In vitro systems for coupling RNAP II transcription to splicing and polyadenylation

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 (32)P-UTP to generate the nascent transcript. Transcription is then stopped by addition of α-amanitin followed by continued incubation to allow RNA processing.