The Physiology of Prothrombin Gene Expression Integrates RNA Polyadenylation and Splicing in a Novel Regulatable 3′ RNP-Complex.

The functional analysis of the common prothrombin (F2) 20210*A allele has recently revealed gain-of-function of 3′end processing as a novel genetic mechanism predisposing to human disease. The general susceptibility of the F2 mRNA for gain-of-function is further exemplified by F2 20209*T and F2 20221*T, and can be explained by an unusual architecture of non-canonical 3′end formation sequence elements: Specifically, the F2 3′ untranslated region (3′UTR) contains a stimulatory upstream sequence element (USE) that compensates for the weak functional activities of the cleavage site and the downstream U-rich element in the F2 3′ flanking sequence.

We now show that the F2 USE promotes 3′end formation in a position- and sequence-dependent manner, stimulating the step of mRNA polyadenylation rather than cleavage, and identify specific proteins that interact with the USE. Unexpectedly, the USE RNP includes splicing factors, components of the 3′end processing machinery and AU-rich sequence element-binding proteins (ARE-BP). We demonstrate that the splicing factors U2AF35 and U2AF65, hnRNPI/PTB, PSF/SFPQ and p54nrb/NonO promote 3′end formation via the USE contained in the 3′UTR uncovering a novel and more general functional link between these splicing factors and mRNA 3′ end formation. We propose a model of USE-directed 3′ end processing that involves a novel mRNP that integrates different nuclear pre-mRNA processing steps. Furthermore, the involvement of ARE-BP in this mRNP reveals an intriguing potential for a post-transcriptional regulation of prothrombin gene expression through external stimuli. Our data thus implicate USE-dependent RNP-complex formation in the regulated physiology of prothrombin gene expression specifically and in hemostasis (and other thrombin-dependent processes) more generally.