A potential regulatory role for mRNA secondary structures within the prothrombin 3′UTR

The distal 3′UTR of prothrombin mRNA exhibits significant sequence heterogeneity reflecting an inexact 3′ cleavage/polyadenylation reaction. This same region encompasses a single-nucleotide polymorphism that enhances the normal post-transcriptional processing of nascent prothrombin transcripts. Both observations indicate the importance of 3′UTR structures to physiologically relevant properties of prothrombin mRNA. Using a HepG2-based model system, we mapped both the primary structures of reporter mRNAs containing the prothrombin 3′UTR, as well as the secondary structures of common, informative 3′UTR processing variants. A chromatographic method was subsequently employed to assess the effects of structural heterogeneities on the binding of candidate trans -acting regulatory factors. We observed that prothrombin 3′UTRs are constitutively polyadenylated at seven or more positions, and can fold into at least two distinct stem-loop conformations. These alternate structures expose/sequester a consensus binding site for hnRNP-I/PTB-1, a trans -acting factor with post-transcriptional regulatory properties. hnRNP-I/PTB-1 exhibits different affinities for the alternate 3′UTR secondary structures in vitro , predicting a corresponding regulatory role in vivo . These analyses demonstrate a critical link between the structure of the prothrombin 3′UTR and its normal function, providing a basis for further investigations into the molecular pathophysiology of naturally occurring polymorphisms within this region.