Figure 1. General strategy to study cotranscriptional AS of the fibronectin EDI exon using reporter minigenes transiently transfected in mammalian cells. (a) Overall structure of the human fibronectin gene, showing three regions of AS named, from 5′ to 3′, EDII (also known as EDB), EDI (also known as EDA), and IIICS; (b) A fibronectin/α‐globin hybrid minigene under the control of different promoters (pSVEDA/FN or pSVEDA Tot minigenes bearing fibronectin or α‐globin promoter, respectively) is transfected into mammalian cells as described in Ref. [ 2]; (c) Pre‐mRNA expressed from the minigene is alternatively spliced inside the transfected cells. Total RNA is extracted and subjected to semiquantitative radioactive RT‐PCR using the same pair of PCR primers to amplify both isoforms; (d) RT‐PCR products are separated in native 6% PAGE and identified using autoradiography.

Figure 2. Plasmids used in the inducible minigene reporter system. (a) Scheme of the EDI AS reporter minigene (pUHC‐EDA) under the control of a minimal CMV promoter and seven tetracycline operators; (b) Schemes of the constructs expressing fusion proteins between the tetracycline regulator and the transactivation domains of the eukaryotic transcriptional activators SP1 (left) and VP16 (right). Expression of both constructs is controlled by the constitutive CMV promoter.

Figure 3. Strategy to transiently express AS reporter minigenes using different RNA polymerase II mutants. Cells are cotransfected with the reporter minigene and a plasmid expressing α‐amanitin‐resistant variants of RNA polymerase II large subunit, following the method originally developed in Ref. [ 8]. The Asn 792 Asp of Pol II (pAT7Rpb1 αAmr) mutation confers α‐amanitin resistance. Incubation of the transfected cells with α‐amanitin inhibits endogenous Pol II and allows the minigene to be transcribed by the recombinant Pol II variant.

Figure 4. Effect of a slow (hC4) polymerase on EDI AS. Hep3B cells were transfected with pUHC‐EDA (inducible minigene), plasmids expressing tTA‐VP16, and either a WT (WT ^res) or a slow (hC4) α‐amanitin‐resistant polymerase. AS was assessed by radioactive semiquantitative RT‐PCR, followed by native 6% PAGE. Mean values (± standard deviation) are shown (n = 3) in the histogram.