Binding of a 40-kDa protein to the N-myc 3'-untranslated region correlates with enhanced N-myc expression in human neuroblastoma

Subclones of neuronal (N) and non neuronal (S) cells established from neuroblastoma tumors cultured in vitro differ in their growth characteristics and N-myc expression. N (W-N) cells derived from the NBL-W cell line express 5-fold higher levels of N-myc mRNA and 10-12-fold higher levels of protein than S cells (W-S), despite having the same N-myc copy number. This study demonstrates that the steady-state levels of N-myc are largely determined by differences in N-myc mRNA stability. The half-life of N-myc mRNA in the W-N cells is similar to 35 min compared with similar to 6 min in the W-S cells. Turnover of labile mRNAs is thought to be mediated in part by the interactions of trans-acting factors with elements within the 3'-untranslated region. RNA UV cross-linking assays using W-N cell lysate demonstrate abundant quantities of a protein complex that is 40 kDa in size (p40) that binds to the N-myc 3'-untranslated region, p40 is barely detectable in W-S cells. We have mapped two distinct regions within the 3'-UTR that specifically bind p40 (base pairs 5694-5715 and 6465-6482). Analysis of nine additional neuroblastoma cell lines shows that p40 activity correlates with enhanced expression of N-myc. p40 activity is also detected in 5 of 19 primary neuroblastomas, and activity is associated with clinically aggressive disease. In the accompanying study, we identify p40 as a member of the embryonic lethal abnormal vision (ELAV)-like family of RNA binding proteins. Our studies suggest that ELAV-like proteins may play a role in the regulation of N-myc mRNA turnover and thereby modulate the steady-state levels of N-myc expression and tumor cell phenotype.