Abstract
Nephroblastoma overexpressed (Nov) inhibits osteoblastogenesis in part because it binds bone morphogenetic protein (BMP)-2. In the present study, we investigated whether Nov regulated the expression of the BMP antagonist gremlin. Overexpression of Nov increased gremlin mRNA levels in ST-2 cells, and its downregulation by RNA interference decreased gremlin mRNA. Nov did not affect Grem1 transcription, but prolonged the half-life of gremlin mRNA in ST-2 cells, demonstrating that Nov acts by post-transcriptional mechanisms. This was confirmed by demonstrating that downregulation of Nov destabilizes gremlin transcripts. To assess whether the 3'-untranslated region (UTR) of gremlin mRNA mediated the effect of Nov, the decay of a chimeric cfos gremlin 3'-UTR construct was compared to that of cfos in ST-2 cells. The presence of the gremlin 3'-UTR prolonged the half-life of cfos and was responsible for the effect of Nov. To examine the binding of the gremlin 3'-UTR to ribonucleoproteins, radiolabeled gremlin RNA fragments were incubated with cytosolic extracts from Nov overexpressing and control cells. RNA electrophoretic mobility analysis revealed that Nov enhanced the binding of cytosolic proteins to the fragments spanning the 3'-UTR of gremlin between bases 1,358-1,557 and 1,158-1,357 from the transcriptional start. Mutations of AU-rich elements in these two RNA fragments prevented the formation of RNA-protein complexes induced by Nov. Nov did not alter the binding of cytosolic extracts to sequences present in the 5'-UTR or coding region of gremlin. In conclusion, Nov stabilizes gremlin transcripts, and this effect is possibly mediated by AU-rich elements present in the 3'-UTR of gremlin.