The largest human gene (2400 kb) encodes dystrophin. This gene would require ~16 h to be transcribed, and it was demonstrated that its pre-mRNA is spliced cotranscriptionally (Tennyson et al. 1995
). Cotranscriptional splicing appears here as a very intuitive concept. In fact, it would be very difficult to conceive that the splicing of the dozens of dystrophin introns would "wait" until the synthesis of a huge 2400-kb pre-mRNA substrate molecule were completed. In agreement with this observation, direct visualization of nascent transcripts of early Drosophila embryo genes by electron microscopy clearly demonstrated that splicing occurs cotranscriptionally with a reasonable frequency and that splice site selection precedes polyadenylation (Beyer and Osheim 1988). Nevertheless most biology (and even molecular biology) textbooks keep showing drawings in which a fully transcribed primary transcript, with all its introns, appears as the substrate for splicing (Fig. 1). Indeed, for years gene transcription and pre-mRNA processing were thought to be independent events until a series of biochemical, cytological, and functional experiments demonstrated that all three processing reactions (capping, splicing, and cleavage/polyadenylation) can be tightly coupled to RNA polymerase II (pol II) transcription (excellent reviews have been recently published by Bentley 2002; Howe 2002; Maniatis and Reed 2002; Neugebauer 2002; Proudfoot et al. 2002; Proudfoot 2003).
In the case of splicing, cotranscriptionality seems to be a reasonable prerequisite for coupling, but the existence of cotranscriptionality per se does not necessarily imply that transcription and pre-mRNA splicing are coupled. It is worth noting that cotranscriptional splicing is not obligatory. As Karla Neugebauer pointed out clearly in her recent review, "...the time that it takes for pol II to synthesize each intron defines a minimal time and distance along the gene in which splicing factors can be recruited and spliceosomes formed. The time that it takes for pol II to reach the end of the transcription unit defines the maximal time in which splicing could occur cotranscriptionally..." (Neugebauer 2002). In a long gene, for example, some introns could be spliced out cotranscriptionally, whereas others could be processed well after transcription has been completed. In most cases, we do not know which introns follow each pattern. We do not even know if a particular intron always follows the same pattern of processing. In certain cases, the position of the intron along the gene seems to be relevant to the excision pattern. For example, in the Balbiani ring 1 (BR1) gene, intron 3, located 3 kb from the 5'-end of the 40-kb pre-mRNA, is excised cotranscriptionally. However, intron 4, located 0.6 kb from the poly(A) site, is excised cotranscriptionally in ~10% of the molecules, but posttranscriptionally in the remaining molecules (Bauren and Wieslander 1994). Further studies on another Balbiani ring gene, BR3, allowed the investigators to propose that spliceosomes assemble rapidly as introns appear in the pre-mRNA, but intron-specific constraints result in cotranscriptional excision of some introns, preferentially those located in the 5'-part of the primary transcript, and posttranscriptional excision of other introns, preferentially those located in the 3'-part. (Wetterberg et al. 1996). It is worth noting that if splicing were strictly cotranscriptional, that is, if the elimination of one intron would necessarily be completed before the transcription of the following intron has begun, widely distributed mechanisms such as exon definition (Robberson et al. 1990) or alternative splicing by exon skipping would simply not exist.
This review focuses on discussing the evidence supporting the existence of functional links between transcription and splicing. Both processes are extremely complex, involving thousands of protein factors, RNA molecules, and DNA sequences. The added complexity of both processes probably hinders any attempt at generalization and simplification. The reader should bear in mind that certain molecular interactions or kinetic constraints might be relevant for a particular gene or set of genes but not for others. Part of the evidence discussed, although robust, is rather indirect. Nevertheless, because the regulation of transcription and splice site selection are paramount events in eukaryotic cell regulation and differentiation, such indirect evidence sets the necessary framework for more direct investigation in a dynamic emerging field.
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