Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-11-27
2003-06-17
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100
Reexamination Certificate
active
06579681
ABSTRACT:
DESCRIPTION
The present invention relates to a test system comprising
(a) one or more identical or different immobilized nucleic acid(s) having at least one spliceable nucleic acid.
(b) at least one gel-free detection system for detecting a splicing reaction, where appropriate
(c) at least one composition comprising splicing components, and preferably
(d) suitable detection probes, and, where appropriate,
(e) further aids.
Most of the protein-encoding genes in eukaryotes are interrupted in their genomic form by one or more sequences not coding for the protein (introns). When transcribing the genomic DNA into messenger RNA (mRNA), these non-coding regions (introns) are incorporated into the primary transcript. In order to generate the correct mRNA, this pre-mRNA has to be processed.
The pre-mRNA is processed by removing the introns and fusion of the coding regions (exons). Only then is it possible to provide a nucleotide strand which can be read in an interrupted manner for translation in the cytoplasm. mRNA formation in eukaryotes therefore requires a “splicing process” in which the non-coding gene regions (introns) are removed from the primary gene transcript.
Splicing occurs in the nucleus, before the mRNA is transported out of the nucleus. It is generally carried out in a two-stage mechanism in which in each case a transesterification step is involved (Moore, J. M. et al., (1993) Splicing of precursors to mesenger RNAs by the Spliceosome. In The RNA world, Edited by Gesteland R. F., Gesteland, J. F., Cold Spring Harbor Laboratory Press, 303-358). The first step generates a free 5′ exon and a “lariat structure” of the intron which is still linked to the 3′ exon. The lariat structure comprises a branched RNA which is produced by esterification of the 5′ end of the intron with a 2′-hydroxyl group of a ribose in an adenosine which is located approx. 20-40 nucleotides upstream of the 3′ end of the intron. The second catalytic step leads to ligation of the exons and liberation of the intron. Although no nucleotides are incorporated during these reactions, an energy source, for example ATP, is necessary for this catalysis (Guthrie, C. (1991) Science, 253, 157).
A plurality of factors is involved in the process of mRNA splicing. Two classes of splicing factors are distinguished at the moment. The first class comprises four evolutionarily highly conserved protein-RNA particles (small nuclear ribonucleoprotein particles=snRNPs); U1, U2, U4/U6 and U5, which comprise either one (U1, U2, U5) or two (U4/U6) snRNA components (Moore, J. M. et al., (1993) supra; Guthrie, (1991) supra; Green, M. R. (1991), Annu. Rev. Cell Biol., 7, 559). The second class comprises proteins which have not been characterized much up until now and which are not tightly bound to the snRNPs and are therefore called non-snRNP splicing factors (Lamm, G. M. & Lamond, A. J. (1993) Biochim. Biophys. Acta, 1173, 247; Beggs, J. D. (1995), Yeast splicing factors and genetic strategies for their analysis, In: Lamond, A. I. (ed) Pre-mRNA Processing Landes, R.G. Company, Texas, pp. 79-95. K ämer A. (1995), The biochemistry of pre-mRNA splicing. In: Lamond, A. I. (ed), Pre-mRNA Processing. Landes, R.G. Company, Texas, pp. 35-64).
The composition of snRNPs has been studied most successfully in HeLa cells (Will, C. L. et al., (1995) Nuclear pre-mRNA splicing. In: Eckstein, F. and Lilley, D. M. J. (eds). Nucleic Acids and Molecular Biology. Springer Verlag, Berlin, pp. 342-372). At relatively low salt concentrations at which it is possible for nuclear extracts from HeLa cells to promote splicing of pre-mRNA in vitro, the snRNPs are present in a 12S U1 snRNP, a 17S U2 snRNP and a 25S [U4/U6.U5] tri-snRNP complex. At higher salt concentrations (approx. 350-450 mM) the tri-snRNP complex dissociates into a 20S U5 particle and a 12S U4/U6 particle. In the U4/U6 snRNP, the U4 and U6 RNAs are present base-paired via two intermolecular helices (Bringmann, P. et al. (1984) EMBO J., 3, 1357; Hashimoto, C. & Steitz, J. A. (1984) Nucleic Acids Res., 12, 3283; Rinke, J. et al., (1985) J. Mol. Biol., 185, 721; Brow. D. A. & Guthrie, C. (1988) Nature, 334, 213).
The snRNPs comprise two groups of proteins. All snRNPs comprise the group of general proteins (B/B′, D1, D2, D3, E, F and G). In addition, each snRNP comprises specific proteins which are present only in said snRNP.
Thus, according to the prior state of research, U1 snRNP comprises three additional proteins (70K, A and C) and U2 snRNP eleven further proteins. According to prior knowledge, 20S U5 snRNP carries nine further proteins having molecular weights of 15, 40, 52, 100, 102, 110, 116, 200 and 220 kDa, while 12S U4/U6 snRNP comprises two additional proteins having molecular weights of approx. 60 and 90 kDa. 25S tri-snRNP [U4/U6.U5] comprises five additional proteins having molecular weights of approx. 15.5, 20, 27, 61 and 63 kDa (Behrens, S. E. & Lührmann, R. (1991) Genes Dev., 5, 1439; Utans, U. et al., (1992) Genes Dev., 6, 631; Lauber, J. et al., (1996) EMBO J., 15, 4001; Will, C. L. et al. (1995), supra, Will, C. L. & Lührmann, R. (1997) Curr. Opin. Cell Biol., 9, 320-328).
The composition of splicing components in
Saccharomyces cerevisiae
has not yet been studied in detail. Biochemical and genetic studies, however, indicate that the sequences of both the snRNAs and the snRNP proteins are evolutionarily highly conserved (Fabrizio, P. et al., (1994) Science, 264, 261; Lauber, J. et al., (1996), supra, Neubauer, G. et al., (1997) Proc. Natl. Acad. Sci. USA, 94, 385; Krämer, A. (1995), supra; Beggs, J. D. (1995); supra, Gottschalk, A. et al. (1998) RNA, 4, 374-393).
In order to form a functional splicing complex (spliceosome), the individual components (pre-mRNA, snRNPs and non-snRNP proteins) are combined in a stage-wise process. This is achieved not only by interactions of the pre-mRNA with the protein-containing components but also by numerous interactions between the protein-containing components themselves (Moore, J. M. (1993) supra; Madhani, H. D. & Guthrie, C. (1994) Annu. Rev. Genetics, 28, 1; Nilsen, T. W. (1994) Cell, 65, 115). The pre-mRNA sequence carries specific recognition sequences for the different splicing components. Firstly, U1 snRNP binds via said recognition sequences to the 5′ splicing region of the pre-mRNA intron. At the same time, an as yet unspecified number of various other factors (e.g. SF2/ASF, U2AF, SC35, SF1) is taken up by this complex and cooperates with the snRNAs in the continued formation of the pre-spliceosome. The U2 snRNP particle interacts with the “branch site” in the intron region (Krämer, A. & Utans, U. (1991) EMBO J., 10, 1503; Fu, X. D. & Maniatis, T. (1992) Proc. Natl. Acad. Sci USA, 89, 1725; Krämer, A. (1992) Mol. Cell Biol., 12, 4545; Zamore, P. D. et al. (1992) Nature, 355, 609; Eperon, J. C. et al. (1993) EMBO J., 12, 3607; Zuo, P. (1994) Proc. Natl. Acad. Sci. USA, 91, 3363; Hodges, P. E. & Beggs, J. D. (1994) Curr. Biol. 4, 264; Reed, R. (1996) Curr. Op. Gen. Dev., 6, 215). In a last step of spliceosome formation, [U4/U6.U5] tri-snRNP and a number of proteins not yet characterized in detail interact with the pre-spliceosome, in order to form the mature spliceosome (Moore, J. M. et al., (1993) supra).
For the splicing process, various interactions between pre-mRNA, snRNAs and sn-RNP are removed and new ones are formed. Thus it is known that before or during the first catalytic step of the splicing reaction two helices are separated from one another in the interacting structures of U4 and U6 and that new interactions form base pairs between U2 RNAs and U6 RNAs (Datta, B. & Weiner, A. M. (1991) Nature, 352, 821; Wu, J. A. & Manley, J. L. (1991) Nature, 352, 818; Madhani, H. D. & Guthrie, C. (1992) Cell, 71, 803; Sun, J. S. & Manley, J. L. (1995) Genes Dev., 9, 843). At the same time, binding of U1 to the 5′ splicing site is removed and pre-mRNA binds to the recognition sequence ACAGAG of U6 snRNA (Fabrizio, P. & Abelson, J. (1990), Science, 25
Achsel Tilmann
Bauer Bettina
Hüls Christoph
Lührmann Reinhard
Simandi Claus
Aventis Research and Technologies GmbH & Co. KG
Connolly Bove & Lodge & Hutz LLP
Horlick Kenneth R.
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