title: Structural determinants of human ζ-globin mRNA stability.
abstract: BACKGROUND
  The normal accumulation of adult α and β globins in definitive erythrocytes is critically dependent upon processes that ensure that the cognate mRNAs are maintained at high levels in transcriptionally silent, but translationally active progenitor cells. The impact of these post-transcriptional regulatory events on the expression of embryonic ζ globin is not known, as its encoding mRNA is not normally transcribed during adult erythropoiesis. Recently, though, ζ globin has been recognized as a potential therapeutic for α thalassemia and sickle-cell disease, raising practical questions about constitutive post-transcriptional processes that may enhance, or possibly prohibit, the expression of exogenous or derepresssed endogenous ζ-globin genes in definitive erythroid progenitors.
  
  
  METHODS
  The present study assesses mRNA half-life in intact cells using a pulse-chase approach; identifies cis-acting determinants of ζ-globin mRNA stability using a saturation mutagenesis strategy; establishes critical 3'UTR secondary structures using an in vitro enzymatic mapping method; and identifies trans-acting effector factors using an affinity chromatographical procedure.
  
  
  RESULTS
  We specify a tetranucleotide 3'UTR motif that is required for the high-level accumulation of ζ-globin transcripts in cultured cells, and formally demonstrate that it prolongs their cytoplasmic half-lives. Surprisingly, the ζ-globin mRNA stability motif does not function autonomously, predicting an activity that is subject to structural constraints that we subsequently specify. Additional studies demonstrate that the ζ-globin mRNA stability motif is targeted by AUF1, a ubiquitous RNA-binding protein that enhances the half-life of adult β-globin mRNA, suggesting commonalities in post-transcriptional processes that regulate globin transcripts at all stages of mammalian development.
  
  
  CONCLUSIONS
  These data demonstrate a mechanism for ζ-globin mRNA stability that exists in definitive erythropoiesis and is available for therapeutic manipulation in α thalassemia and sickle-cell disease.
authors.forename: Zhenning
authors.initials: Z
authors.lastname: He
authors.forename: Decheng
authors.initials: D
authors.lastname: Song
authors.forename: Sebastiaan
authors.initials: S
authors.lastname: van Zalen
authors.affiliation: Department of Medicine (Hematology/Oncology), Perelman School of Medicine at the University of Pennsylvania, Biomedical Research Building, Room 808, 421 Curie Boulevard, Philadelphia, PA 19104, USA. jeruss@mail.med.upenn.edu.
authors.forename: J Eric
authors.initials: JE
authors.lastname: Russell
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D000328:Adult; D001483:Base Sequence; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006207:Half-Life; D006367:HeLa Cells; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs / Q000235:genetics; D011485:Protein Binding; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D055543:zeta-Globins / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; C000630986:HNRNPD protein, human; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D012333:RNA, Messenger; D055543:zeta-Globins
references.citation: Genomics. 1998 Mar 1;48(2):195-202
references.pmid: 9521873
references.citation: EMBO J. 1995 Sep 1;14(17):4357-64
references.pmid: 7556077
references.citation: J Biol Chem. 2001 Dec 21;276(51):47922-9
references.pmid: 11598129
references.citation: Blood. 2012 Jan 26;119(4):1045-53
references.pmid: 22134169
references.citation: Mol Cell Biol. 2001 Aug;21(15):4900-8
references.pmid: 11438647
references.citation: Blood. 1998 Nov 1;92(9):3057-63
references.pmid: 9787139
references.citation: Mol Cell Biol. 2001 Oct;21(20):6960-71
references.pmid: 11564879
references.citation: Mol Cell Biol. 1986 Apr;6(4):1108-16
references.pmid: 3785159
references.citation: Cell. 1986 Aug 29;46(5):659-67
references.pmid: 3488815
references.citation: Dev Biol. 2001 Aug 1;236(1):230-43
references.pmid: 11456457
references.citation: Blood. 1997 Aug 1;90(3):1275-82
references.pmid: 9242562
references.citation: Br J Haematol. 2000 Jun;109(4):882-90
references.pmid: 10929046
references.citation: Electrophoresis. 2007 Nov;28(21):3862-7
references.pmid: 17922510
references.citation: Nat Med. 2004 Apr;10(4):365-7
references.pmid: 15034572
references.citation: Hum Genet. 1986 Jul;73(3):221-4
references.pmid: 3015767
references.citation: J Biol Chem. 2003 May 23;278(21):19534-40
references.pmid: 12642582
references.citation: Mol Cell Biol. 1999 Jul;19(7):4572-81
references.pmid: 10373506
references.citation: Blood. 2001 Feb 15;97(4):1099-105
references.pmid: 11159543
references.citation: J Biochem. 2001 Dec;130(6):737-40
references.pmid: 11726272
references.citation: Mol Cell Biol. 2001 Sep;21(17):5879-88
references.pmid: 11486027
references.citation: Blood. 2007 Jan 15;109(2):795-801
references.pmid: 17003365
references.citation: Mol Cell Biol. 1997 Sep;17(9):5201-9
references.pmid: 9271398
references.citation: Nat Struct Mol Biol. 2007 Jul;14(7):670-6
references.pmid: 17572684
references.citation: J Interferon Cytokine Res. 2005 Jan;25(1):1-10
references.pmid: 15684617
references.citation: J Mol Biol. 1996 Mar 29;257(2):276-89
references.pmid: 8609623
references.citation: J Mol Biol. 1999 May 21;288(5):911-40
references.pmid: 10329189
references.citation: Blood. 1993 Mar 15;81(6):1636-40
references.pmid: 7680924
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
references.pmid: 12824337
references.citation: EMBO J. 1999 Apr 15;18(8):2218-28
references.pmid: 10205175
references.citation: Thromb Res. 2010 Aug;126(2):130-6
references.pmid: 20553951
references.citation: Prenat Diagn. 2001 Jul;21(7):529-39
references.pmid: 11494285
references.citation: Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547-51
references.pmid: 1319065
references.citation: Mol Cell Biol. 1996 Jun;16(6):2637-46
references.pmid: 8649371
references.citation: Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2410-4
references.pmid: 9122208
references.citation: Hemoglobin. 1989;13(1):45-65
references.pmid: 2703365
references.citation: Mol Cell Biol. 1994 Dec;14(12):8123-32
references.pmid: 7969150
references.citation: Nucleic Acids Res. 2006 Jan 03;33(22):7138-50
references.pmid: 16391004
references.citation: Eur J Haematol. 2007 Dec;79(6):516-25
references.pmid: 17976188
references.citation: Cell. 1989 Jul 28;58(2):373-82
references.pmid: 2752428
references.citation: Blood. 1989 Aug 1;74(2):629-37
references.pmid: 2752137
references.citation: N Engl J Med. 1986 Jan 9;314(2):76-9
references.pmid: 3941693
references.citation: Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14669-74
references.pmid: 9843947
references.citation: J Biol Chem. 1996 Feb 9;271(6):3293-9
references.pmid: 8621733
references.citation: J Clin Invest. 1983 Mar;71(3):461-6
references.pmid: 6826718
references.citation: PLoS One. 2012;7(7):e40827
references.pmid: 22808270
references.citation: Mol Cell Biol. 1995 Mar;15(3):1769-77
references.pmid: 7862166
references.citation: Mol Cell Biol. 1998 Apr;18(4):2173-83
references.pmid: 9528789
references.citation: Mol Cell Biol. 2006 Mar;26(6):2419-29
references.pmid: 16508016
references.citation: Mol Cell Biol. 1987 Dec;7(12):4557-9
references.pmid: 3437896
references.citation: J Thromb Haemost. 2010 Nov;8(11):2580-3
references.pmid: 20723024
references.citation: J Biol Chem. 2004 Mar 26;279(13):12974-9
references.pmid: 14711832
references.citation: Biochem Biophys Res Commun. 2004 Dec 24;325(4):1376-82
references.pmid: 15555579