Objective: To analyze the distribution and subcellular localization of NOR1 alternative splice isoforms in human tissues and cell lines.
Methods: NOR1 open reading frame(ORF) was amplified from human fetal brain cDNA library and subcloned into pCMV/myc vector. The level of NOR1 mRNA in human tissues was determined by real-time RT-PCR. Region spanning exon 2 was amplified from cDNA or genomic DNA by specific primers and sequenced. The expression plasmids of NOR1 were transfected into cells and immunofluoresence assay was performed to determine the subcellular localization of NOR1 protein isoforms in human cells.
Results: The expression of NOR1 mRNA was high in human adult testis, moderate in human fetus nasopharynx, trachea, brain, and kidney tissues, and weak or undetectable in other tissues. Two splice variants of human NOR1 gene resulted from alternative splicing at exon 2 were identified. Both of isoform 1 and isoform 2 mRNA were detected in human fetus brain. Isoform 2 was the sole isoform in other tissues but brain. Only isoform 2 mRNA was detected in cell lines used in this study, though no genomic deletion of exon 2 could be found in all these cell lines. Immunofluoresence assay showed both isoform 1 and isoform 2 proteins were distributed in cytoplasm.
Conclusion: Alternative splice isoforms of tumor suppressor gene NOR1 are identified. NOR1 isoform 1 and isoform 2 are both detected in fetus brain. NOR1 isoform 2 lacking of exon 2 is the sole isoform in multiple tissues except for brain. The exon 2 encoded peptide does not affect the subcellular location of NOR1 protein.