Abstract
The development of in vitro assays to analyze pre-mRNA splicing resulted in the discovery of many fundamental features characterizing splicing signals and the machinery that completes this process. Because in vitro assays can be manipulated by various biochemical approaches, the versatility of investigating alternative pre-mRNA splicing in the test tube appears endless. Importantly, modifications in reaction conditions can lead to the accumulation, isolation, and characterization of reaction intermediates, a prerequisite for gaining mechanistic insights into how the spliceosome carries out intron removal, and how regulatory elements assist the general splicing machinery in defining splice sites and alternative exons. These considerable experimental advantages have made the in vitro splicing system a standard assay, even though this approach is independent from RNA transcription and other RNA processing events, and in some respects deviates from the natural process of mRNA biogenesis. Here, we describe the tools and techniques necessary to carry out in vitro splicing assays. Analyses of various experimental designs are presented to highlight the approaches taken to gain insights into the mechanisms by which splice site recognition and activation are communicated with the general splicing machinery. Methods to measure the kinetics of splicing, to observe the formation of the pre-spliceosomal complexes, and to manipulate and modify the in vitro system to resolve the regulatory influences in alternative splicing are presented.
Highlights
The analysis of adenovirus gene expression demonstrated that intron removal occurs post-transcriptionally at the pre-mRNA level [1,2,3]
The use of the in vitro splicing assay has led to the discovery of many fundamental characteristics of the splicing reaction
The standard in vitro splicing assay limits the investigation to RNA splicing of minigenes uncoupled from transcription and other RNA processing events, the in vitro splicing assay has emerged as a powerful tool to investigate splicing mechanisms, chemistry, and structural rearrangements of the spliceosome
Summary
The analysis of adenovirus gene expression demonstrated that intron removal occurs post-transcriptionally at the pre-mRNA level [1,2,3]. It was shown that single gene transcripts resulted in various mRNA products, which later were revealed to be alternative spliced isoforms [4,5,6]. It was the advent of the in vitro splicing reaction using whole cell extract, and exclusively nuclear extract, that eventually led to the description of many fundamental rules that define how exons and introns are recognized and to detailed molecular and structural insights into the machinery that completes this process [7]. H., unpublished] [see Natalizio and Garcia-Blanco in this volume], will allow measurements in a system presumably closer related to the in vivo condition
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