Abstract
Xenopus oocytes have been utilized in a number of laboratories as an experimental system to study a variety of biological processes. Here, we describe its application to functional studies of spliceosomal small nuclear RNAs (snRNAs) in pre-messenger RNA (pre-mRNA) splicing, a process that occurs extremely efficiently in Xenopus oocytes. A DNA oligonucleotide complementary to an snRNA of interest is injected into the oocyte cytoplasm. The oligonucleotide subsequently diffuses into the nucleus and hybridizes to the target snRNA, thereby triggering snRNA degradation via endogenous RNase H activity. By the time the endogenous snRNA is depleted, the DNA oligonucleotide itself is degraded by endogenous deoxyribonuclease (DNase) activity. In principle, this procedure enables one to quantitatively deplete any snRNA of choice. Subsequently, a rescuing snRNA that is constructed in vitro may be injected into the snRNA-depleted oocytes to restore the splicing function. After reconstitution, a radiolabeled splicing substrate is injected into the nuclei of the oocytes. These oocyte nuclei are then manually isolated and used to prepare both nuclear RNA for splicing assays and nuclear extract for spliceosome assembly assays. The ability of an injected rescuing snRNA to reconstitute splicing can therefore be tested. Because all types of rescuing snRNAs (e.g., mutant snRNAs, snRNAs with or without modified nucleotides) can be constructed readily, the results obtained from this procedure provide valuable information on the function of a particular snRNA of interest in pre-mRNA splicing.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sharp, P. A. (1994) Split genes and RNA splicing. (Nobel Lecture). Cell 77, 805–815.
Burge, C. B., Tuschl, T. H., and Sharp. P. A. (1999) Splicing of precursors to mRNAs by the spliceosome, in The RNA World, 2nd ed. (Gesteland, R. F., Cech, T. R., and Atkins, J. F., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 525–560.
Yu, Y.-T., Scharl, E. C, Smith, C. M., and Steitz, J. A. (1999) The growing world of small nuclear ribonucleoprotein particles, in The RNA World, 2nd ed. (Gesteland, R. F., Cech, T. R., and Atkins, J. F., eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 487–524.
Staley, J. P. and Guthrie, C. (1998) Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 92, 315–326.
Guthrie, C. (1988) Genetic analysis of yeast snRNAs, in Structure and Function of Major and Minor snRNPs (Birnstiel, M., ed.), Springer-Verlag, Berlin, Germany, pp. 196–211.
Lin, R. J., Newman, A. J., Cheng, S. C, and Abelson, J. (1985) Yeast mRNA splicing in vitro. J. Biol. Chem. 260, 14,780–14,792.
Padgett, R. A., Hardy, S. F., and Sharp, P. A. (1983) Splicing of adenovirus RNA in a cell-free transcription system. Proc. Natl. Acad. Sci. USA. 80, 5230–5234.
Grabowski, P. J., Padgett, R. A., and Sharp, P. A. (1984) Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate. Cell 37, 415–427.
Krainer, A. R., Maniatis, T., Ruskin, B., and Green, M. R. (1984) Normal and mutant human β-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell 36, 993–1005.
Ruskin, B., Krainer, A. R., Maniatis, T., and Green, M. R. (1984) Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell 38, 317–331.
Rio, D. C. (1988) Accurate and efficient pre-mRNA splicing in Drosophila cell-free extracts. Proc. Nad. Acad. Sci. USA 85, 2904–2908.
Spikes, D. and Bingham, P. M. (1992) Analysis of spliceosome assembly and the structure of a regulated intron in Drosophila in vitro splicing extracts. Nucleic Acids Res. 20, 5719–5727.
Pan, Z. Q. and Prives, C. (1989) U2 snRNA sequences that bind U2-specific proteins are dispensable for the function of U2 snRNP in splicing. Genes Dev. 3, 1887–1898.
Hamm, J., Dathan, N. A., and Mattaj, I. W. (1989) Functional analysis of mutant Xenopus U2 snRNAs. Cell 59, 159–169.
Hannon, G. J., Maroney, P. A., Denker, J. A., and Nilsen, T. W. (1990) Trans splicing of nematode pre-messenger RNA in vitro. Cell 61, 1247–1255.
Black, D. L. (1992) Activation of c-src neuron-specific splicing by an unusual RNA element in vivo and in vitro. Cell 69, 795–807.
Yu, Y.-T., Shu, M.-D., and Steitz, J. A. (1998) Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing. EMBO J. 17, 5783–5795.
Evans, J. P. and Kay, B. K. (1991) Biochemical fractionation of oocytes. Methods Cell Biol. 36, 133–148.
Forbes, D. J., Kornberg, T. B., and Kirschner, M. W. (1983) Small nuclear RNA transcription and ribonucleoprotein assembly in early Xenopus development. J. Cell Biol. 97, 62–72.
Hamm, J. and Mattaj, 1. W. (1989) An abundant U6 snRNP found in germ cells and embryos of Xenopus laevis. EMBO J. 8, 4179–4187.
Peculis, B. A. and Steitz, J. A. (1994) Sequence and structural elements critical for U8 snRNP function in Xenopus oocytes are evolutionary conserved. Genes Dev. 8, 2241–2255.
Peculis, B. A. and Steitz, J. A. (1993) Disruption of U8 nucleolar snRNA inhibits 5.8S and 28S rRNA processing in the Xenopus oocyte. Cell 73, 1233–1245.
Tycowski, K. T., Shu, M.-D., and Steitz, J. A. (1994) Requirement for intron-encoded U22 small nucleolar RNA in 18S ribosomal RNA maturation. Science 266, 1558–1561.
Mattaj, I. W. (1986) Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding. Cell 46, 905–911.
Zhao, X. and Yu, Y.-T. (2004) Pseudouridines in and near the branch site recognition region of U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing in Xenopus oocytes. RNA 10, 681–690.
Kornarska, M. M. (1990) Analysis of splicing complexes and small nuclear ribonucleoprotein particles by native gel electrophoresis. Methods Enzymol. 180, 442–453.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Moon, K.H., Zhao, X., Yu, YT. (2006). Pre-mRNA Splicing in the Nuclei of Xenopus Oocytes. In: Liu, X.J. (eds) Xenopus Protocols. Methods in Molecular Biology™, vol 322. Humana Press. https://doi.org/10.1007/978-1-59745-000-3_11
Download citation
DOI: https://doi.org/10.1007/978-1-59745-000-3_11
Publisher Name: Humana Press
Print ISBN: 978-1-58829-362-6
Online ISBN: 978-1-59745-000-3
eBook Packages: Springer Protocols