Coupling of Transcription with Alternative Splicing Paula Cramer, Javier F Cáceres, Demián Cazalla, Sebastián Kadener, Andrés F Muro, Francisco E Baralle, Alberto R Kornblihtt  Molecular Cell  Volume 4, Issue 2, Pages 251-258 (August 1999) DOI: 10.1016/S1097-2765(00)80372-X

Figure 1 The SF2/ASF Effect on Alternative Splicing Depends on Promoter Structure (A) Schemes of the plasmid constructs carrying the different promoters. Empty boxes, human globin sequences; dashed boxes, human FN sequences; black box, Gal5-HIV-2 sequence. Arrows indicate transcription initiation sites. (B) Effect of SF2/ASF on alternative splicing in the context of different promoters. Hep3B cells were transfected with 800 ng of the corresponding minigene plasmid and 200 ng of g10 SF2/ASF wild-type plasmid (even lanes) or pBS SK+ (odd lanes). alpha-gb, human α-globin promoter; wt FN, −220 fragment of wild-type human FN promoter; mut FN, −220 fragment of mutant (CRE−/CCAAT−) human FN promoter. RNA splicing variants were detected by radioactive RT-PCR and analyzed in 6% native polyacrylamide gels. Histograms display the ratios between radioactivity in EDI+ bands and radioactivity in EDI− bands. Numbers below each pair of bars indicate the fold increase of EDI+/EDI− ratios in presence/absence of the SF2/ASF plasmid. (C) Dose response curve of SF2/ASF on alternative splicing of minigenes driven by the α-globin, mutant FN, and HIV-2 promoters in vivo. Hep3B cells were transfected with 800 ng of pSVEDA/FN mutant plasmid, pSVEDAtot, or pSVEDA/Gal5-HIV-2 and varying amounts of g10 SF2/ASF wild-type plasmid: 0 ng, lanes 1, 6, and 11; 25 ng, lanes 2, 7, and 12; 50 ng, lanes 3, 8, and 13; 100 ng, lanes 4, 9, and 14; 200 ng, lanes 5, 10, and 15. Cotransfection of varying amounts of pBS SK+ allowed keeping the same mass of total DNA in every well. Lane 14 has the double amount of both isoforms compared to lanes 11, 12, 13, and 15. Molecular Cell 1999 4, 251-258DOI: (10.1016/S1097-2765(00)80372-X)

Figure 2 Promoter Modulation Is Not Affected by the Amounts of Pre-mRNA nor by the Identity of Its 5′ Noncoding Region (A) Dose response curve of SF2/ASF on alternative splicing of minigenes driven by the α-globin and mutant FN promoters in vivo, in the context of the FN 5′ noncoding regions (squares) or an artificial 5′ noncoding region of the same length (circles). (B) Top: effects of varying the amounts of reporter plasmid on the alternative splicing of the EDI exon elicited by α-globin or mutant FN promoter constructs with or without overexpression of SF2/ASF. Hep3B cells were transfected with the indicated amounts of reporter plasmid (squares, α-globin; circles, mutant FN), without (black) or with (empty) cotransfection with 50 ng of gt10 SF2/ASF plasmid. EDI+/EDI− ratios were determined through RT-PCRs of 23 (dotted lines) or 30 cycles. Bottom: variations in mRNA levels do not affect EDI+/EDI− ratios characteristic of each promoter. Total amounts of mRNAs (black bars) expressed by minigenes were estimated directly by Northern blots, using a probe that also detects endogenous FN mRNA (Cramer et al. 1997). mRNA levels were normalized by dividing the radioactivity of the transfected mRNA bands by the radioactivity of the endogenous FN mRNA bands. Gray bars, EDI+/EDI− ratios. Each independent experiment is indicated by a number. Molecular Cell 1999 4, 251-258DOI: (10.1016/S1097-2765(00)80372-X)

Figure 3 Dose Response Curves of 9G8 and hTra2β on Alternative Splicing of Minigenes Driven by the α-Globin and Mutant FN Promoters Hep3B cells were transfected with 800 ng of pSVEDAtot or pSVEDA/FN mutant plasmids and varying amounts of g10 9G8 (left) or pEGFP-Tra2 (right) plasmids. Amounts of the corresponding plasmids: 0 ng, lanes 1, 6, 11, and 18; 25 ng, lanes 2, 7, 12, and 19; 50 ng, lanes 3, 8, 13, and 20; 100 ng, lanes 4, 9, 14, and 21; 200 ng, lanes 5, 10, 15, 17, 23, and 25; 500 ng, lanes 16 and 24. Molecular Cell 1999 4, 251-258DOI: (10.1016/S1097-2765(00)80372-X)

Figure 4 SF2/ASF and 9G8 Act through the Splicing Enhancer Present in EDI Hep3B cells were transfected with 800 ng plasmid bearing different versions of the α-globin/FN minigene and 200 ng of either pBS SK+ (odd lanes), g10 SF2/ASF wild type (lanes 2, 4, 6, 8, and 10), or g10 9G8 (lanes 12, 14, 16 and 18). Two versions of the minigene differing on the promoter were used: human α-globin promoter (lanes 1–6 and lanes 11–14) or a −220 fragment of mutant (CRE−/CCAAT−) human FN promoter (lanes 7–10 and 15–18) (for schemes of the constructs, see Figure 1). Minigenes carrying intact splicing enhancer (ESE) and splicing silencer (ESS) (wt) were used in lanes 1 and 2, 7 and 8, 11 and 12, and 15 and 16; minigenes carrying mutant ESE and intact ESS (A−) were used in lanes 3 and 4, 13 and 14, and 17 and 18; minigenes carrying intact ESE and mutant ESS (B−) were used in lanes 5 and 6. Histograms display the ratios between radioactivity in EDI+ bands and radioactivity in EDI− bands. Molecular Cell 1999 4, 251-258DOI: (10.1016/S1097-2765(00)80372-X)

Figure 5 Model for Promoter Modulation of SF2/ASF Control of Fibronectin EDI Alternative Splicing Molecular Cell 1999 4, 251-258DOI: (10.1016/S1097-2765(00)80372-X)