Hemostasis, Thrombosis, and Vascular Biology Running head: Determinants of 3’end formation of the prothrombin (F2) pre mRNA The prothrombin 3’end formation signal reveals a unique architecture that is sensitive to thrombophilic gain-of-function mutations

The functional analysis of the common prothrombin 20210 G A (F2 20210*A) mutation has recently revealed gain-of-function of 3’end processing as a novel genetic mechanism predisposing to human disease. We now show that the physiological G at the cleavage site at position 20210 is the functionally least efficient nucleotide to support 3’end processing but has evolved to be physiologically optimal. Furthermore, the F2 3’end processing signal is characterized by a weak downstream CstF binding site with a low uridine density and the functional efficiency of F2 3’end processing can be enhanced by the introduction of additional uridine-residues. The recently identified thrombosis related mutation (F2 20221*T) within the CstF binding site up-regulates F2 3’end processing and prothrombin biosynthesis in vivo. F2 20221*T thus represents the first example of a likely pathologically relevant mutation of the putative CstF binding site in the 3’flanking sequence of a human gene. Finally, we show that the low-efficiency F2 cleavage and CstF binding sites are balanced by a stimulatory upstream uridine-rich element in the 3’UTR. The architecture of the F2 3’end processing signal is thus characterized by a delicate balance of positive and negative signals. This balance appears to be highly susceptible to be disturbed by clinically relevant gain-of-function mutations.