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Nucleic Acids Research, 2000, Vol. 28, No. 7 E20-e20
© 2000 Oxford University Press

RNA-binding analyses of HuC and HuD with the VEGF and c-myc 3'-untranslated regions using a novel ELISA-based assay

Peter H. King*

Department of Neurology, University of Alabama, 1235 Jefferson Tower, 625 South 19th Street, Birmingham, AL 35294-0007, USA

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.

* Tel: +1 205 975 8116; Fax: +1 205 934 0928; Email: pking@uab.edu


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