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.

[1]  W. Keller,et al.  Human Fip1 is a subunit of CPSF that binds to U‐rich RNA elements and stimulates poly(A) polymerase , 2004, The EMBO journal.

[2]  N. von Ahsen,et al.  The intronic prothrombin 19911A>G polymorphism influences splicing efficiency and modulates effects of the 20210G>A polymorphism on mRNA amount and expression in a stable reporter gene assay system. , 2004, Blood.

[3]  C. C. Spaargaren-van Riel,et al.  G20210A is a functional mutation in the prothrombin gene; effect on protein levels and 3′‐end formation , 2004, Journal of thrombosis and haemostasis : JTH.

[4]  B. Kosova,et al.  Budd–Chiari syndrome in a patient heterozygous for the point mutation C20221T of the prothrombin gene , 2003, Journal of thrombosis and haemostasis : JTH.

[5]  M. Hentze,et al.  Y14 and hUpf3b form an NMD-activating complex. , 2003, Molecular cell.

[6]  T. Cooper,et al.  Pre-mRNA splicing and human disease. , 2003, Genes & development.

[7]  Jeffrey Wilusz,et al.  Upstream Elements Present in the 3′-Untranslated Region of Collagen Genes Influence the Processing Efficiency of Overlapping Polyadenylation Signals* , 2002, The Journal of Biological Chemistry.

[8]  Bosiljka Tasic,et al.  Alternative pre-mRNA splicing and proteome expansion in metazoans , 2002, Nature.

[9]  E. Pollak,et al.  The G20210A mutation does not affect the stability of prothrombin mRNA in vivo. , 2002, Blood.

[10]  A. Shyu,et al.  RNA surveillance by nuclear scanning? , 2002, Nature Cell Biology.

[11]  Kathryn A. O’Donnell,et al.  An mRNA Surveillance Mechanism That Eliminates Transcripts Lacking Termination Codons , 2002, Science.

[12]  M. Hentze,et al.  Abnormally spliced beta-globin mRNAs: a single point mutation generates transcripts sensitive and insensitive to nonsense-mediated mRNA decay. , 2002, Blood.

[13]  M. O’Connell,et al.  The many roles of an RNA editor , 2001, Nature Reviews Genetics.

[14]  Matthias W. Hentze,et al.  Increased efficiency of mRNA 3′ end formation: a new genetic mechanism contributing to hereditary thrombophilia , 2001, Nature Genetics.

[15]  K. Wielckens,et al.  A Novel Point Mutation in the 3’ Region of the Prothrombin Gene at Position 20221 in a Lebanese/Syrian Family , 2001, Thrombosis and Haemostasis.

[16]  L. Almasy,et al.  Linkage analysis demonstrates that the prothrombin G20210A mutation jointly influences plasma prothrombin levels and risk of thrombosis , 2000 .

[17]  N. Proudfoot,et al.  Recruitment of a Basal Polyadenylation Factor by the Upstream Sequence Element of the Human Lamin B2 Polyadenylation Signal , 2000, Molecular and Cellular Biology.

[18]  J. Manley,et al.  Functional interaction of BRCA1-associated BARD1 with polyadenylation factor CstF-50. , 1999, Science.

[19]  Jing Zhao,et al.  Formation of mRNA 3′ Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis , 1999, Microbiology and Molecular Biology Reviews.

[20]  M. Hentze,et al.  A Perfect Message RNA Surveillance and Nonsense-Mediated Decay , 1999, Cell.

[21]  C. Dieckmann,et al.  Regulation of poly(A) site choice of several yeast mRNAs. , 1998, Nucleic acids research.

[22]  M. Wollerton,et al.  The upstream sequence element of the C2 complement poly(A) signal activates mRNA 3' end formation by two distinct mechanisms. , 1998, Genes & development.

[23]  M. Hentze,et al.  Binary specification of nonsense codons by splicing and cytoplasmic translation , 1998, The EMBO journal.

[24]  J. Rommens,et al.  Short GCG expansions in the PABP2 gene cause oculopharyngeal muscular dystrophy , 1998, Nature Genetics.

[25]  H. Lou,et al.  Alternative RNA processing--its role in regulating expression of calcitonin/calcitonin gene-related peptide. , 1998, The Journal of endocrinology.

[26]  M. Makris,et al.  Co-inheritance of the 20210A Allele of the Prothrombin Gene Increases the Risk of Thrombosis in Subjects with Familial Thrombophilia , 1997, Thrombosis and Haemostasis.

[27]  J. Manley,et al.  Mechanism and regulation of mRNA polyadenylation. , 1997, Genes & development.

[28]  T. Dandekar,et al.  RNA Ligands Selected by Cleavage Stimulation Factor Contain Distinct Sequence Motifs That Function as Downstream Elements in 3′-End Processing of Pre-mRNA* , 1997, The Journal of Biological Chemistry.

[29]  J. Manley,et al.  RNA recognition by the human polyadenylation factor CstF , 1997, Molecular and cellular biology.

[30]  G. Edwalds-Gilbert,et al.  Alternative poly(A) site selection in complex transcription units: means to an end? , 1997, Nucleic acids research.

[31]  L. Minvielle-Sebastia,et al.  A comparison of mammalian and yeast pre-mRNA 3'-end processing. , 1997, Current opinion in cell biology.

[32]  W. Li,et al.  Apobec-1 and apolipoprotein B mRNA editing. , 1997, Biochimica et biophysica acta.

[33]  P. Reitsma,et al.  A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. , 1996, Blood.

[34]  D. Bell,et al.  Polyadenylation polymorphism in the acetyltransferase 1 gene (NAT1) increases risk of colorectal cancer. , 1995, Cancer research.

[35]  J. Wilusz,et al.  Cleavage site determinants in the mammalian polyadenylation signal. , 1995, Nucleic acids research.

[36]  A. Harris,et al.  Prevalence of common mutations in the arylsulphatase A gene in metachromatic leukodystrophy patients diagnosed in Britain , 1993, Human Genetics.

[37]  H. Kazazian,et al.  Two mutations in the beta-globin polyadenylylation signal reveal extended transcripts and new RNA polyadenylylation sites. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[38]  G S Stein,et al.  Regulation of histone gene expression. , 1992, Current opinion in cell biology.

[39]  S. Orkin,et al.  Thalassemia due to a mutation in the cleavage‐polyadenylation signal of the human beta‐globin gene. , 1985, The EMBO journal.

[40]  A. Carter,et al.  Prothrombin G20210A is a Bifunctional Gene Polymorphism , 2002, Thrombosis and Haemostasis.

[41]  H. Dietz,et al.  Nonsense-mediated mRNA decay in health and disease. , 1999, Human molecular genetics.

[42]  E. Taioli,et al.  The G20210A mutation of the prothrombin gene in patients with previous first episodes of deep-vein thrombosis: prevalence and association with factor V G1691A, methylenetetrahydrofolate reductase C677T and plasma prothrombin levels. , 1999, Thrombosis research.

[43]  W. Richards,et al.  Assaying the polyadenylation state of mRNAs. , 1999, Methods.

[44]  R. Kraaijenhagen,et al.  Clinical Expression of a Rare β-Globin Gene Mutation Co-Inherited with Haemoglobin E-Disease , 1996, European journal of clinical chemistry and clinical biochemistry : journal of the Forum of European Clinical Chemistry Societies.

[45]  M. Losekoot,et al.  A novel polyadenylation signal mutation in the alpha 2-globin gene causing alpha thalassaemia. , 1994, British Journal of Haematology.

[46]  T. Huisman,et al.  Two novel polyadenylation mutations leading to beta(+)-thalassemia. , 1990, British journal of haematology.

[47]  S. Goodbourn,et al.  Alpha-thalassaemia caused by a polyadenylation signal mutation. , 1983, Nature.