The function of HuD, a neuronal-specific RNA -binding protein, in regulation of N-myc in human neuroblastoma cells

Kathryn Brooke Grandinetti, Fordham University


Amplification of the proto-oncogene N-myc is an important indicator of aggressiveness in human neuroblastoma. One factor that influences the expression and amplification of N-myc is HuD, one member of the Hu family of neuronal-specific RNA binding proteins. Previous work has shown that HuD binds to the first intron of the N- myc transcript as well as the 3′ untranslated region, and N-myc mRNA and protein levels are dependent upon levels of HuD. The studies described here sought to determine how HuD regulates N-myc expression and how such regulation might affect gene amplification. In N-myc amplified, non-neuronal LA1-5s neuroblastoma cells stably transfected with sense HuD, HuD enhances the stability of nascent N-myc transcripts. Relative to vector controls, nuclear Hu protein levels increased 7-fold and N-myc mRNA and protein levels increased as much as 8-fold. The increase in N- myc levels is not due to an increase in the half-life of the message or to a difference in the rate of transcription. The 2-fold increase in levels of splice intermediates suggests that the increase results from stabilization of N-myc pre-mRNA by HuD. A second line of research has implicated reduced levels of HuD as a selective factor in amplification of the N-myc gene. Transfection of HuD into N-myc amplified, BE(2)-M17 cells with only one HuD allele decreases their N-myc gene copy number, while increasing expression of N-myc. Additionally, in order to directly test the involvement of HuD in N-myc gene amplification, N-myc nonamplified SH-SY5Y cells were stably transfected with an antisense HuD construct. Hu protein levels are reduced 4-fold and consequently, N-myc mRNA levels are decreased 2-fold. Analysis of N-myc gene copy number in these cells by semi-quantitative PCR as well as fluorescence in situ hybridization reveals additional gene copies in the antisense-HuD transfected cells. My studies support a vital role for HuD in N-myc expression and provide evidence that the loss of HuD can lead to at least low level amplification of the N-myc gene in human neuroblastoma cells.

Subject Area

Molecular biology|Cellular biology

Recommended Citation

Grandinetti, Kathryn Brooke, "The function of HuD, a neuronal-specific RNA -binding protein, in regulation of N-myc in human neuroblastoma cells" (2005). ETD Collection for Fordham University. AAI3169382.