Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon - PubMed (original) (raw)

Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon

Silvia Spena et al. RNA. 2006 Jun.

Abstract

In this work we report the identification of a strong SF2/ASF binding site within exon 7 of the human fibrinogen Bbeta-chain gene (FGB). Its disruption in the wild-type context has no effect on exon recognition. However, when the mutation IVS7 + 1G>T--initially described in a patient suffering from congenital afibrinogenemia--is present, this SF2/ASF binding site is critical for cryptic 5'ss (splice site) definition. These findings, besides confirming and extending previous results regarding the effect of SF2/ASF on cryptic splice site activation, identify for the first time an enhancer sequence in the FGB gene specific for cryptic splice site usage. Taken together, they suggest the existence of a splicing-regulatory network that is normally silent in the FGB natural splicing environment but which can nonetheless influence splicing decisions when local contexts allow. On a more general note, our conclusions have implications for the evolution of alternative splicing processes and for the development of methods to control aberrant splicing in the context of disease-causing mutations.

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Figures

FIGURE 1.

(A) (Upper panel) Schematic diagram of the human FGB minigene spanning from exon 7 to exon 8 (white and black boxes, respectively) indicating the position (drawn to scale) and sequence of the three cryptic splice sites (c1, c2, and c3) used when the natural donor splice site is abolished following the IVS7 + 1G>T mutation (black circle) and of the two potential donor sites (p1 and p2) identified by in silico analysis but never used in vivo. (Lower panel) Individual scores for each of these cryptic and potential splice sites obtained by a panel of donor site prediction programs (NN, MAXENT, MDD, and MM); np denotes not predicted sites. (B) (Upper panel) Outline of the wild-type FGB minigene used in transfection experiments. Rectangles, drawn to scale, indicate exons (gray, exon 6; white, exon 7; and black, exon 8); single lines, introns. Dotted lines indicate splicing products; arrows, the primer couple used to amplify the splicing products by RT-PCR. (Middle panel) FGB genomic region (numbered according to GenBank [accession no. M64983]) containing exon 7 (in uppercase letters) with the mutations (numbered above the sequence) introduced in its wild-type sequence to remove all potential SR binding sites identified by the ESEfinder program. In mutant 1 potential binding sites were disrupted by point mutations (reported below the sequence); in mutants 2–8, by deletion (the deleted sequence is underlined in each case). Sequences of the three cryptic sites (c1, c2, and c3) are bold and shaded in gray. (Lower panel) Effect of all these mutations (lanes 1_–_8) with respect to the wild-type minigene (wt) following transfection of each minigene in HeLa cells. RT-PCR products were run on a 2%-agarose gel electrophoresis. M indicates molecular weight marker (pUC8_Hae_III).

FIGURE 2.

(A) (Upper panel) Schematic diagram of FGB exon 7 with the three sequences (A, B, and C) into which it has been divided. Borders of fragments, drawn to scale, are indicated and numbered according to GenBank (accession no. M64983). (Lower panel) UV cross-linking profiles (panel 1) of the RNAs obtained from the four sequences (7, A, B, and C). The electrophoretic mobility of prestained marker (Broad Range, New England Biolabs) is shown on the left. Immunoprecipitation profiles of each UV-cross-linked sample shown at left, with specific monoclonal antibodies against different SR proteins: SF2/ASF (mAb 96, panel 2), the phosphorylated RS domain (mAb 1H4, panel 3), and SC35 (anti-SC35, panel 4). The mobility of the SR proteins is indicated on the left. (B) Western blot panels of SR proteins bounded to exon 7 resulting to pull-down analysis using coated and uncoated (as control) adipic acid beads. SR proteins were detected by their respective monoclonal antibodies against SF2/ASF (mAb 96, panel 1), the phosphorylated RS domain (mAb 1H4, panel 2), and SC35 (anti-SC35, panel 3). Input represents 20% of the total nuclear extract load during each pull-down experiment. (C) (Left panel) Schematic representation of the three sequences (C1, C2, and C3) used to map the SF2/ASF binding site (hatched) on fragment C of exon 7. Ends of fragments, drawn to scale, are indicated and numbered according to GenBank (accession no. M64983). Sequence of the 36-bp binding region is reported. (Middle panel) Total UV cross-linking analysis of these three RNAs. (Right panel) Immunoprecipitation analysis of these samples with mAb 96 (specific for SF2/ASF).

FIGURE 3.

(A) Agarose (2%) gel electrophoresis of RT-PCR products amplified from total RNA extracted from HeLa cells after transfection either with the wild-type FGB minigene (wt, lane 1), the wild-type minigene carrying the deletion of the SF2/ASF binding region (Δwt, lane 2), the minigene carrying the +1G>T substitution in the natural donor splice site (IVS7 + 1G>T, lane 3), and the minigene carrying this mutation together with the deletion of the SF2/ASF binding region (ΔIVS7 + 1G>T, lane 4). M indicates molecular weight marker (pUC8_Hae_III). (B) (Upper panel) GeneScan Analysis windows of the capillary-electrophoretic runs of the same fluorochrome labeled RT-PCR products showed in A. Molecular weight standard peaks are in gray, while peaks corresponding to the labeled fragments, indicated by arrows, are in black. Lengths of molecular weight fragments (bp) are reported on the left. (Lower panel) Size (bp) and relative amounts (%) of the fluorochrome labeled RT-PCR products indicated in the upper panel, evaluated by means of the GeneScan software. The relative amounts represent the average of three independent experiments with standard deviation values. Levels of c1 and Δc1 products are shaded in gray. (C) Functional specificity of SF2/ASF binding region tested by evaluating the effect of mutations 1–4 (see Fig. 1B, middle panel) on cryptic splice site usage in the IVS7 + 1G>T minigene with respect to the same minigene following transfection of each construct in HeLa cells. RT-PCR products were run on a 2%-agarose gel electrophoresis. M indicates molecular weight marker (pUC8_Hae_III). (D) (Left panel) Schematic representation of the IVS7 + 1G>T minigene used to replace the deleted (Δ) binding region of SF2/ASF by the heterologous fibronectin EDA ESE sequence (EDA hTot) and the deleted EDA ESE sequence (EDA Δ2e) used as a control. Black circle indicates the IVS7 + 1G>T mutation; arrows, the primer couple used to amplify the splicing products by RT-PCR. (Right panel) Agarose (2%) gel electrophoresis of RT-PCR products amplified from total RNA extracted from HeLa cells expressing the heterologous ESE-carrying minigene (ΔIVS7 + 1G>T + EDA hTot) as compared with the effects of the same minigene carrying the GAAGAAGA deletion (ΔIVS7 + 1G>T + EDA Δ2e) and the IVS7 + 1G>T and ΔIVS7 + 1G>T minigenes. The relative amounts (%) of c1 usage for each minigene are indicated on the bottom. M indicates molecular weight marker (pUC8_Hae_III). (E) Schematic diagram of the switch in relative cryptic splice site usage (depicted by thicker dotted lines) following the deletion (Δ) of the SF2/ASF binding sites in the IVS7 + 1G>T and ΔIVS7 + 1G>T constructs.

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Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon - PubMed (original) (raw)