HuD, a neuronal-specific RNA-binding protein, is a potential regulator of MYCN expression in human neuroblastoma cells.
HuD is one of a family of neural antigens recognised by the sera of patients with antibody-associated paraneoplastic encephalomyelitis. Localised exclusively to neurons, these proteins are among the earliest markers of the developing nervous system. Sequence analysis suggests that HuD is an RNA-binding protein. Hu protein levels were determined for the three cell types characterising human neuroblastoma cell lines: sympathoadrenal neuroblasts (N), substrate-adherent Schwann/glial/melanoblastic precursors (S) and stem cells (I) which can give rise to both N and S cells. Western blot analysis showed similar levels of protein in three N-type cell lines; S cells have no detectable Hu protein. Northern blot analysis indicated that N cells express all three Hu genes, HuD, HuC and Hel-N1. N cells, mostly from MYCN-amplified cell lines, have consistently higher steady-state levels of MYCN mRNA than S cell counterparts. Nuclear run-on and mRNA half-life experiments revealed no differences in transcription rate or mRNA stability between N and S cells from the LA-N-1 cell line, implicating differences in post-transcriptional regulation. HuD is postulated to be instrumental in splicing/processing and/or stabilisation of mRNAs involved in cell growth and neuronal differentiation. As determined by gel-mobility shift assays, HuD fusion protein binds to the 3'UTR of human MYCN mRNA. Analysis of HuD deletion mutants has demonstrated that the first and second RNA-recognition motifs (RRMs) are required for binding. Whether HuD regulates MYCN expression and thereby influences tumour aggressiveness is of major interest.
Ross
R A
RA
Department of Biological Sciences, Fordham University, Bronx, New York 10458, USA.
Lazarova
D L
DL
Manley
G T
GT
Smitt
P S
PS
Spengler
B A
BA
Posner
J B
JB
Biedler
J L
JL
D001665:Binding Sites; D015152:Blotting, Northern; D051959:ELAV Proteins; D000067779:ELAV-Like Protein 4; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009419:Nerve Tissue Proteins; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D051959:ELAV Proteins; D000067779:ELAV-Like Protein 4; C498010:ELAVL4 protein, human; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D016601:RNA-Binding Proteins