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Nephroblastoma overexpressed (Nov) induces gremlin in ST-2 stromal cell lines by post-transcriptional mechanisms
Anna Smerdel-Ramoya1, Stefano Zanotti, Ernesto Canalis
1Department of Research, Saint Francis Hospital, Medical Center, Hartford, Connecticut 06105-1299, USA.
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.
Insights
Nephroblastoma overexpressed (Nov) stabilizes gremlin mRNA through post-transcriptional mechanisms. This regulation involves the 3'-untranslated region (UTR) of gremlin mRNA and AU-rich elements, impacting bone morphogenetic protein (BMP) signaling.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nephroblastoma overexpressed (Nov) is known to inhibit osteoblastogenesis.
- Nov exerts its inhibitory effects partly by binding to bone morphogenetic protein (BMP)-2.
- The precise mechanisms by which Nov influences bone formation pathways require further elucidation.
Purpose of the Study:
- To investigate the regulatory role of Nov in the expression of gremlin, a known antagonist of BMP signaling.
- To determine whether Nov affects gremlin expression at the transcriptional or post-transcriptional level.
- To identify the specific RNA elements and mechanisms involved in Nov-mediated regulation of gremlin.
Main Methods:
- Overexpression and RNA interference (RNAi) of Nov in ST-2 cells.
- Quantitative analysis of gremlin mRNA levels and mRNA half-life.
- Construction and analysis of chimeric cfos-gremlin 3 -untranslated region (UTR) reporter assays.
- RNA electrophoretic mobility shift assays (EMSA) using radiolabeled RNA fragments and cytosolic extracts.
Main Results:
- Nov overexpression increased gremlin mRNA levels, while Nov downregulation decreased them.
- Nov did not alter Grem1 transcription but significantly prolonged gremlin mRNA half-life, indicating post-transcriptional regulation.
- The gremlin 3 -UTR was identified as the critical region mediating Nov's effect on mRNA stability.
- Nov enhanced the binding of cytosolic proteins to specific AU-rich elements within the gremlin 3 -UTR, stabilizing the transcript.
Conclusions:
- Nov stabilizes gremlin transcripts through post-transcriptional mechanisms.
- This stabilization is mediated by the 3 -UTR of gremlin mRNA, involving interactions with specific ribonucleoproteins.
- AU-rich elements within the gremlin 3 -UTR are likely key targets for Nov-induced stabilization, influencing BMP signaling pathways.
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