HuD, a neuronal-specific RNA-binding protein, is a putative regulator of N-myc pre-mRNA processing/stability in malignant human neuroblasts

Abstract

N-myc gene copy numbers and transcription rates are similar in N (neuroblastic, tumorigenic) and S (nonneuronal, non-tumorigenic) neuroblastoma cells with chromosomally integrated ampli®ed N-myc genes. However, N cells show signi®cantly higher N-myc mRNA levels than S cells. Therefore, post-transcriptional control of N-myc gene expression must di er between these cell types. Since no di erences in N-myc mRNA half-life were found between N and S cells from two cell lines, steady-state levels of N-myc pre-mRNA processing intermediates were analysed. Results suggest that the di erences in N-myc expression arise primarily at the nuclear post-transcriptional level. The neuronal-speci®c RNA-binding Hu proteins are present in cytoplasmic and nuclear fractions of N cells and one of them, HuD, binds speci®cally to both exonic and intronic N-myc RNA sequences. In sense and antisense HuD-transfected N cells, there are coordinate changes in HuD and N-myc expression levels. Thus, we propose that HuD plays a role in the nuclear processing/stability of N-myc premRNA in N-type neuroblastoma cells.

Role of the Aspartyl-Asparaginyl-β-Hydroxylase Gene in Neuroblastoma Cell Motility

“…The Sy5y cell line was cloned from a human peripheral neuroblastoma and exhibits high levels of MYCN expression in the absence of gene amplification (Sadee et al, 1987). In a subset of neuroblastomas that lack MYCN gene amplification, increased MYCN expression is mediated by HuD stabilization of MYCN mRNA transcripts (Lazarova et al, 1999). Western blot analysis with the FB-50 mAb detected the expected~86-kd AAH protein in cell lysates and higher levels of AAH in Sy5y cells compared with normal brain (Fig.…”

Nestin Is a Potential Mediator of Malignancy in Human Neuroblastoma Cells

“…Cells were cultured according to published methods (22). Growth rates were determined by previously described methods (21).…”

“…Nuclear and cytoplasmic fractions were isolated as previously described (22). Western blot procedures have been published (22,24).…”

“…Nuclear and cytoplasmic fractions were isolated as previously described (22). Western blot procedures have been published (22,24). Primary antibodies included polyclonal antibodies to nestin (331b), homing cell adhesion molecule (HCAM) (Santa Cruz Biotechnology, Santa Cruz, CA), secretogranin II (gift of Dr. Jonathan G. Scammell, Department of South Alabama College of Medicine, Mobile, AL), tissue plasminogen activator (tPA) (ICN Pharmaceuticals, Inc., Aurora, OH), and vimentin (Chemicon International, Temecula, CA) and monoclonal antibodies to neurofilaments (NFs) 70 and 160, chromogranin A, ␤-tubulin, and actin (clone C4) (Roche Applied Science).…”

Characterization of the Interaction between Neuronal RNA-binding Protein HuD and AU-rich RNA

“…In vitro studies would help elucidate cooperativity and sequential occupation of sequences and would yield detailed mechanistic insight that could be applied, among others, to the design of small molecule inhibitors. Such inhibitors might be of clinical importance in situations in which improper mRNA stabilization leads to overexpression of labile mRNAs, such as tumor necrosis factor ␣ overexpression in inflammatory disease (1,5) and proto-oncogene overexpression in cancer cells inappropriately expressing Hu proteins (34,35,47,68). Recent experiments in which TTP was overexpressed demonstrate that the modulation of mRNA decay can be used to suppress oncogenesis (67).…”