Alternative splicing and nonsense-mediated decay in the F5 gene

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.