Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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3' Untranslated Regions / metabolism*
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Antibodies / immunology
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Base Sequence
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Binding, Competitive
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ELAV Proteins
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ELAV-Like Protein 3
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ELAV-Like Protein 4
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Endothelial Growth Factors / genetics*
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Enzyme-Linked Immunosorbent Assay / methods*
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Humans
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Lymphokines / genetics*
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Molecular Sequence Data
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / immunology
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Nerve Tissue Proteins / metabolism*
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Paraneoplastic Syndromes, Nervous System / immunology
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Poly U / metabolism
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Protein Binding
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Proto-Oncogene Proteins c-myc / genetics*
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / immunology
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RNA-Binding Proteins / metabolism*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Regulatory Sequences, Nucleic Acid
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Tumor Cells, Cultured
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Vascular Endothelial Growth Factor A
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Vascular Endothelial Growth Factors
Substances
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3' Untranslated Regions
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Antibodies
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ELAV Proteins
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ELAV-Like Protein 3
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ELAV-Like Protein 4
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ELAVL3 protein, human
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ELAVL4 protein, human
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Endothelial Growth Factors
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Lymphokines
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Nerve Tissue Proteins
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Proto-Oncogene Proteins c-myc
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RNA-Binding Proteins
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Recombinant Fusion Proteins
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Vascular Endothelial Growth Factor A
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Vascular Endothelial Growth Factors
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Poly U