Abstract
Splicing of eukaryotic precursor messenger RNAs (pre-mRNAs) excises the intron from the precursor and ligates the two exons together to produce the mature mRNA. It occurs via a two-step mechanism (Fig. 1) (reviewed in ref. 1). In the first step the 2′ hydroxyl group of an intronic adenylyl residue initiates a transesterfication reaction at the 5′ splice site. The result is the cleavage of the 5′ splice site phosphodiester bond and the formation of a new, 2′–5′ phosphodiester bond between the adenylyl residue and the 5′ end of the intron. Because the intronic adenylyl residue has both 2′–5′ and 3′–5′ phosphodiester bonds, it is commonly referred to as the branch point nucleotide. The first splicing step yields two intermediates, “free” exon 1 and the lariat intermediate, which are not covalently linked together. In the second step, the 3′ hydroxyl of the “free” exon 1 initiates a second transesterification reaction at the 3′ splice site, resulting in the ligation of the two exons together to form the mRNA and the release of the lariat intron.
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References
Moore, M. J., Query, C. C., and Sharp, P. A. (1993) Splicing of precursors to mRNA by the spliceosome, in The RNA World (Gesteland, R. F. and J. F. Atkins, eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 303–358.
Guthrie, C. (1991) Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science 253, 157–163.
Ruby, S. W. and Abelson, J. (1991) Pre-mRNA splicing in yeast. Trends Genet. 7, 79–85.
Kramer, A. (1996) The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu. Rev. Biochem. 65, 367–409.
Chamberlin, M., Kingston, R., Gilman, M., Wiggs, J., and deVera, A. (1983) Isolation of bacterial and bacteriophage RNA polymerases and their use in synthesis of RNA in vitro. Methods Enzymol. 101, 540–568.
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.
Seraphin, B. and Rosbash, M. (1989) Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell 59, 349–358.
Umen, J. and Guthrie, C. (1995) A novel role for a U5 snRNP protein in 3′ splice site selection. Genes Dev. 9, 855–868.
Jones, E. W. (1991) Three proteolytic systems in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 266, 7963–7966.
Lustig, A. J., Lin, R. J., and Abelson, J. (1986) The yeast RNA gene products are essential for mRNA splicing in vitro. Cell 47, 953–963.
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.
Zavanelli, M. I. and Ares, M. (1991) Efficient association of U2 snRNPs with pre-mRNA requires an essential U2 RNA structural element. Genes Dev. 5, 2521–2533.
Cheng, S.-C. and Abelson, J. (1986) Fractionation and characterization of a yeast mRNA splicing extract. Proc. Natl. Acad. Sci. USA 83, 2387–2391.
McPheeters, D. S., Fabrizio, P., and Abelson, J. (1989) In vitro reconstitution of functional yeast U2 snRNPs. Genes Dev. 3, 2124–2136.
Cheng, S. C., Newman, A. N., Lin, R. J., McFarland, G. D., and Abelson, J. N. (1990) Preparation and fractionation of yeast splicing extract. Methods Enzymol. 181, 89–96.
Fabrizio, P., Esser, S., Kastner, B., and Luhrmann, R. (1994) Isolation of S. cerevisiae snRNPs: comparison of U1 and U4/U6. U5 to their human counterparts. Science 264, 261–265.
Cheng, S.-C. and Abelson, J. (1987) Splicesome assembly in yeast. Genes Dev. 1, 1014–1027.
Werner-Washburne, M., Braun, E., Johnston, G. C., and Singer, R. A. (1993) Stationary phase in the yeast Saccharomyces cerevisiae. Microbiol. Rev. 57, 383–401.
Vijayraghavan, U., Company, M., and Abelson, J. (1989) Isolation and characterization of pre-mRNA splicing mutants of Saccharyomces cerevisiae. Genes Dev. 3, 1206–1216.
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Ruby, S.W. (1999). Yeast Pre-mRNA Splicing Extracts. In: Haynes, S.R. (eds) RNA-Protein Interaction Protocols. Methods in Molecular Biology™, vol 118. Humana Press. https://doi.org/10.1385/1-59259-676-2:323
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DOI: https://doi.org/10.1385/1-59259-676-2:323
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