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Regulation of the NPT gene by a naturally occurring antisense transcript.

Cell Biochem. Biophys.2002;36(2-3):241-52
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摘要


The epithelial Na/Pi cotransporter (NaPi-II) is instrumental in maintaining phosphate (Pi) homeostasis in vertebrates. Hormones and metabolic factors (PTH, Pi availability) that acutely influence renal Pi excretion have been demonstrated to target NaPi-II expression. Upon stimulation, newly synthesized transporter molecules become integrated into the brush-border membrane to increase the Vmax of Pi uptake; reduction of Pi reabsorption is achieved by endocytosis of NaPi-II followed by lysosomal degradation of the protein. The long-term regulation of the protein is less well studied. Only recently, regulatory elements for vitamin D3 and Pi have been identified in the promoter region of the npt gene. However, signaling pathways leading to the activation of these regulatory sequences need to be established. Other reports suggested messenger RNA stability to play a role in the medium range regulation of NaPi-II expression. Recent findings in our laboratory added to the complex picture of npt gene regulation. We have identified npt-related endogenous antisense transcripts from mouse, zebrafish, and winter flounder. The two fish transcripts have been cloned and characterized; the mouse homolog has only very recently been detected. The transcripts are devoid of an open reading frame and appear in different splice forms. The evolutionary conservation of bidirectional transcription of the npt gene implies a regulatory function for the antisense transcript. In order to test the functional consequences of bidirectional transcription, we coexpressed sense and the antisense transcripts from zebrafish in Xenopus oocytes. Pi transport activity was reduced as a result of the presence of antisense RNA. Re-extraction of the RNA from injected oocytes followed by Northern blot revealed that the coexpression had no significant effect on the stability of either transcript. We concluded that the antisense mRNA interfered with the translation of the transporter if coexpressed in the Xenopus system. However, the regulatory mechanism(s) involving the npt-related antisense transcript is expected to be much more complicated in vivo, (i.e., requiring supplementary factors like double-stranded RNA recognizing proteins or specific RNases). It is planned to test this hypothesis by a transgenic zebrafish approach and/or knockout mice.

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