Protein-RNA Interactions Determine the Stability of the Renal NaPi-2 Cotransporter mRNA and Its Translation in Hypophosphatemic Rats

Abstract

Hypophosphatemia leads to an increase in type II Na(+)-dependent inorganic phosphate cotransporter (NaPi-2) mRNA and protein levels in the kidney and increases renal phosphate reabsorption. Nuclear transcript run-on experiments showed that the effect of a low phosphate diet was post-transcriptional. In an in vitro degradation assay, renal proteins from hypophosphatemic rats stabilized the NaPi-2 transcript 6-fold compared with control rats and this was dependent upon an intact NaPi-2 3'-untranslated region (UTR). To determine an effect of hypophosphatemia upon NaPi-2 protein synthesis, the incorporation of injected [(35)S]methionine into renal proteins was studied in vivo. Hypophosphatemia led to increased [(35)S]methionine incorporation only into NaPi-2 protein. The effect of hypophosphatemia on translation was studied in an in vitro translation assay, where hypophosphatemic renal proteins led to increased translation of NaPi-2 and other transcripts. NaPi-2 RNA interaction with cytosolic proteins was studied by UV cross-linking and Northwestern gels. Hypophosphatemic proteins led to increased binding of renal cytosolic proteins to the 5'-UTR of NaPi-2 mRNA. Therefore, hypophosphatemia increases NaPi-2 gene expression post-transcriptionally, which correlates with a more stable transcript mediated by the 3'-UTR, and an increase in NaPi-2 translation involving protein binding to the 5'-UTR. These findings show that phosphate regulates gene expression by affecting protein-RNA interactions in vivo.