Regulation of urokinase receptor mRNA stability by hnRNP C in lung epithelial cells.
Increased urokinase receptor (uPAR) expression as well as stabilisation of uPAR mRNA contribute to the pathogenesis of lung inflammation and neoplasia. Post-transcriptional regulation of uPAR mRNA involves interaction of both coding and 3'-UTR sequences with regulatory uPAR mRNA binding proteins (Bps). In order to identify novel regulatory interactions, we performed gel mobility shift and UV cross-linking assays and found two distinct uPAR mRNA-protein complexes. We identified a rapidly migrating 40 kDa uPAR mRNABp that selectively bound a 110 nucleotide (nt) fragment of the uPAR mRNA 3'UTR. Chimeric beta-globin/uPAR mRNA containing the 110 nt 40 kDa protein binding fragment destabilised stable beta-globin mRNA with a rate of decay identical to that of chimeric beta-globin/uPAR containing the full uPAR 3'UTR. The 40 kDa uPAR 3'UTR Bp was purified using poly (U) sepharose and identified as heterogeneous nuclear ribonucleoprotein C (hnRNPC). Finally, we confirmed its interaction with the uPAR mRNA 3' UTR by gel mobility supershift assay using an anti-hnRNPC antibody. Direct in vivo interaction of hnRNPC with the uPAR mRNA 3'UTR was demonstrated by immunoprecipitation and combined RT PCR-Southern blotting assay. Co-transfection of hnRNPC cDNA in Beas2B cells reversed destabilisation of chimeric beta-globin/uPAR 3'UTR mRNA and its over-expression also induced uPAR protein and mRNA expression through stabilisation of uPAR mRNA. These observations indicate a novel mechanism of uPAR gene regulation in lung epithelial cells in which cis elements within a 110 nt uPAR mRNA 3'UTR sequence interact with hnRNPC to regulate uPAR mRNA stability.
Shetty
Sreerama
S
Department of Specialty Care Services, The University of Texas Health Center at Tyler, Tyler, TX 75708, USA. sreerama.shetty@uthct.edu
D020413:3' Untranslated Regions / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D005914:Globins / Q000235:genetics; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000378:metabolism*; D008969:Molecular Sequence Data; D026721:RNA 3' End Processing; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics*; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins / Q000235:genetics
D020413:3' Untranslated Regions; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; C524898:PLAUR protein, human; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; D005914:Globins