Abstract
The in vitro splicing assay is a valuable technique that can be used to study the mechanism and machinery involved in the splicing process. The ability to investigate various aspects of splicing and alternative splicing appears to be endless due to the flexibility of this assay. Here, we describe the tools and techniques necessary to carry out an in vitro splicing assay. Through the use of radiolabeled pre-mRNA and crude nuclear extract, spliced mRNAs can be purified and visualized by autoradiography for downstream analysis.
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
Cooper TA (2005) Use of minigene systems to dissect alternative splicing elements. Methods 37:331–340
Beyer AL, Osheim YN (1988) Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. Genes Dev 2:754–765
Hicks MJ, Lam BJ, Hertel KJ (2005) Analyzing mechanisms of alternative pre-mRNA splicing using in vitro splicing assays. Methods 37:306–313
Hernandez N, Keller W (1983) Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts. Cell 35:89–99
Padgett RA, Hardy SF, Sharp PA (1983) Splicing of adenovirus RNA in a cell-free transcription system. Proc Natl Acad Sci USA 80:5230–5234
Hardy SF, Grabowski PJ, Padgett RA et al (1984) Cofactor requirements of splicing of purified messenger RNA precursors. Nature 308:375–377
Lee KA, Bindereif A, Green MR (1988) A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene Anal Tech 5:22–31
Reichert V, Moore MJ (2000) Better conditions for mammalian in vitro splicing provided by acetate and glutamate as potassium counterions. Nucleic Acids Res 28:416–423
Krainer AR, Maniatis T, Ruskin B, Green MR (1984) Normal and mutant human β-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell 36:993–1005
Furdon PJ, Kole R (1986) Inhibition of splicing but not cleavage at the 5′ splice site by truncating human beta-globin pre-mRNA. Proc Natl Acad Sci USA 83:927–931
Dirksen ML, Chrambach A (1972) Studies on the redox state in polyacrylamide gels. Separ Sci 7:747–772
Gelfi C, Righetti PG (1981) Polymerization kinetics of polyacrylamide gels. I. Effect of different cross-linkers. Electrophoresis 2: 213–219
Righetti PG, Gelfi C, Bosisio AB (1981) Polymerization kinetics of polyacrylamide gels. III. Effect of catalysts. Electrophoresis 2:291–295
Rio DC, Ares M, Hannon GJ, Nilsen TW (2010) Polyacrylamide gel electrophoresis of RNA. Cold Spring Harb Protoc 2010:1–6
Voytas D, Ke N (1999) Current protocols in molecular biology – detection and quantitation of radiolabeled proteins and DNA in gels and blots. Curr Protoc Mol Biol 48:A.3A.1–A.3A.10
Johnston RF, Pickett SC, Barker DL (1990) Autoradiography using storage phosphor technology. Electrophoresis 11:355–360
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 29:1072–1082
Acknowledgment
Research in the Hertel laboratory is supported by NIH (RO1 GM62287 and R21 CA149548).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Movassat, M., Mueller, W.F., Hertel, K.J. (2014). In Vitro Assay of Pre-mRNA Splicing in Mammalian 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_11
Download citation
DOI: https://doi.org/10.1007/978-1-62703-980-2_11
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-979-6
Online ISBN: 978-1-62703-980-2
eBook Packages: Springer Protocols