Introduction: The nonsense-mediated mRNA decay (NMD) is a quality-control mechanism that selectively detects and degrades mRNA species carrying premature termination codons (PTCs) resulting from nonsense mutations, out-of-frame alternative splicing (AS), or transcription errors. Coagulation factor V (FV) is encoded by a 72-kb gene (F5) composed of 25 exons, whose pattern of splicing was here analyzed in detail for the first time. Methods: A 5097-bp F5 region was PCR amplified and inserted into the pTARGET vector, and transiently transfected in HeLa cells. Total RNA was isolated and RT-PCR amplified. NMD was studied by real-time RT-PCR on RNA extracted from HepG2 cells treated with three known inhibitors of NMD. Long-range PCR was used to further analyze F5 splicing pattern. Results: Mutational screening of F5 in a FV-deficient patient identified the splicing mutation IVS8+6T>C. To demonstrate its pathogenic role, transfections of F5 minigene constructs (either wild type or mutant) were carried out. RT-PCR analysis demonstrated that IVS8+6T>C causes the skipping of exon 8 (D8). Interestingly, D8 was detected also in the wild-type. Subsequent RT-PCR experiments (performed on RNA from human liver and HepG2 cells) demonstrated that the exon-8 skipping takes place constitutively. Since D8 F5 mRNA bears a PTC, we investigated whether this transcript is subjected to NMD degradation. The obtained results confirmed the involvement of NMD in the degradation of F5 PTC+ mRNAs. On the basis of the identification of a physiologic AS, F5 splicing pattern was further analyzed, leading to the identification of 2 additional splicing variants, resulting from the skipping of exons 3 and 22+23. Conclusions: In conclusion we present the identification of the first AS in F5. Since it has been proposed that the coupling of AS and NMD could be a way to regulate gene expression, we suggest that this mechanism could act also on the F5 gene.

Alternative splicing and nonsense-mediated decay in the F5 gene / C. Dall’Osso, S. Duga, N. Locatelli, F. Peyvandi, M.L. Tenchini, R. Asselta. - In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS. - ISSN 1538-7933. - 5:suppl. 2(2007), p. O-W-089. ((Intervento presentato al 21. convegno Congress of the International Society on Thrombosis and Haemostasis tenutosi a Ginevra nel 2007.

Alternative splicing and nonsense-mediated decay in the F5 gene

S. Duga
Secondo
;
N. Locatelli;F. Peyvandi;M.L. Tenchini
Penultimo
;
R. Asselta
Ultimo
2007

Abstract

Introduction: The nonsense-mediated mRNA decay (NMD) is a quality-control mechanism that selectively detects and degrades mRNA species carrying premature termination codons (PTCs) resulting from nonsense mutations, out-of-frame alternative splicing (AS), or transcription errors. Coagulation factor V (FV) is encoded by a 72-kb gene (F5) composed of 25 exons, whose pattern of splicing was here analyzed in detail for the first time. Methods: A 5097-bp F5 region was PCR amplified and inserted into the pTARGET vector, and transiently transfected in HeLa cells. Total RNA was isolated and RT-PCR amplified. NMD was studied by real-time RT-PCR on RNA extracted from HepG2 cells treated with three known inhibitors of NMD. Long-range PCR was used to further analyze F5 splicing pattern. Results: Mutational screening of F5 in a FV-deficient patient identified the splicing mutation IVS8+6T>C. To demonstrate its pathogenic role, transfections of F5 minigene constructs (either wild type or mutant) were carried out. RT-PCR analysis demonstrated that IVS8+6T>C causes the skipping of exon 8 (D8). Interestingly, D8 was detected also in the wild-type. Subsequent RT-PCR experiments (performed on RNA from human liver and HepG2 cells) demonstrated that the exon-8 skipping takes place constitutively. Since D8 F5 mRNA bears a PTC, we investigated whether this transcript is subjected to NMD degradation. The obtained results confirmed the involvement of NMD in the degradation of F5 PTC+ mRNAs. On the basis of the identification of a physiologic AS, F5 splicing pattern was further analyzed, leading to the identification of 2 additional splicing variants, resulting from the skipping of exons 3 and 22+23. Conclusions: In conclusion we present the identification of the first AS in F5. Since it has been proposed that the coupling of AS and NMD could be a way to regulate gene expression, we suggest that this mechanism could act also on the F5 gene.
Settore BIO/11 - Biologia Molecolare
Settore MED/09 - Medicina Interna
Settore BIO/13 - Biologia Applicata
International Society on Thrombosis and Haemostasis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/69514
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