Findings that splicing factors increase transcriptional elongation (Fong and Zhou 2002) and that introns are necessary for efficient pol II transcription (Furger et al. 2002) suggest that the strong connection between transcription and splicing might be the consequence of a combination of both recruitment of factors to the sites of transcription and elongation control by RNA pol II. Although evidence for the elongation mechanism is stronger in terms of the variety of molecular approaches that support it, certain data allow us to speculate that recruitment and elongation might be interconnected. For instance, the CTD is preferentially phosphorylated at Ser 5 when pol II is recruited at promoter sites but becomes phosphorylated at Ser 2 when located at the coding region (Cho et al. 2001). This change in phosphorylation quality might be relevant for the recruitment of splicing factors. Simultaneously, it would be important to determine whether a pausing pol II has the same phosphorylation status and recruitment properties of a fast-elongating pol II. Chromatin immunoprecipitation experiments with antibodies to different kinds of phosphopol II and characterization of protein complexes throughout different segments of transcribed regions should help to test the combined hypothesis.
Studies on the yeast Spt5 factor also suggest a combined mechanism. This factor has been proposed to regulate pol II elongation through nucleosomes. General elongation factors such as TFIIF and TFIIS coimmunopurify with Spt5, which, in turn, is able to interact with capping enzymes. Lindstrom et al. (2003) found that spt5 mutations lead to accumulation of unspliced pre-mRNAs. Such an inhibition of splicing may occur because splicing factors fail to interact with the transcription machinery in the absence of Spt5.
A complex panorama emerges when trying to summarize the factors involved in the regulation of splice site selection. On the "cis" side, we should take into account not only the specific sequences acting at the RNA level (splice sites, splicing enhancers and silencers, and determinants of pre-mRNA secondary structures) but also those acting at the DNA level such as promoters, transcriptional enhancers, and the pol II pausing architecture of a gene. On the "trans" side, the abundance, cell localization, and phosphorylation state of SR and hnRNP proteins should be complemented with those of transcription factors, coactivators, chromatin factors, CTD kinases, transcriptional elongation factors, and factors with dual activities in both transcription and splicing.
Alternative splicing has been associated with increased evolutionary change in vertebrates. Comparative genomic analysis has shown that whereas constitutive exons are strongly conserved in the mouse and human genomes, alternative exons are mostly not conserved and are the product of recent exon creation or loss events (Modrek and Lee 2003). Selection of the cis- and trans-acting regulatory factors of splicing, involving multiple links with the transcription machinery, must have occurred concomitantly in a short evolutionary time, contributing to the high adaptive benefit of alternative splicing.