Protein-RNA interactions determine the stability of the renal NaPi-2 cotransporter mRNA and its translation in hypophosphatemic rats.
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.
Moz
Y
Y
Minerva Center for Calcium and Bone Metabolism, Nephrology Services, Hadasssah University Hospital, Jerusalem 91120, Israel.
Silver
J
J
Naveh-Many
T
T
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D017674:Hypophosphatemia / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D008297:Male; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D002352:Carrier Proteins; D012333:RNA, Messenger; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters