Limits...
SR protein-mediated inhibition of CFTR exon 9 inclusion: molecular characterization of the intronic splicing silencer.

Buratti E, Stuani C, De Prato G, Baralle FE - Nucleic Acids Res. (2007)

Bottom Line: We have also performed a functional analysis of the ISS element using a variety of unrelated SR-binding sequences and different splicing systems.Our results suggest that SR proteins mediate CFTR exon 9 exclusion by providing a 'decoy' sequence in the vicinity of its suboptimal donor site.The results of this study give an insight on intron 'exonization' mechanisms and provide useful indications for the development of novel therapeutic strategies aimed at the recovery of exon inclusion.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB) 34012 Trieste, Italy.

ABSTRACT
The intronic splicing silencer (ISS) of CFTR exon 9 promotes exclusion of this exon from the mature mRNA. This negative influence has important consequences with regards to human pathologic events, as lack of exon 9 correlates well with the occurrence of monosymptomatic and full forms of CF disease. We have previously shown that the ISS element interacts with members of the SR protein family. In this work, we now provide the identification of SF2/ASF and SRp40 as the specific SR proteins binding to this element and map their precise binding sites in IVS9. We have also performed a functional analysis of the ISS element using a variety of unrelated SR-binding sequences and different splicing systems. Our results suggest that SR proteins mediate CFTR exon 9 exclusion by providing a 'decoy' sequence in the vicinity of its suboptimal donor site. The results of this study give an insight on intron 'exonization' mechanisms and provide useful indications for the development of novel therapeutic strategies aimed at the recovery of exon inclusion.

Show MeSH
(A) Shows a schematic diagram of the splicing controlling regions of CFTR exon 9, both within the exon (CERES element) and in the flanking IVS8 and IVS9 sequences: TG(m)T(n), PCE and ISS. h3′int defines the IVS9 region that includes both the PCE and ISS controlling elements. (B) Shows the trans-acting factors identified up to now that bind to these elements. (C) Shows an immunoprecipitation analysis of the h3′int region and of the fibronectin EDA ESE element both in its wild-type (hTot) and mutated form (h▵2e). The left, central and right panels show the immunoprecipitation profiles obtained from each RNA using mAb 96 (specific against SF2/ASF), mAb 1H4 (specific against the phosphorylated RS domain) and an anti-SC35 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1935002&req=5

Figure 1: (A) Shows a schematic diagram of the splicing controlling regions of CFTR exon 9, both within the exon (CERES element) and in the flanking IVS8 and IVS9 sequences: TG(m)T(n), PCE and ISS. h3′int defines the IVS9 region that includes both the PCE and ISS controlling elements. (B) Shows the trans-acting factors identified up to now that bind to these elements. (C) Shows an immunoprecipitation analysis of the h3′int region and of the fibronectin EDA ESE element both in its wild-type (hTot) and mutated form (h▵2e). The left, central and right panels show the immunoprecipitation profiles obtained from each RNA using mAb 96 (specific against SF2/ASF), mAb 1H4 (specific against the phosphorylated RS domain) and an anti-SC35 antibody.

Mentions: The regulation of CFTR exon 9 splicing has been extensively studied in recent years because of its clear connection with CF disease (1–6). At present, several splicing controlling regions have been characterized near the 3′ and 5′ boundaries of this exon. These include a polymorphic TG(m)T(n) region near the 3′ss, a suboptimal donor site and a Polypyrimidine-rich Controlling Element just downstream of the 5′ss (PCE) (7–9). Moreover, additional controlling regions have been identified inside the exon itself in the form of CERES elements (10) and as an intronic splicing silencer region (ISS) further away in the IVS9 intron sequence (11) (Figure 1A). During the course of these studies several trans-acting elements have also been identified as binding specifically to these regulatory elements: TDP-43 to the (TG)m region near the 3′ss of the exon, which has been recently shown to recruit hnRNP proteins near the 3′ss (6,12,13), TIA-1 to the PCE that promotes exon inclusion (9), and unidentified members of the SR protein family to the ISS sequence (11) (Figure 1B).Figure 1.


SR protein-mediated inhibition of CFTR exon 9 inclusion: molecular characterization of the intronic splicing silencer.

Buratti E, Stuani C, De Prato G, Baralle FE - Nucleic Acids Res. (2007)

(A) Shows a schematic diagram of the splicing controlling regions of CFTR exon 9, both within the exon (CERES element) and in the flanking IVS8 and IVS9 sequences: TG(m)T(n), PCE and ISS. h3′int defines the IVS9 region that includes both the PCE and ISS controlling elements. (B) Shows the trans-acting factors identified up to now that bind to these elements. (C) Shows an immunoprecipitation analysis of the h3′int region and of the fibronectin EDA ESE element both in its wild-type (hTot) and mutated form (h▵2e). The left, central and right panels show the immunoprecipitation profiles obtained from each RNA using mAb 96 (specific against SF2/ASF), mAb 1H4 (specific against the phosphorylated RS domain) and an anti-SC35 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935002&req=5

Figure 1: (A) Shows a schematic diagram of the splicing controlling regions of CFTR exon 9, both within the exon (CERES element) and in the flanking IVS8 and IVS9 sequences: TG(m)T(n), PCE and ISS. h3′int defines the IVS9 region that includes both the PCE and ISS controlling elements. (B) Shows the trans-acting factors identified up to now that bind to these elements. (C) Shows an immunoprecipitation analysis of the h3′int region and of the fibronectin EDA ESE element both in its wild-type (hTot) and mutated form (h▵2e). The left, central and right panels show the immunoprecipitation profiles obtained from each RNA using mAb 96 (specific against SF2/ASF), mAb 1H4 (specific against the phosphorylated RS domain) and an anti-SC35 antibody.
Mentions: The regulation of CFTR exon 9 splicing has been extensively studied in recent years because of its clear connection with CF disease (1–6). At present, several splicing controlling regions have been characterized near the 3′ and 5′ boundaries of this exon. These include a polymorphic TG(m)T(n) region near the 3′ss, a suboptimal donor site and a Polypyrimidine-rich Controlling Element just downstream of the 5′ss (PCE) (7–9). Moreover, additional controlling regions have been identified inside the exon itself in the form of CERES elements (10) and as an intronic splicing silencer region (ISS) further away in the IVS9 intron sequence (11) (Figure 1A). During the course of these studies several trans-acting elements have also been identified as binding specifically to these regulatory elements: TDP-43 to the (TG)m region near the 3′ss of the exon, which has been recently shown to recruit hnRNP proteins near the 3′ss (6,12,13), TIA-1 to the PCE that promotes exon inclusion (9), and unidentified members of the SR protein family to the ISS sequence (11) (Figure 1B).Figure 1.

Bottom Line: We have also performed a functional analysis of the ISS element using a variety of unrelated SR-binding sequences and different splicing systems.Our results suggest that SR proteins mediate CFTR exon 9 exclusion by providing a 'decoy' sequence in the vicinity of its suboptimal donor site.The results of this study give an insight on intron 'exonization' mechanisms and provide useful indications for the development of novel therapeutic strategies aimed at the recovery of exon inclusion.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB) 34012 Trieste, Italy.

ABSTRACT
The intronic splicing silencer (ISS) of CFTR exon 9 promotes exclusion of this exon from the mature mRNA. This negative influence has important consequences with regards to human pathologic events, as lack of exon 9 correlates well with the occurrence of monosymptomatic and full forms of CF disease. We have previously shown that the ISS element interacts with members of the SR protein family. In this work, we now provide the identification of SF2/ASF and SRp40 as the specific SR proteins binding to this element and map their precise binding sites in IVS9. We have also performed a functional analysis of the ISS element using a variety of unrelated SR-binding sequences and different splicing systems. Our results suggest that SR proteins mediate CFTR exon 9 exclusion by providing a 'decoy' sequence in the vicinity of its suboptimal donor site. The results of this study give an insight on intron 'exonization' mechanisms and provide useful indications for the development of novel therapeutic strategies aimed at the recovery of exon inclusion.

Show MeSH