Abstract: BACKGROUND ＆ AIM: This study was aimed at the detection of novel alternative splicing variants of NRDR in human neuroblastoma. MATERIAL AND METHODS: We obtained 635 bp and 429 bp PCR fragments using NRDR specific primer from human neuroblastoma cell lines SK-N-SH and SK-SY-5Y. The two fragments were then respectively confirmed as NRDR and a novel isoform by alternative splicing. We performed 3'RACE and 5'RACE to obtain the cDNA full sequence of the spliced isoform, and tried to determine its subcellular location by GFP-humNRDRA2 fusion protein. RESULTS: We named the alternative splicing variant as humNRDRA2 with Genbank accesstion No.AY616182. Comparing with NRDR which is composed of eight exons, the novel variant NRDR A2 lost the exon4 and exon6 by alternative splicing, leading to frameshift in coding region starting from exon 5 and emergence of a premature stop codon. Thus, it produced an 188 aa protein with an altered 3'terminal sequence starting from 137 aa. The frameshift generated a bipartite nuclear targeting sequence at 160～176 aa, with the loss of the original peroxisomal targeting signal -SRL tripeptide as in NRDR. Absence of this splice variant in EST pool suggested that NRDR A2 probably was unique to human neuroblastoma. Comparing with the successful subcellular location of another splice variant of NRDR in the control experiment, we found HeLa cells that were transformed by GFP-NRDR A2 fusion protein all floaing rather than sticking to the bottom of flask, suggesting that NRDR A2-encoded protein was probably toxic to cells. CONCLUSION: Human neuroblastoma produces a novel alternative splicing isoform NRDRA2 with frameshift and a bipartite nuclear targeting sequence. Its translated protein is probably cytotoxic.

Key words: NADP(H)-dependent retinol dehydrogenase/reductase, alternative splicing, frameshift