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.