FRET-detectable interactions between the ARE binding proteins, HuR and p37AUF1.

A number of highly regulated gene classes are regulated post-transcriptionally at the level of mRNA stability. A central feature in these mRNAs is the presence of A+U-rich elements (ARE) within their 3' UTRs. Two ARE binding proteins, HuR and AUF1, are associated with mRNA stabilization and destabilization, respectively. Previous studies have demonstrated homomultimerization of each protein and the capacity to bind simultaneous or competitively to a single ARE. To investigate this possibility further, cell biological and biophysical approaches were undertaken. Protein-protein interaction was monitored by fluorescence resonance energy transfer (FRET) and by immunocytochemistry in live and fixed cells using fluorescently labeled CFP/YFP fusion proteins of HuR and p37AUF1. Strong nuclear FRET between HuR/HuR and AUF1/AUF1 homodimers as well as HuR/AUF1 heterodimers was observed. Treatment with the MAP kinase activator, anisomycin, which commonly stabilizes ARE-containing mRNAs, caused rapid nuclear to cytoplasmic shuttling of HuR. AUF1 also underwent shuttling, but on a longer time scale. After shuttling, HuR/HuR, AUF1/AUF1, and HuR/AUF1, FRET was also observed in the cytoplasm. In further studies, arsenite rapidly induced the formation of stress granules containing HuR and TIA-1 but not AUF1. The current studies demonstrate that two mRNA binding proteins, HuR and AUF1, are colocalized and are capable of functional interaction in both the nucleus and cytoplasm. FRET-based detection of AUF1/HuR interaction may serve as a basis of opening up new dimensions in delineating the functional interaction of mRNA binding proteins with RNA turnover.

[1]  J S Morris,et al.  Adhesion-dependent regulation of an A+U-rich element-binding activity associated with AUF1 , 1997, Molecular and cellular biology.

[2]  J. Port,et al.  Reciptrocal regulation of beta-adrenergic receptor mRNA stability by mitogen activated protein kinase activation and inhibition , 2004, Molecular and Cellular Biochemistry.

[3]  G. M. Wilson,et al.  Assembly of AUF1 Oligomers on U-rich RNA Targets by Sequential Dimer Association* , 1999, The Journal of Biological Chemistry.

[4]  Wei Li,et al.  RNA-Binding Proteins Tia-1 and Tiar Link the Phosphorylation of Eif-2α to the Assembly of Mammalian Stress Granules , 1999, The Journal of cell biology.

[5]  Jack D. Keene,et al.  Post-transcriptional operons and regulons co-ordinating gene expression , 2005, Chromosome Research.

[6]  N. Cherradi,et al.  Antagonistic functions of tetradecanoyl phorbol acetate-inducible-sequence 11b and HuR in the hormonal regulation of vascular endothelial growth factor messenger ribonucleic acid stability by adrenocorticotropin. , 2006, Molecular endocrinology.

[7]  J. Arthur,et al.  Posttranslational Regulation of Tristetraprolin Subcellular Localization and Protein Stability by p38 Mitogen-Activated Protein Kinase and Extracellular Signal-Regulated Kinase Pathways , 2006, Molecular and Cellular Biology.

[8]  J. Port,et al.  Correlation between intrinsic mRNA stability and the affinity of AUF1 (huRNP D) and HuR for A+U-rich mRNAs , 2002, Molecular and Cellular Biochemistry.

[9]  H. Azmi,et al.  Selective Cytoplasmic Translocation of HuR and Site-specific Binding to the Interleukin-2 mRNA Are Not Sufficient for CD28-mediated Stabilization of the mRNA* , 2004, Journal of Biological Chemistry.

[10]  Vladislav V Verkhusha,et al.  Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells , 2004, Nature Methods.

[11]  D. Morello,et al.  Synchronous and regulated expression of two AU-binding proteins, AUF1 and HuR, throughout murine development , 2000, Oncogene.

[12]  P. Blackshear,et al.  Evidence that tristetraprolin is a physiological regulator of granulocyte-macrophage colony-stimulating factor messenger RNA deadenylation and stability. , 2000, Blood.

[13]  J. Port,et al.  Regulation of the mRNA-binding Protein AUF1 by Activation of the -Adrenergic Receptor Signal Transduction Pathway (*) , 1996, The Journal of Biological Chemistry.

[14]  L. Dwyer-Nield,et al.  Differential Expression and Localization of the mRNA Binding Proteins, AU‐Rich Element mRNA Binding Protein (AUF1) and Hu Antigen R (HuR), in Neoplastic Lung Tissue , 2000, Molecular carcinogenesis.

[15]  M. Gorospe,et al.  Concurrent versus individual binding of HuR and AUF1 to common labile target mRNAs , 2004, The EMBO journal.

[16]  R. Quaife,et al.  Coordinate Changes in Myosin Heavy Chain Isoform Gene Expression Are Selectively Associated With Alterations in Dilated Cardiomyopathy Phenotype , 2002, Molecular medicine.

[17]  J. Steitz,et al.  Protein ligands mediate the CRM1-dependent export of HuR in response to heat shock. , 2001, RNA.

[18]  K. Mahtani,et al.  The 3′ Untranslated Region of Tumor Necrosis Factor Alpha mRNA Is a Target of the mRNA-Stabilizing Factor HuR , 2001, Molecular and Cellular Biology.

[19]  G. M. Wilson,et al.  Folding of A+U-rich RNA Elements Modulates AUF1 Binding , 2001, The Journal of Biological Chemistry.

[20]  M. Fechir,et al.  Involvement of KSRP in the post-transcriptional regulation of human iNOS expression–complex interplay of KSRP with TTP and HuR , 2005, Nucleic acids research.

[21]  Marco Colombi,et al.  Functional cloning of BRF1, a regulator of ARE‐dependent mRNA turnover , 2002, The EMBO journal.

[22]  R. Schneider,et al.  Tissue Distribution of AU-rich mRNA-binding Proteins Involved in Regulation of mRNA Decay* , 2004, Journal of Biological Chemistry.

[23]  J. Dean,et al.  p38 Mitogen-activated Protein Kinase Stabilizes mRNAs That Contain Cyclooxygenase-2 and Tumor Necrosis Factor AU-rich Elements by Inhibiting Deadenylation* , 2003, Journal of Biological Chemistry.

[24]  R. Schneider,et al.  Alternate exon insertion controls selective ubiquitination and degradation of different AUF1 protein isoforms. , 2002, Nucleic acids research.

[25]  Klaus Resch,et al.  The p38 MAP kinase pathway signals for cytokine‐induced mRNA stabilization via MAP kinase‐activated protein kinase 2 and an AU‐rich region‐targeted mechanism , 1999, The EMBO journal.

[26]  Anne Wright,et al.  Cloning and Characterization of HuR, a Ubiquitously Expressed Elav-like Protein (*) , 1996, The Journal of Biological Chemistry.

[27]  Shelly C. Lu,et al.  S-adenosylmethionine regulates cytoplasmic HuR via AMP-activated kinase. , 2006, Gastroenterology.

[28]  P. Stoeckle,et al.  Cellular mutants define a common mRNA degradation pathway targeting cytokine AU-rich elements. , 2001, RNA.

[29]  P. Blackshear,et al.  Tristetraprolin Down-Regulates IL-2 Gene Expression through AU-Rich Element-Mediated mRNA Decay1 , 2005, The Journal of Immunology.

[30]  T. Akagi,et al.  Identification of the Nucleocytoplasmic Shuttling Sequence of Heterogeneous Nuclear Ribonucleoprotein D-like Protein JKTBP and Its Interaction with mRNA* , 2002, The Journal of Biological Chemistry.

[31]  J. Hauber,et al.  Analysis of Nucleocytoplasmic Trafficking of the HuR Ligand APRIL and Its Influence on CD83 Expression* , 2007, Journal of Biological Chemistry.

[32]  C. Demaria,et al.  Increased granulocyte-macrophage colony-stimulating factor mRNA instability in cord versus adult mononuclear cells is translation-dependent and associated with increased levels of A + U-rich element binding factor. , 1996, Blood.

[33]  B. Herman,et al.  Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. , 1998, Biophysical journal.

[34]  U. Certa,et al.  Roles of AUF1 isoforms, HuR and BRF1 in ARE-dependent mRNA turnover studied by RNA interference. , 2004, Nucleic acids research.

[35]  I. Lafon,et al.  Developmental expression of AUF1 and HuR, two c-myc mRNA binding proteins , 1998, Oncogene.

[36]  G. Kollias,et al.  MK2 Targets AU-rich Elements and Regulates Biosynthesis of Tumor Necrosis Factor and Interleukin-6 Independently at Different Post-transcriptional Levels* , 2002, The Journal of Biological Chemistry.

[37]  R. Schneider,et al.  Selective Degradation of AU-Rich mRNAs Promoted by the p37 AUF1 Protein Isoform , 2003, Molecular and Cellular Biology.

[38]  David E. Golan,et al.  Dynamic Shuttling of Tia-1 Accompanies the Recruitment of mRNA to Mammalian Stress Granules , 2000, The Journal of cell biology.

[39]  J. Hauber,et al.  Expression of CD83 Is Regulated by HuR via a Novel cis-Active Coding Region RNA Element* , 2006, Journal of Biological Chemistry.

[40]  S. Esnault,et al.  The peptidyl-prolyl isomerase Pin1 regulates the stability of granulocyte-macrophage colony-stimulating factor mRNA in activated eosinophils , 2005, Nature Immunology.

[41]  C. Demaria,et al.  AUF1 Binding Affinity to A+U-rich Elements Correlates with Rapid mRNA Degradation (*) , 1996, The Journal of Biological Chemistry.

[42]  B. Haynes,et al.  A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. , 1996, Immunity.

[43]  Y. Totsuka,et al.  Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism , 2005, The Journal of cell biology.

[44]  Myriam Gorospe,et al.  HuR Regulates p21 mRNA Stabilization by UV Light , 2000, Molecular and Cellular Biology.

[45]  G. Dreyfuss Structure and function of nuclear and cytoplasmic ribonucleoprotein particles. , 1986, Annual review of cell biology.

[46]  Th. Förster Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[47]  G. M. Wilson,et al.  Phosphorylation of p40AUF1 Regulates Binding to A + U-rich mRNA-destabilizing Elements and Protein-induced Changes in Ribonucleoprotein Structure* , 2003, Journal of Biological Chemistry.

[48]  Haiwei Song,et al.  The enzymes and control of eukaryotic mRNA turnover , 2004, Nature Structural &Molecular Biology.

[49]  E. Galperin,et al.  Visualization of Rab5 activity in living cells by FRET microscopy and influence of plasma-membrane-targeted Rab5 on clathrin-dependent endocytosis , 2003, Journal of Cell Science.

[50]  G. M. Wilson,et al.  Structural Remodeling of an A + U-rich RNA Element by Cation or AUF1 Binding* , 2001, The Journal of Biological Chemistry.

[51]  M. Gorospe,et al.  RNA-binding protein HuR enhances p53 translation in response to ultraviolet light irradiation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[52]  B. Séraphin,et al.  Cytoplasmic foci are sites of mRNA decay in human cells , 2004, The Journal of cell biology.

[53]  M. Karin,et al.  A KH domain RNA binding protein, KSRP, promotes ARE-directed mRNA turnover by recruiting the degradation machinery. , 2004, Molecular cell.

[54]  J. Steitz,et al.  Overexpression of HuR, a nuclear–cytoplasmic shuttling protein, increases the in vivo stability of ARE‐containing mRNAs , 1998, The EMBO journal.

[55]  G. M. Wilson,et al.  Regulation of A + U-rich Element-directed mRNA Turnover Involving Reversible Phosphorylation of AUF1* , 2003, Journal of Biological Chemistry.

[56]  R. Cuesta,et al.  Control of mRNA decay by heat shock-ubiquitin-proteasome pathway. , 1999, Science.

[57]  Tala Bakheet,et al.  ARED 3.0: the large and diverse AU-rich transcriptome , 2005, Nucleic Acids Res..

[58]  Heinfried H Radeke,et al.  Protein Kinase C-dependent Phosphorylation of the mRNA-stabilizing Factor HuR : Implications for Posttranscriptional Regulation of Cyclooxygenase-2 , 2007 .

[59]  Roy Parker,et al.  Movement of Eukaryotic mRNAs Between Polysomes and Cytoplasmic Processing Bodies , 2005, Science.

[60]  O. Mühlemann,et al.  Turnover in the Alps: an mRNA perspective , 2006 .

[61]  E. Izaurralde,et al.  P bodies: at the crossroads of post-transcriptional pathways , 2007, Nature Reviews Molecular Cell Biology.

[62]  P. Anderson,et al.  MK2‐induced tristetraprolin:14‐3‐3 complexes prevent stress granule association and ARE‐mRNA decay , 2004, The EMBO journal.

[63]  A. MacMillan,et al.  FRET Analysis of in Vivo Dimerization by RNA-editing Enzymes* , 2006, Journal of Biological Chemistry.

[64]  P. Anderson,et al.  Stress granules: sites of mRNA triage that regulate mRNA stability and translatability. , 2002, Biochemical Society transactions.

[65]  R. Schneider,et al.  Nuclear Import and Export Functions in the Different Isoforms of the AUF1/Heterogeneous Nuclear Ribonucleoprotein Protein Family* , 2003, Journal of Biological Chemistry.

[66]  J. Port,et al.  Purification and Characterization of β-Adrenergic Receptor mRNA-binding Proteins* , 2000, The Journal of Biological Chemistry.

[67]  T. Nagase,et al.  CRM1-dependent, but not ARE-mediated, nuclear export of IFN-α1 mRNA , 2004, Journal of Cell Science.

[68]  G. M. Wilson,et al.  Structure and genomic organization of the human AUF1 gene: alternative pre-mRNA splicing generates four protein isoforms. , 1998, Genomics.

[69]  Kotb Abdelmohsen,et al.  Phosphorylation of HuR by Chk2 regulates SIRT1 expression. , 2007, Molecular cell.

[70]  E. Chan,et al.  GW Bodies, MicroRNAs and the Cell Cycle , 2006, Cell cycle.

[71]  Myriam Gorospe,et al.  HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation , 2000, The EMBO journal.

[72]  S. Tenenbaum,et al.  Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays. , 2002, Methods.

[73]  M. Moore From Birth to Death: The Complex Lives of Eukaryotic mRNAs , 2005, Science.

[74]  G. M. Wilson,et al.  A Hairpin-like Structure within an AU-rich mRNA-destabilizing Element Regulates trans-Factor Binding Selectivity and mRNA Decay Kinetics* , 2005, Journal of Biological Chemistry.

[75]  K. White,et al.  ELAV Multimerizes on Conserved AU4-6 Motifs Important for ewg Splicing Regulation , 2005, Molecular and Cellular Biology.

[76]  K. Mahtani,et al.  Mitogen-Activated Protein Kinase p38 Controls the Expression and Posttranslational Modification of Tristetraprolin, a Regulator of Tumor Necrosis Factor Alpha mRNA Stability , 2001, Molecular and Cellular Biology.

[77]  C. Demaria,et al.  Structural Determinants in AUF1 Required for High Affinity Binding to A + U-rich Elements* , 1997, The Journal of Biological Chemistry.

[78]  C. Y. Chen,et al.  mRNA decay mediated by two distinct AU-rich elements from c-fos and granulocyte-macrophage colony-stimulating factor transcripts: different deadenylation kinetics and uncoupling from translation , 1995, Molecular and cellular biology.

[79]  Randal J. Kaufman,et al.  Stress granules and processing bodies are dynamically linked sites of mRNP remodeling , 2005, The Journal of cell biology.

[80]  B. Monsarrat,et al.  A "liaison dangereuse" between AUF1/hnRNPD and the oncogenic tyrosine kinase NPM-ALK. , 2006, Blood.

[81]  A. Shyu,et al.  RNA stabilization by the AU‐rich element binding protein, HuR, an ELAV protein , 1998, The EMBO journal.

[82]  Yusuke Tomozoe,et al.  Two separate regions essential for nuclear import of the hnRNP D nucleocytoplasmic shuttling sequence , 2005, The FEBS journal.

[83]  M. Magnasco,et al.  Decay rates of human mRNAs: correlation with functional characteristics and sequence attributes. , 2003, Genome research.

[84]  T. Jenuwein,et al.  Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component , 2002, Nature Genetics.

[85]  G. Stoecklin,et al.  Mitogen-Activated Protein Kinase Phosphatidylinositol 3-Kinase and p 38 Interleukin-3 mRNA Turnover by Parallel and Independent Regulation of , 2001 .

[86]  J. Steitz,et al.  HNS, a nuclear-cytoplasmic shuttling sequence in HuR. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[87]  K. White,et al.  The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages. , 1988, Developmental biology.

[88]  B. Hemmings,et al.  The ARE‐dependent mRNA‐destabilizing activity of BRF1 is regulated by protein kinase B , 2004, The EMBO journal.

[89]  S. Kuersten,et al.  Linking nuclear mRNP assembly and cytoplasmic destiny , 2005, Biology of the cell.

[90]  Myriam Gorospe,et al.  Role of HuR in Skeletal Myogenesis through Coordinate Regulation of Muscle Differentiation Genes , 2003, Molecular and Cellular Biology.

[91]  U. Atasoy,et al.  ELAV protein HuA (HuR) can redistribute between nucleus and cytoplasm and is upregulated during serum stimulation and T cell activation. , 1998, Journal of cell science.

[92]  P. Blackshear,et al.  Zinc inhibits turnover of labile mRNAs in intact cells , 1995, Journal of cellular physiology.