Abstract: TH-PO1055

3’UTR Regulates the Baseline Protein Abundance and Activity of Mouse NaPi-IIa

Session Information

Category: Mineral Disease

  • 1201 Mineral Disease: Ca/Mg/PO4

Authors

  • Amlal, Hassane, University of Cincinnati, Cincinnati, Ohio, United States
  • Sheriff, Sulaiman, University of Cincinnati, Cincinnati, Ohio, United States
  • Alam, Perwez, University of Cincinnati, Cincinnati, Ohio, United States
Background

The apical sodium-phosphate cotransporter NaPi-IIa plays an important role in the control of phosphate balance by regulating the rate of inorganic phosphate reabsorption in the kidney proximal tubule. We have previously shown that the 3’-untranslated region (3’UTR) of NaPi-IIa mRNA transcript plays an important role in the post-transcriptional regulation of NaPi-IIa in response to estrogen. However, whether 3’UTR regulates the baseline expression of NaPi-IIa has not been studied.

Methods

We have studied the role of 3’UTR in mNaPi-IIa expression and activity using OK cells transfected with mammalian expression plasmids containing the open-reading frame (ORF) 3'-UTR or 5'-UTR ORF or the full-length (FL) mouse NaPi-IIa transcript. Immunoblotting, Real-time PCR and 32P uptake studies were performed. Further, the role of microRNAs (miRNA) in the function of mNaPi-IIa-3’UTR was examined by luciferase assay using the miRNA target expression vector. Full-length mNaPi-IIa 3’UTR or 4 overlapping (~250bp) fragments (F1-F4 from stop codon) were generated by PCR and individually sub-cloned into the miRNA target expression vector. Subsequently, chimeras of 5’UTR-ORF-(F1 to F4) were generated in mammalian expression vector and used to examine the expression of mNaPi-IIa protein in OK cells by immunoblotting.

Results

The protein abundance of mNaPi-IIa is increased by 3-fold in OK cells transfected with 3’UTR-free plasmid (i.e. 5’-ORF), as compared to FL or ORF-3’UTR. This correlates with 50% increase in Pi transport activity, as shown by a sharp increase in Na+-dependent 32P uptake in cells expressing 5’-ORF vs. ORF-3’UTR. Real-time PCR data showed no difference in the mRNA expression levels between the 3 plasmid constructs. Normalized luciferase activity was increase by 36%, 74%, 54% and 27% for F1 to F4 fragments, respectively, compared to 3’UTR. Interestingly, the protein abundance of NaPi-IIa was significantly reduced in cell transfected with all F1 to F4 chimeras, with a more profound reduction in chimera 5’UTR-ORF-F4, as compared to 5’UTR-ORF construct.

Conclusion

3’UTR regulates the baseline expression of mouse NaPi-IIa protein abundance and activity in OK cells. This phenomenon is mediated through a sequence specific post-transcriptional mechanism involving microRNAs, which interact with cis-acting elements distributed throughout the 3’UTR.

Funding

  • NIDDK Support