The RNA binding protein HuD: rat cDNA and analysis of the alternative spliced mRNA in neuronal differentiating cell lines P19 and PC12.
HuD belongs to a family of neurospecific RNA binding proteins found in man, frog and fly [49]. To investigate whether this protein is involved in regulation of neuronal differentiation of rodent cells in vivo and in vitro, the cDNA of the rat homolog gene (r-HuD) was cloned, its expression was studied in rat brain and in neurogenic cell lines, and the splicing of its RNA was analyzed. Coding sequences of HuD from man and rat were found to be 99.5 and 95% identical at protein and DNA level, respectively. In rat brain r-HuD transcripts 3.7 and 4.2 kb in length were detected by Northern blot analysis. RT-PCR and in situ hybridization revealed that rodent homologues of HuD transcripts are present in P19 mouse embryo carcinoma and in PC12 rat pheochromocytoma cell lines both able to differentiate into neurons. In contrast, r-HuD transcripts were not detectable in the rat glioma cell line C6. In P19 cells a strong induction of HuD mRNA was observed after triggering neuronal differentiation by retinoic acid, whereas in PC12 cells the mRNA was present before and after nerve growth factor (NGF) induced neuronal differentiation. In both neuronal cell lines and in brain of adult rat and mouse HuD mRNA is alternatively spliced in a region which encodes a proline rich linker domain between the second and third RNA recognition motif. This RNA processing event seems to be differently regulated in PC12 cells on the one hand, and in P19 cells and brain of rat and mouse on the other.