DNA sequences can be linked to other DNA sequences, in particular sequences which enable expression of the protein in a desired host organism. Such sequences are known in the prior art. These are, for example, regulatory sequences such as promoter sequences, Shine-Dalgamo sequences,

Transcription termination signals, polyadenylation signals or enhancer elements. In this way, the desired protein can be obtained inexpensively in large quantities.

The invention therefore also relates to recombinant DNA molecules which contain the DNA sequences according to the invention.

The recombinant DNA molecules can either be directly inserted into the desired one

Host organism are introduced or first incorporated into vectors with which the host organisms are then transformed in a manner known per se. Such vectors are also the subject of the invention. The vectors customary in the prior art, for example plasmids, bacteriophages or viruses, can be used as vectors. Preferred vectors are

Expression vectors. The invention also relates to host organisms which contain the recombinant DNA molecules or vectors according to the invention.

Suitable host organisms are, for example, prokaryotic or eukaryotic microorganisms, for example bacteria such as Escherichia coli,

Yeast or tissue cells.

The DNA sequences according to the invention or fragments thereof can be used to find homologous DNA sequences in different organisms or tissue types which have a similar or the same function as the spliceosome protein according to the invention.

The protein according to the invention and the DNA sequences coding for this protein can also be used advantageously as diagnostics, for example for autoimmune diseases and diseases which are related to disorders in the

Splicing mechanism.

It is known that spliceosome components can act as autoantigens. For example, patients affected by the autoimmune disease systemic lupus erythematosus often produce antibodies with which most snRNPs can be precipitated. The protein according to the invention now opens up a possibility for the rapid diagnosis of autoimmune diseases based on the formation of antibodies against proteins which are specific for U12-dependent spliceosome by means of a simple immunoassay.

Diseases that can be traced back to disorders in the splicing mechanism of the U12-dependent spliceosome that are based on a defect in the 35kD protein can be diagnosed, for example, with the aid of complementation tests in in-vitro splicing systems known in the art. Here, for example, a pre-mRNA with an independent intron is first produced by in vitro transcription, which contains all structural elements which are necessary for the recognition of the pre-mRNA by the spliceosome and for the splicing process. For example, the RNA is radiolabelled, so that later Separation on a denaturing urea-polyacrylic amide gel based on the characteristic band pattern can be used to assess whether a splicing reaction has taken place. Subsequently, samples from patient tissue are tested with and without the addition of the 35 kD protein according to the invention. Complementation then detects the defect in said protein.

Another usable in vitro splicing system is described in PCT / EP 00/01595.

The DNA sequences according to the invention can be carried out in the usual way

Hybridization tests are used to diagnose defects in the gene for the 35 kD protein described, in particular in prenatal diagnostics.

The protein according to the invention can also be used as a therapeutic agent in diseases based on splice defects.

Furthermore, the spliceosome protein according to the invention can advantageously be used to find or develop splice modulators, e.g. Use splice inhibitors, which are then suitable for the treatment of other diseases. For example, a U12-dependent intron was recently identified in the mutant genes that are responsible for the autosomal recessive Hermansky-Pudlak syndrome (HPS). It is to be expected that such introns will also be found in other genes in the future, which can be ascribed a role in genetically caused diseases. A targeted inhibition of the splicing of the introns present in such genes and thus the expression of the harmful mutated genes therefore represents a possible route to the therapy of the diseases mentioned. Because of the rare occurrence of the U12-dependent introns, such splice inhibitors can also be therapeutic in the case of U12-dependent spliceosomes are used more effectively than with U2-dependent spliceosomes.

To find the splicing modulators sought, the known in vitro test systems already mentioned above can be used to investigate splicing mechanisms. Splice modulators or inhibitors that Monoclonal antibodies can be analyzed in this way. The influencing of splicing processes in the cell with the aid of antisera or monocional antibodies, for example the generation of mature mRNA, has already been described [RA Padgett et al, Cell 35:10 (1983); R. Gattoni et al, Nucleic Acid Res. 24: 2535 (1996)].

The identification of the regulatory processes during splicing by the minor spliceosome is also important for virology. Recently (Hibbert et al., 1999) a sequence of the U1 5 'splice site was identified in a region of the negative splice regulator element (NRS) in the Rous Sarcoma virus.

The binding of the U1 snRNP to the NRS inhibits the splicing process, thus increasing the activity of the NRS.

The U1 binding site overlaps with a U11 binding site which is identical to a sequence at the NRS in the 5 'splice site of the U12-dependent spliceosome.

The U11 snRNP binding to the NRS promotes splicing, thus reducing NRS activity. The competition between U1 and U1 snRNPs helps to establish the balance between spliced and unspliced viral RNAs for the purpose of optimal viral replication.

We like the studies by Hibbert et al. (1999) show that targeted mutations at the U1 or U11 binding domain could contribute to influencing and possibly even controlling the viral life cycles. A strategy to inhibit the U11 / NRS interaction could be developed using the information obtained in this work regarding the pre-spliceosomal character.

The invention therefore also relates to a medicament comprising a nucleic acid according to the invention and / or a spliceosome protein according to the invention and optionally pharmaceutically acceptable additives and / or auxiliaries.

The invention further relates to a method for producing a medicament for the treatment of cancer, autoimmune diseases, in particular Grave's disease, spinal muscular atrophy, β'-thalassemia, cancer related to the c-erb oncogene, hepatitis C infection, herpes simplex virus infection, systemic lupus erythematosus, Hermansky-Pudlak syndrome, wherein a nucleic acid according to the invention or a spliceosome protein according to the invention is formulated.

The invention also relates to a diagnostic agent containing a nucleic acid according to the invention and / or a spliceosome protein according to the invention and optionally pharmaceutically acceptable additives and / or auxiliaries.

The invention further relates to a method for producing a

Diagnostic for the diagnosis of Grave's disease, spinal muscle atrophy, ß'-thalassemia, cancer related to the c-erb oncogene, hepatitis C infection, herpes simplex virus infection, systemic lupus erythematosus, Hermansky-Pudlak syndrome, a nucleic acid according to the invention and / or a spliceosome protein according to the invention with a pharmaceutically acceptable one

Carrier is moved.

Description of the figures and most important sequences:

1 shows the snRNA composition of purified snRNPs.

2 shows the protein composition of U11 / U12 snRNPs selected with oligonucleotides.

SEQ ID No. Figure 17 shows the genomic DNA sequence for the U12-associated 35kD

Protein including non-coding sequences. The coding sequence is shown by the inferred derived amino acid sequence and corresponds to SEQ ID No 18.

SEQ ID No. 18 shows the amino acid sequence of the U12-associated 35kD protein consisting of 246 amino acids. The isolation of the U11 / U12-associated 35kD protein according to the invention is explained below by way of example.

The following examples serve to explain the invention in more detail without restricting it to these examples.

Examples

Preparation of HeLa core extracts

For the production of nuclear extracts from HeLa cells, cell cultures with Heia cells were cultivated. The cells were then sedimented from the culture medium by centrifugation (1000 × g, 10 min.) And washed with phosphate buffer. The cell sediment was then taken up in five times the volume of buffer A (10 mM HEPES, 1.5 mM MgCl ₂ , 10 mM KCI, 0.5 mM DTT, pH 7.9, 4 ° C.) and incubated for 10 minutes. The cells were sedimented again and buffer A was taken up in twice the volume. This suspension was digested with a dounce homogenizer (pestle B) (moving the pestle up and down 10 times). The cores were sedimented by centrifugation. The cores were then taken up again in buffer A and centrifuged for 20 minutes at 25,000 xg. The sediment was dissolved in 3 ml of buffer B (20 mM HEPES, 25% (v / v) glycerol, 0.42 M NaCl, 1, 5 mM MgCl _2, 0.2 mM EDTA, 0.5 mM Phenylmethylsulfonylfluoπ ^'d (PMSF ), 0.5 mM DTT, pH 7.9) and digested again with the Dounce homogenizer. The resulting suspension was incubated for 30 minutes on a magnetic stirrer and then centrifuged at 25,000 xg for 30 minutes. Another closed

Centrifugation at 25,000 x g (30 min). The supernatant was compared to 50 times the volume of buffer C (20 mM HEPES, 20% (v / v) glycerol, 0.1 M KCI, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, pH 7.9) dialyzed. The dialysate was centrifuged (25,000 x g, 20 min.). The resulting supernatant can be stored as a core extract in liquid nitrogen (Dignam, J.D. et al. (1983) Nucleic Acid Res., 11, 1475).

Isolation and analysis of snRNPs Spliceosomal snRNPs, which had previously been provided with a trimethylguanosine (m3G) cap, were purified from core extracts of HeLa cells by immunoaffinity chromatography with anti-m3G antibodies and fractionated by sedimentation through a 10-30% glycerol gradient [B. Laggerbauer, J. Lauber and R.Lührmann, Nucleic Acids Res. 24: 868 (1996)].

Fractions containing the 18 S U11 / U12 snRNP complexes were pooled and the KCI concentration adjusted to 250 mM. The snRNPs of 2.4 ml of the pooled 18S gradient fractions were 16 h at 4 ° C with 12 μg oligonucleotide, which either to the nucleotides 2-18 of human U11 snRNA, 5'ACGACAGAAGCCCUUUUdT * dt * dT * dT * -3 '( U11 oligo), or to the nucleotides

11-28 of human U12 snRNA, 5'-AUUUUCCUUACUCAUAAGdT ^* dT ^* dT ^* dT ^* -3 '(U12-Oligo), complementary, incubated, where * is a biotinylated 2'-deoxythymidine and A, U, G and C 2 '-O-methylribonucleotides mean. The oligonucleotide-bound snRNPs were precipitated with streptavidin agarose in a manner known per se [Al Lamond and BS Sproat, in RNA Processing: A Practical Approach,

D. Rickwood and B.D. Harnes, ed. (Oxford University Press, Oxford, 1996) p. 103]. The RNA from 1/5 of the agarose-precipitated snRNPs was eluted to 10% by incubation for 30 minutes at 95 ° C. in 100 μl of DH buffer (15 mM NaCl, 1.5 mM Na citrate, 0.1% SDS) Polyacryiamide 7M urea gels fractionated and visualized by silver staining. The identity of the selected RNAs as U11 and U12 was confirmed by Northern blotting. The protein was removed from the remaining beads by incubation for 5 minutes at 95 ° C. in 200 μl S buffer (60 mM Tris, pH 6.8, 1 mM EDTA, 17.5% glycerol, 2% SDS, 0.2 M DTE) eluted and precipitated with 5 volume units of acetone. The proteins were fractionated by SDS-PAGE on gels with 10% (upper half) and 13% (lower half) polyacrylamide and visualized by Coomasie staining. For comparison, RNA and protein from 50 μl of the starting material (pooled 18S gradient fractions) were also analyzed.

The results for the U11 oligo are shown in FIG. 1, lane 2, those for the U12 oligo in FIG. 1, lane 3. Lane 1 shows the snRNAs of the starting material (input); Lane 4 shows control of precipitation in the absence of oligonucleotides (mock). The coselection of U12 with one directed against U11 Oligonucleotide and vice versa showed that mainly U11 / U12 snRNP complexes and not U11 or U12 monoparticles had been selected. Accordingly, FIG. 2, lanes 2 and 3, show identical protein patterns of the U11 / U12 snRNPs, regardless of the oligonucleotide with which they were selected. The molecular weight of the proteins (in kD) is shown on the right. Lane 1 again shows the proteins of the starting material (input) and their identity (left side). Lane 4: control.

Identification of proteins associated with the U11 / U12 snRNP complex

Of the 20 different proteins found in the U11 / U12 complex, 8 migrated with the Sm snRNP core proteins B ', B, D3, D2, D1, E, F, and G, which are present in the snRNPs of the major spliceosome (Fig. 2, lanes 1-3). Antibodies that reacted specifically with B7B, D3, D2, F or G also recognized the proteins of identical molecular weight on immunoblots of the U11 / U12 complex.

U11 / U12 therefore contains the same 8 Sm proteins that are also found in the major spliceosome.

Of the 12 remaining U11 / U12 proteins, the 160kD, 150kD, 130kD and 49kD proteins of the U 11 / U 12 complex migrated with four of the proteins specific for the 17S U2 complex that make up the essential splice factor SF3b, namely U2-160, U2-150, U2-120 and U2-53. Antibodies directed against U2-160, U2-150 and U2-120 also reacted strongly with the 160kD, 150kD and 130kD proteins of the U11 / U12 complex. Peptide sequences obtained by microsequencing the 160kD, 150kD, 130kD and 49kD proteins of the U11 / U12

Complexes obtained (Table I) were found to be essentially identical to the known sequences of SF3b. The four proteins mentioned are therefore very likely to be identical to the proteins known from SF3b, with deviations in the apparent molecular weights of some of the comigrating proteins being due to differences in the electrophoresis conditions that were used in the original identification of the U2 snRNP proteins. table

U11 / U12 peptide protein

160kD KMNARTYMDVMREQHLTK

KLTATPTPLGGMTGF

KAIVNVIGMH

150kD KRIFEAFK KLRRMNRFTVAE KRTGIQEMREALQEK KLTIHGDLYYEG

130kD KLGAVFNQVAFPLQYT KLLRVYDLGK KNVSEELDRTPPEVSK KLENIAQRYAF

49kD KVSEPLLXELFLQ KDRVTGQHQGYGFVEFLSEE

X here means any amino acid

Characterization of the 35kD associated with the U11 / U12 snRNP complex

Protein

The 35kD protein associated with the U11 / U12 snRNP complex was characterized from the remaining proteins. For this purpose, the peptides KEYDPLK and KRWQEREPTRVWPDND were obtained from the gel-fractionated, trypsin-digested 35 kD protein by microsequencing. These peptides were used to search for cDNAs using the TBLASTN program in the National Center for Biotechnology Information's EST database. Both peptides were in one ORF of a cDNA from human macrophage cells (Genbank Accession No. U44798) which encoded for an unknown protein. A second cDNA from human muscle cells with an identical ORF (Genbank Accession No. AA211268) in pBluescript SK was completely sequenced with an ABI PRISM sequence analyzer after transformation into E. coli HB101. The DNA sequence coding for the 35 kD protein is shown in SEQ ID No. 17 together with the amino acid sequence derived therefrom. In SEQ ID No. 17, the complete cDNA sequence, including non-coding 5 'and 3' sequences, is inherently shown.

Protein was produced from the cDNA by in vitro translation (TNT (Coupled Transcription / Translation) kit from Promega). The protein migrated on an SDS polyacrylamide gel along with the purified 35kD protein, showing that the DNA encoded the complete protein.

The U11 / U12-35kD protein has an RNA recognition motif (RRM; amino acids 51-129). This region and the neighboring glycine-rich region are very similar to a region of the U1-70K protein. Antisera against the 35 kD protein also efficiently precipitated U11 from a mixture of gradient fractionated snRNPs containing U11 monoparticles. Analogous to U1-

70K, the U11-35kD protein should therefore facilitate the detection of the 5 'splice site. Furthermore, the protein may be involved in the exon linkage in interaction with components of the major spliceosome.

Claims

Patent claims

1 DNA sequence coding for a spliceosome protein with the function of the 35kD protein associated with the U11/U12 snRNP complex of the U12 spliceosome, selected from the group consisting of

a) DNA sequences that encode a protein with the amino acid sequence according to SEQ ID No. 18,

b) DNA sequences that hybridize with the sequences complementary to the sequences under a) and are capable of encoding a protein with the functional properties of the 35kD protein of the U11/U12 snRNP complex, and

c) DNA sequences that are degenerated in their genetic code with respect to the sequences mentioned under a) or b),

as well as fragments of these sequences and the sequences complementary to the sequences mentioned under a), b) and c) or the fragments thereof

2 DNA sequence according to claim 1, comprising the nucleotide sequence according to SEQ ID No 17, complementary sequences and fragments of these sequences

3 Recombinant DNA molecule containing a DNA sequence according to claim 1 or 2

4 Recombinant DNA molecule according to claim 3, wherein the DNA encoding the spliceosome protein is linked to regulatory sequences that enable expression of the protein in prokaryotic or eukaryotic cells

5. Vector containing a sequence according to claim 1 or 2 or a recombinant DNA molecule according to one of claims 3 or 4.

6. Host organism containing a recombinant DNA molecule according to any one of claims 3 or 4 or a vector according to claim 5.

7 Word organism according to claim 6, which is a prokaryotic microorganism.

8. Host organism according to claim 6, which is a eukaryotic

microorganism is.

9 spliceosome protein with the function of the 35kD protein associated with the U11/U12 snRNP complex of the U12 spliceosome, which is encoded by one of the sequences according to claim 1 or 2.

10 spliceosome protein according to claim 9, selected from the group consisting of

a) a polypeptide with the amino acid sequence according to SEQ ID No. 18;

b) a polypeptide which, in comparison with the sequence according to a), has one or more amino acid deletions, amino acid exchanges, amino acid additions and / or ammo acid insertions

11 Use of a DNA sequence according to claim 1 or 2 or a fragment thereof for isolating homologous DNA or RNA sequences

12. Use of a spliceosome protein according to claim 9 for finding spiking modulators

3. Medicaments containing a nucleic acid according to one of claims 1 to 4 and/or a spliceosome protein according to one of claims 9 or 10 and optionally pharmaceutically acceptable additives and/or auxiliary substances.

14. Process for producing a drug for treating cancer,

Autoimmune diseases, in particular Grave's disease, spinal muscular atrophy, ß'-thalassemia, cancers related to the c-erb oncogene, hepatitis C infection, herpes simplex virus infection, systemic lupus erythematosus, Hermansky-Pudlak syndrome, characterized in that a nucleic acid according to one of claims 1 to 4 and / or a

Spliceosome protein according to one of claims 9 or 10 is formulated with a pharmaceutically acceptable additive and / or excipient.

15. Diagnostic agent containing nucleic acid according to one of claims 1 to 4 and / or a spliceosome protein according to one of claims 9 or 10 and optionally pharmaceutically acceptable additives and / or auxiliary substances.

16. Method for producing a diagnostic for diagnosing Grave's disease, spinal muscular atrophy, ß'-thalassemia, cancer diseases related to the c-erb oncogene, hepatitis C infection, herpes simplex virus

Infection, systemic lupus erythematosus, Hermansky-Pudlak syndrome, characterized in that a nucleic acid according to one of claims 1 to 4 and/or a spliceosome protein according to one of claims 9 or 10 is mixed with a pharmaceutically acceptable carrier.