Stabilization of Oncostatin-M mRNA by Binding of Nucleolin to a GC-Rich Element in Its 3'UTR.

Abstract

Oncostatin-M (OSM) is a patho-physiologically important pleiotropic, multifunctional cytokine. OSM mRNA sequence analysis revealed that its 3'UTR contains three highly conserved GC-rich cis-elements (GCREs) whose role in mRNA stability is unidentified. In the present study, the functional role of the proximal GC-rich region of osm 3'-UTR (GCRE-1) in post-transcriptional regulation of osm expression in U937 cells was assessed by transfecting construct containing GCRE-1 at 3'-end of a fairly stable reporter gene followed by analysis of the expression of the reporter. GCRE-1 showed mRNA destabilizing activity; however, upon PMA treatment the reporter message containing GCRE-1 was stabilized. This stabilization is owing to a time-dependent progressive binding of trans-factors (at least five proteins) to GCRE-1 post-PMA treatment. Nucleolin was identified as one of the proteins in the RNP complex of GCRE-1 with PMA-treated U937 cytosolic extracts by oligo-dT affinity chromatography of poly-adenylated GCRE-1. Immuno-blot revealed time-dependent enhancement of nucleolin in the cytoplasm which in turn directly binds GCRE-1. RNA co-immunoprecipitation confirmed the GCRE-1-nucleolin interaction in vivo. To elucidate the functional role of nucleolin in stabilization of osm mRNA, nucleolin was overexpressed in U937 cells and found to stabilize the intrinsic osm mRNA, where co-transfection with the reporter containing GCRE-1 confirms the role of GCRE-1 in stabilization of the reporter mRNA. Thus, in conclusion, the results asserted that PMA treatment in U937 cells leads to cytoplasmic translocation of nucleolin that directly binds GCRE-1, one of the major GC-rich instability elements, thereby stabilizing the osm mRNA.