Functional association between promoter structure and transcript alternative splicing

Abstract

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...

E2F1 controls alternative splicing pattern of genes involved in apoptosis through upregulation of the splicing factor SC35

“…It has now emerged from the literature that splicing not only depends on the interaction of splicing factors with their target pre-mRNAs, but is also coupled to transcription. 25 Indeed, variations of pol II promoter structure can lead to differences in alternative splicing of the transcript [26][27] and components of the spliceosome such as the p54nrb (p54 nuclear RNAbinding protein) and PSF (polypyrimidine tract-binding protein-associated splicing factor) RNA-binding proteins are involved in both transcription and splicing processes. 28 (for review).…”

“…The anti-E2F1 (C-20), anti-SC35 (H-55) and anti-Bcl-x L (H5) antibodies were purchased from Santa Cruz, the anti-Bcl-x S (Ab-1) from Oncogene Research, the anti-E2F1 (KH95) and anti-procaspase-3 from Pharmingen, the anti-FLIP (NF6) from Alexis, the anti-actin (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33) from Sigma, the anti-SC35 (4F-11) from Euromedex and the anti-SRp20 (7B4) and anti-SF2/ASF from Zymed. Cleaved caspase-3 (Asp175) was from Cell Signaling.…”

BARX2 and estrogen receptor-α (ESR1) coordinately regulate the production of alternatively spliced ESR1 isoforms and control breast cancer cell growth and invasion

“…This indicates that BARX2 can bind directly to upstream promoters E1 and F, whereas ESR1 is recruited to promoter F primarily. As promoter use is known to affect alternative splicing events (Cramer et al, 1997(Cramer et al, , 1999, the differential binding of BARX2 and ESR1 to the E1 and F promoters of ESR1 might function to regulate the splicing events that determine which ESR1 isoform is produced.…”

Targeted disruption of maebl in Plasmodium falciparum

“…This strong bidirectional promoter is likely driving mRNA synthesis of the 3′ end of maebl after the disruption instead of the resident maebl promoter, which would be over 20,000 nt upstream [14]. In higher eukaryotes, transcription and pre-mRNA processing are functionally linked so analogous experimental changes in promoter structure have been shown to alter splicing patterns, presumably through altering associated co-factors responsible for splicing [15][16][17][18]. Our results show that definition of exon structure, including recognition of suboptimal alternative splicing junctions, occurs independently of promoter usage.…”