Abstract
Studies over the past several years have revealed that steps in gene expression are extensively coupled to one another both physically and functionally. Recently, in vitro systems were developed for understanding the mechanisms involved in coupling transcription by RNA polymerase II to RNA processing. Here we describe an efficient two-way system for coupling transcription to splicing and a robust three-way system for coupling transcription, splicing, and polyadenylation. In these systems a CMV-DNA construct is incubated in HeLa cell nuclear extracts in the presence of 32P-UTP to generate the nascent transcript. Transcription is then stopped by addition of α-amanitin followed by continued incubation to allow RNA processing.
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Acknowledgement
This work was supported by NIH grant GM043375.
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Folco, E.G., Reed, R. (2014). In Vitro Systems for Coupling RNAP II Transcription to Splicing and Polyadenylation. 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_13
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DOI: https://doi.org/10.1007/978-1-62703-980-2_13
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-979-6
Online ISBN: 978-1-62703-980-2
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