Expression, binding affinity, and function of HuD, a neuronal-specific RNA-binding protein, in neuroblastoma cells
Neuroblastoma is a cancer of neural crest origin. In one-third of neuroblastoma tumors, the N-myc proto-oncogene is amplified, and its over-expression correlates with advanced disease stage. Therefore, it is essential to understand the mechanisms responsible for overexpression of N-myc. Neuroblastoma cell lines such as LA1-55n (N-type) and LA1-5s (S-type), derived from a common cell line, have similar N-myc gene copy numbers and transcription rates; however, N cells exhibit higher N-myc mRNA steady-state levels. Therefore, N-myc expression is regulated differently at the post-transcriptional level in N and S cells. The Hu proteins HuD and Hel-N1, neuronal-specific RNA-binding proteins, have been implicated in post-transcriptional control of N-myc. In this thesis, I report that N cells, expressing high levels of N-myc, are Hu-positive and express more than one Hu mRNA, while S cells, with low or undetectable N-myc expression are Hu-negative. Of the three Hu antigens, only HuD binds long N-myc RNA sequences shown to regulate proto-oncogene's expression in a tissue-specific manner (Babiss and Friedman, 1990; Sivak et al., 1997). The binding of HuD to intronic, as well as exonic, N-myc RNA sequences and the Hu-immunoreactivity in nuclei of N cells suggest that HuD may function in the nuclear processing or stability of N-myc pre-mRNA.^ Analysis of the steady-state levels of N-myc pre-mRNA processing intermediates suggests that cleavage/polyadenylation and intron 2 removal of N-myc transcripts may be more efficient in N than in S cells. Both processing steps may be controlled by trans-acting factors, such as HuD, in a lineage-specific fashion. Results from in vivo experiments confirm this hypothesis. Antisense HuD-transfected N cell clones with decreased HuD mRNA levels exhibit proportionally decreased N-myc expression. Transfections with a HuD expression construct also confirm the direct linear relation between HuD protein levels and N-myc expression levels. Thus, these studies implicate HuD as a regulator of nuclear processing or stability of N-myc pre-mRNA. ^
Biology, Molecular|Biology, Cell
Darina Lazarova Lazarova,
"Expression, binding affinity, and function of HuD, a neuronal-specific RNA-binding protein, in neuroblastoma cells"
(January 1, 1998).
ETD Collection for Fordham University.