title: The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets.
abstract: Human antigen R (HuR) is a 32 kDa protein with 3 RNA Recognition Motifs (RRMs), which bind to Adenylate and uridylate Rich Elements (AREs) of mRNAs. Whereas the N-terminal and central domains (RRM1 and RRM2) are essential for AREs recognition, little is known on the C-terminal RRM3 beyond its implication in HuR oligomerization and apoptotic signaling. We have developed a detergent-based strategy to produce soluble RRM3 for structural studies. We have found that it adopts the typical RRM fold, does not interact with the RRM1 and RRM2 modules, and forms dimers in solution. Our NMR measurements, combined with Molecular Dynamics simulations and Analytical Ultracentrifugation experiments, show that the protein dimerizes through a helical region that contains the conserved W261 residue. We found that HuR RRM3 binds to 5'-mer U-rich RNA stretches through the solvent exposed side of its β-sheet, located opposite to the dimerization site. Upon mimicking phosphorylation by the S318D replacement, RRM3 mutant shows less ability to recognize RNA due to an electrostatic repulsion effect with the phosphate groups. Our study brings new insights of HuR RRM3 as a domain involved in protein oligomerization and RNA interaction, both functions regulated by 2 surfaces on opposite sides of the RRM domain.
authors.affiliation: a Instituto de Bioquímica Vegetal y Fotosíntesis; cicCartuja ; Sevilla , Spain.
authors.forename: Rafael M
authors.initials: RM
authors.lastname: Scheiba
authors.forename: Alain Ibáñez
authors.initials: AI
authors.lastname: de Opakua
authors.forename: Antonio
authors.initials: A
authors.lastname: Díaz-Quintana
authors.forename: Isabel
authors.initials: I
authors.lastname: Cruz-Gallardo
authors.forename: Luis A
authors.initials: LA
authors.lastname: Martínez-Cruz
authors.forename: María L
authors.initials: ML
authors.lastname: Martínez-Chantar
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Blanco
authors.forename: Irene
authors.initials: I
authors.lastname: Díaz-Moreno
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics*; D001665:Binding Sites; D002942:Circular Dichroism; D051959:ELAV Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D051959:ELAV Proteins; D016601:RNA-Binding Proteins; D012313:RNA
keywords: AREs, Adenylate and uridylate Rich Elements; AU, Analytical Ultracentrifugation; CARM1, Coactivator associated Arginine Methyltransferase 1; CD, Circular Dichroism; Cdk1, Cyclin-dependent kinase 1; Chk2, Checkpoint kinase 2; ELAV1, Embryonic Lethal Abnormal Vision system human homolog 1; EMSA, Electrophoretic Mobility Shift Assay; FIR, FBP-Interacting Repressor; FL, Full-Length, HNS, HuR Nucleocytoplasmic Shuttling Sequence; HSQC, Heteronuclear Single-Quantum Correlation; HuR, Human antigen R; Human antigen R (HuR); MD, Molecular Dynamics; NMR, Nuclear Magnetic Resonance; NOE, Nuclear Overhauser Effect; Nuclear Magnetic Resonance (NMR); PCA, Principal Component Analysis; PKCα, Protein Kinase C α; PKCδ, Protein Kinase C δ; PMSF, PhenylMethylSulfonyl Fluoride; PTB, Polypyrimidine Tract Binding protein; RBPs, RNA Binding Proteins; RNA binding; RNA binding protein (RBP); RNA recognition motif (RRM); RRMs, RNA Recognition Motifs; SPR, Surface Plasmon Resonance; Serine Phosphorylation; WT, Wild-Type; dimerization; hnRNP1, heterogeneous nuclear RiboNucleoprotein C protein