Abstract
Kinetic analysis of in vitro splicing is a valuable technique for understanding splicing regulation. It allows the determination of specific contributions from functional elements for the efficient removal of introns. This chapter will describe the rationale and approach employed to use kinetic analysis to evaluate an in vitro splicing reaction using radiolabeled pre-mRNA incubated in splicing-competent HeLa nuclear extract (NE).
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References
Black DL, Chabot B, Steitz JA (1985) U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell 42(3):737–750
Tarn WY, Steitz JA (1994) SR proteins can compensate for the loss of U1 snRNP functions in vitro. Genes Dev 8(22):2704–2717
Tarn WY, Steitz JA (1995) Modulation of 5′ splice site choice in pre-messenger RNA by two distinct steps. Proc Natl Acad Sci USA 92(7):2504–2508
Krainer AR, Maniatis T, Ruskin B et al (1984) Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell 36(4):993–1005
Ruskin B, Krainer AR, Maniatis T et al (1984) Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell 38(1):317–331
Kotlajich MV, Crabb TL, Hertel KJ (2009) Spliceosome assembly pathways for different types of alternative splicing converge during commitment to splice site pairing in the A complex. Mol Cell Biol 29(4):1072–1082. doi:10.1128/MCB.01071-08, MCB.01071-08 [pii]
Hicks MJ, Lam BJ, Hertel KJ (2005) Analyzing mechanisms of alternative pre-mRNA splicing using in vitro splicing assays. Methods 37(4):306–313
Zhou Z, Sim J, Griffith J, Reed R (2002) Purification and electron microscopic visualization of functional human spliceosomes. Proc Natl Acad Sci USA 99(19):12203–12207
Deckert J, Hartmuth K, Boehringer D et al (2006) Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions. Mol Cell Biol 26(14):5528–5543. doi:10.1128/MCB.00582-06, 26/14/5528 [pii]
Hicks MJ, Mueller WF, Shepard PJ et al (2010) Competing upstream 5′ splice sites enhance the rate of proximal splicing. Mol Cell Biol 30(8):1878–1886. doi:10.1128/MCB.01071-09, MCB.01071-09 [pii]
Audibert A, Weil D, Dautry F (2002) In vivo kinetics of mRNA splicing and transport in mammalian cells. Mol Cell Biol 22(19): 6706–6718
Kornblihtt AR, de la Mata M, Fededa JP et al (2004) Multiple links between transcription and splicing. RNA 10(10):1489–1498
Hicks MJ, Yang CR, Kotlajich MV et al (2006) Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patterns. PLoS Biol 4(6):e147
Erkelenz S, Mueller WF, Evans MS et al (2013) Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms. RNA 19(1):96–102. doi:10.1261/rna.037044.112
Lam BJ, Bakshi A, Ekinci FY et al (2003) Enhancer-dependent 5′-splice site control of fruitless pre-mRNA splicing. J Biol Chem 278(25):22740–22747. doi:10.1074/jbc.M301036200, M301036200 [pii]
Lam BJ, Hertel KJ (2002) A general role for splicing enhancers in exon definition. RNA 8(10):1233–1241
Hertel KJ, Maniatis T (1999) Serine-arginine (SR)-rich splicing factors have an exon-independent function in pre-mRNA splicing. Proc Natl Acad Sci USA 96(6): 2651–2655
Fox-Walsh KL, Dou Y, Lam BJ et al (2005) The architecture of pre-mRNAs affects mechanisms of splice-site pairing. Proc Natl Acad Sci USA 102(45):16176–16181
Lim SR, Hertel KJ (2004) Commitment to splice site pairing coincides with A complex formation. Mol Cell 15(3):477–483
Acknowledgement
Research in the Hertel laboratory is supported by NIH (RO1 GM62287 and R21 CA149548).
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Mueller, W.F., Hertel, K.J. (2014). Kinetic Analysis of In Vitro Pre-mRNA Splicing in HeLa Nuclear Extract. In: Hertel, K. (eds) Spliceosomal Pre-mRNA Splicing. Methods in Molecular Biology, vol 1126. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-980-2_12
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DOI: https://doi.org/10.1007/978-1-62703-980-2_12
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Publisher Name: Humana Press, Totowa, NJ
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