title: Mutant copper-zinc superoxide dismutase associated with amyotrophic lateral sclerosis binds to adenine/uridine-rich stability elements in the vascular endothelial growth factor 3'-untranslated region.
abstract: Vascular endothelial growth factor (VEGF) is a neurotrophic factor essential for maintenance of motor neurons. Loss of this factor produces a phenotype similar to amyotrophic lateral sclerosis (ALS). We recently showed that ALS-producing mutations of Cu/Zn-superoxide dismutase (SOD1) disrupt post-transcriptional regulation of VEGF mRNA, leading to significant loss of expression [Lu et al., J. Neurosci.27 (2007), 7929]. Mutant SOD1 was present in the ribonucleoprotein complex associated with adenine/uridine-rich elements (ARE) of the VEGF 3'-untranslated region (UTR). Here, we show by electrophoretic mobility shift assay that mutant SOD1 bound directly to the VEGF 3'-UTR with a predilection for AREs similar to the RNA stabilizer HuR. SOD1 mutants A4V and G37R showed higher affinity for the ARE than L38V or G93A. Wild-type SOD1 bound very weakly with an apparent K(d) 11- to 72-fold higher than mutant forms. Mutant SOD1 showed an additional lower shift with VEGF ARE that was accentuated in the metal-free state. A similar pattern of binding was observed with AREs of tumor necrosis factor-alpha and interleukin-8, except only a single shift predominated. Using an ELISA-based assay, we demonstrated that mutant SOD1 competes with HuR and neuronal HuC for VEGF 3'-UTR binding. To define potential RNA-binding domains, we truncated G37R, G93A and wild-type SOD1 and found that peptides from the N-terminal portion of the protein that included amino acids 32-49 could recapitulate the binding pattern of full-length protein. Thus, the strong RNA-binding affinity conferred by ALS-associated mutations of SOD1 may contribute to the post-transcriptional dysregulation of VEGF mRNA.
authors.affiliation: Department of Neurology, University of Alabama at Birmingham, Alabama, USA.
authors.forename: Xuelin
authors.initials: X
authors.lastname: Li
authors.forename: Liang
authors.initials: L
authors.lastname: Lu
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Bush
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Zhang
authors.forename: Lei
authors.initials: L
authors.lastname: Zheng
authors.forename: Esther A
authors.initials: EA
authors.lastname: Suswam
authors.forename: Peter H
authors.initials: PH
authors.lastname: King
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism*; D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology* / Q000235:genetics / Q000503:physiopathology; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D051959:ELAV Proteins / Q000235:genetics / Q000378:metabolism; D000067780:ELAV-Like Protein 1; D000067778:ELAV-Like Protein 3; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D020871:RNA Stability / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D000067778:ELAV-Like Protein 3; C098770:ELAVL1 protein, human; C490264:ELAVL3 protein, human; D016209:Interleukin-8; D010446:Peptide Fragments; D016601:RNA-Binding Proteins; D014409:Tumor Necrosis Factor-alpha; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D013482:Superoxide Dismutase