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.
Liu
Xingge
X
Department of Medicine (Hematology-Oncology), University of Pennsylvania School of Medicine and The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Jiang
Yong
Y
Russell
J Eric
JE
D020413:3' Untranslated Regions; D001483:Base Sequence; D056945:Hep G2 Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism; D006801:Humans; D009690:Nucleic Acid Conformation; D038941:Polypyrimidine Tract-Binding Protein / Q000378:metabolism; D011485:Protein Binding; D011516:Prothrombin / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism
D020413:3' Untranslated Regions; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C450006:PTBP1 protein, human; D012333:RNA, Messenger; D038941:Polypyrimidine Tract-Binding Protein; D011516:Prothrombin