A Polar Mechanism Coordinates Different Regions of Alternative Splicing within a Single Gene

Abstract

Alternative splicing plays a key role in generating protein diversity. Transfections with minigenes revealed coordination between two distant, alternatively spliced exons in the same gene. Mutations that either inhibit or stimulate inclusion of the upstream alternative exon deeply affect inclusion of the downstream one. However, similar mutations at the downstream alternative exon have little effect on the upstream one. This polar effect is promoter specific and is enhanced by inhibition of transcriptional elongation. Consistently, cells from mutant mice with either constitutive or null inclusion of a fibronectin alternative exon revealed coordination with a second alternative splicing region, located far downstream. Using allele-specific RT-PCR, we demonstrate that this coordination occurs in cis and is also affected by transcriptional elongation rates. Bioinformatics supports the generality of these findings, indicating that 25% of human genes contain multiple alternative splicing regions and identifying several genes with nonrandom distribution of mRNA isoforms at two alternative regions.

Competing RNA secondary structures are required for mutually exclusive splicing of the Dscam exon 6 cluster

“…However, upon transfection into Drosophila S2 cells, these minigenes did not splice efficiently and did not recapitulate the splicing pattern of the endogenous gene (data not shown). Moreover, given the interplay between the promoter, transcription, and chromatin with splicing (Kornblihtt 2007), as well as potential polar effects, in which the splicing of exons in one portion of a pre-mRNA may influence splicing in other regions (Fededa et al 2005), we felt it critical to use an experimental system that includes the entire Dscam gene and expression was driven by the endogenous Dscam promoter.…”

First come, first served revisited: Factors affecting the same alternative splicing event have different effects on the relative rates of intron removal

“…(B) Mutations introduced in the minigenes shown in A. WT indicates wild-type sequence; GT and DM, G/T and G/T+A/T point mutations, respectively, at the polypyrimidine tract of the 39ss upstream of E33 (Nogues et al 2003); DDRE, deletion of the intronic downstream regulatory element (Gromak et al 2008). The minigenes containing two alternative splicing regions, WT (pFN-EDI WT /IIICS) and DM (pFN-EDI C /IIICS), were previously described (Fededa et al 2005). (C,D) Hep3B cells were transfected with the WT and mutant versions of pUHC-EDA (C) and pFN-EDI/IIICS (D), and analyzed by RT-PCR for E33 inclusion ratios (left), the quantification of E33+/E33À ratios (middle), and the quantification of splicing intermediates (right, black bars) calculated as the %a-intermediate.…”

“…To rule out that the measured %a-intermediate was a consequence of E33 being the penultimate exon within the minigene, an additional control was performed. We used an alternative splicing reporter minigene (pFN-EDI WT /IIICS) (Fededa et al 2005) in which E33 is placed in a position upstream of subsequent constitutive and alternative exons, including the alternative IIICS alternative region ( Fig. 2A, bottom).…”

“…DDRE is based on pUHC-EDA but bears a deletion between sites NcoI-NdeI according to a previous report (Gromak et al 2008). Tandem reporter minigenes bearing a WT EDI exon (pFN-EDI WT /IIICS) or DM EDI exon (pFN-EDI C /IIICS) were previously described (Fededa et al 2005).…”

Distal regulation of alternative splicing by splicing enhancer in equine β-casein intron 1

“…Lately it has been shown that they can act also from a specific weak exon on another weak exon, which are separated by several kilobases (Fededa et al 2005). In this case, their influence is indirect in that they recruit specific splicing activators to the upstream weak exon and these activators are somehow more accessible to the downstream exon.…”