RNA-binding analyses of HuC and HuD with the VEGF and c-myc 3'-untranslated regions using a novel ELISA-based assay.
Human members of the ELAV family, referred to as ELAV-like proteins (ELPs), include HuC, HuD, Hel-N1 and HuR. These proteins bind to AU-rich elements in the 3'-untranslated regions (3'-UTRs) of many growth-related mRNAs, including c-myc and VEGF, and may participate in regulating the stability of these transcripts. Here, I have developed an enzyme-linked immunosorbent assay (ELISA) which can rapidly assess the RNA-protein-binding properties of ELPs. With this assay, I demonstrate that HuC and HuD bind to the VEGF 3'-UTR regulatory segment (VRS) and to the c- myc 3'-UTR in a specific and concentration-dependent pattern, with both proteins showing a greater affinity for the VRS. Further analysis of the VRS indicated that the binding affinity was greater for the 3'-end where the majority of AU motifs reside. Binding to the VRS could be competed by both proteins as well as a poly(U) ribohomopolymer. The binding could not be competed by other ribohomopolymers or serum from patients with high titer anti-HuD antibodies. In summary, this assay provides a rapid analysis of ELP-RNA binding which can be utilized for further characterization of RNA-binding properties and for identification of competitor molecules for in vivo functional analysis of ELPs.
King
P H
PH
Department of Neurology, University of Alabama, 1235 Jefferson Tower, 625 South 19th Street, Birmingham, AL 35294-0007, USA. pking@uab.edu
D020413:3' Untranslated Regions / Q000378:metabolism*; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D001667:Binding, Competitive; D051959:ELAV Proteins; D000067778:ELAV-Like Protein 3; D000067779:ELAV-Like Protein 4; D016228:Endothelial Growth Factors / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D008222:Lymphokines / Q000235:genetics*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020361:Paraneoplastic Syndromes, Nervous System / Q000276:immunology; D011072:Poly U / Q000378:metabolism; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D020413:3' Untranslated Regions; D000906:Antibodies; D051959:ELAV Proteins; D000067778:ELAV-Like Protein 3; D000067779:ELAV-Like Protein 4; C490264:ELAVL3 protein, human; C498010:ELAVL4 protein, human; D016228:Endothelial Growth Factors; D008222:Lymphokines; D009419:Nerve Tissue Proteins; D016271:Proto-Oncogene Proteins c-myc; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D011072:Poly U