Molecular Mechanism of Prothrombin G20210A Variant: Critical New Role of Exon Splicing Enhancer in Poly (A) Tailing.

Although prothrombin G20210A variant was first found in 1996 as a genetic risk factor of thrombosis, the underlying molecular mechanism for elevation of circulatory prothrombin levels due to the variant has remained elusive. The position 20210 is at the poly (A) tailing site, and several research groups reported different, contradictive possibilities including enhancement in poly (A) tailing, change in RNA stability or no difference in prothrombin mRNA level between G (wild type) and A (variant) at position 20210. Such confusions appear to be due to no reliable assay model availability. To overcome these problems, we developed a minigene assay system composed of only autologous components of human prothrombin gene, including its promoter, coding regions, introns and 3′UTR with poly (A) signal.

A minigene containing the first intron and the last intron did not express at any appreciable levels, failing proper splicing of the last intron. Inclusion of upstream other introns was found essential for its appropriate splicing. Although it is generally accepted that the last intron enhances the efficiency of poly (A) tailing, we found that it is not the case for the prothrombin gene. Instead, an exon-splicing enhancer (ESE) of another upstream intron was found to be responsible for efficient poly (A) tailing. ESE, therefore, plays a critical role not only in alternative splicing but also in constitutive RNA processing, including poly (A) tailing. Etiology of prothrombin G20210A variant studied with culture cells and transgenic mice, supported an enhancement of poly (A) tailing, and not a change in mRNA stability.