The prothrombin (F2) 3′ end formation signal is highly susceptible to thrombophilia-associated gain-of-function mutations. In its unusual architecture, the F2 3′ UTR contains an upstream sequence element (USE) that compensates for weak activities of the non-canonical cleavage site and the downstream U-rich element. Here, we address the mechanism of USE function. We show that the F2 USE contains a highly conserved nonameric core sequence, which promotes 3′ end formation in a position- and sequence-dependent manner. We identify proteins that specifically interact with the USE, and demonstrate their function as trans-acting factors that promote 3′ end formation. Interestingly, these include the splicing factors U2AF35, U2AF65 and hnRNPI. We show that these splicing factors not only modulate 3′ end formation via the USEs contained in the F2 and the complement C2 mRNAs, but also in the biocomputationally identified BCL2L2, IVNS and ACTR mRNAs, suggesting a broader functional role. These data uncover a novel mechanism that functionally links the splicing and 3′ end formation machineries of multiple cellular mRNAs in an USE-dependent manner.