Changes in promoter structure and occupation have been shown to modify the splicing pattern of several genes, evidencing a coupling between transcription and alternative splicing. It has been proposed that the promoter effect involves modulation of RNA pol II elongation rates. The C4 point mutation of the Drosophila pol II largest subunit confers on the enzyme a lower elongation rate. Here we show that expression of a human equivalent to Drosophila's C4 pol II in human cultured cells affects alternative splicing of the fibronectin EDI exon and adenovirus E1a pre-mRNA. Most importantly, resplicing of the Hox gene Ultrabithorax is stimulated in Drosophila embryos mutant for C4, which demonstrates the transcriptional control of alternative splicing on an endogenous gene. These results provide a direct proof for the elongation control of alternative splicing in vivo.

It has been assumed that constitutive and regulated splicing of RNA polymerase II transcripts depends exclusively on signals present in the RNA molecule. Here we show that changes in promoter structure strongly affect splice site selection. We investigated the splicing of the ED I exon, which encodes a facultative type III repeat of fibronectin, whose inclusion is regulated during development and in proliferative processes. We used an alternative splicing assay combined with promoter swapping to demonstrate that the extent of ED I splicing is dependent on the promoter structure from which the transcript originated and that this regulation is independent of the promoter strength. Thus, these results provide the first evidence for coupling between alternative splicing and promoter-specific transcription, which agrees with recent cytological and biochemical evidence of coordination between splicing and transcription.Transcriptional activity by RNA polymerase II (pol II) has been shown to occur in association to 20-50 discrete regions within the cell nucleus, known as nuclear speckles. The fact that these clustered domains not only concentrate Poly(A) ϩ -RNAs, but also small nuclear ribonucleoproteins and the nonsmall nuclear ribonucleoprotein splicing factor SC35, suggests that transcription and splicing might not be independent events, but on the contrary, highly coordinated processes both at the functional and structural levels (1).The study of fibronectin (FN) gene transcription confirmed that pre-mRNAs are constrained from free diffusion. FN transcripts are seen accumulated in elongated tracks, spatially coincident with each allele and colocalizing with speckles. Splicing of FN pre-mRNA appears to occur directly within the track, as evidenced by the fluorescence in situ hybridization detection of intron-containing transcripts in a spatially restricted region proximal to the gene. Each track can be considered as an assembly line where transcription and splicing take place sequentially, exhibiting functional and structural association (2).These observations raise the question whether the intimate spatial association between transcription and splicing also implies influence of one process over the other. Misteli et al. (3) showed that splicing factors are recruited to restricted regions of the nucleus where transcription takes place, thus it becomes important to determine whether cell-specific regulatory transcription factors play a role in splicing. Extending these views we decided to investigate whether modifications in RNA pol II promoter architecture could influence alternative splicing of the ED I exon, which encodes a facultative type III repeat of FN (4-5). Alternative splicing of this exon varies during embryo development and aging, in different adult cell types and in proliferative processes such as healing (6). Inclusion of ED I depends on the presence of a 81-bp sequence, known as ''splicing enhancer'' or SE, located within the central region of the exon (7). The SE markedly stimulates the use...