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 pa tients with antibody-associated paraneoplastic encephalomyelitis. Loca lised exclusively to neurons, these proteins are among the earliest ma rkers of the developing nervous system. Sequence analysis suggests tha t HuD is an RNA-binding protein. Hu protein levels were determined for the three cell types characterising human neuroblastoma cell lines: s ympathoadrenal neuroblasts (N), substrate-adherent Schwann/glial/melan oblastic 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. No rthern 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-tran scriptional regulation. HuD is postulated to be instrumental in splici ng/processing and/or stabilisation of mRNAs involved in cell growth an d neuronal differentiation. As determined by gel-mobility shift assays , HuD fusion protein binds to the 3'UTR of human MYCN mRNA. Analysis o f HuD deletion mutants has demonstrated that the first and second RNA- recognition motifs (RRMs) are required for binding. Whether HuD regula tes MYCN expression and thereby influences tumour aggressiveness is of major interest. (C) 1997 Elsevier Science Ltd.