Highly selective actions of HuR in antagonizing AU-rich element-mediated mRNA destabilization.
Human RNA-binding protein HuR, a nucleocytoplasmic shuttling protein, is a ubiquitously expressed member of the family of Hu proteins, which consist of two N-terminal RNA recognition motifs (RRM1 and RRM2), a hinge region, and a C-terminal RRM (RRM3). Although in vitro experiments showed indiscriminate binding of Hu proteins synthesized in bacterial systems to many different AU-rich elements (AREs), in vivo studies have pointed to a cytoplasmic role for HuR protein in antagonizing the rapid decay of some specific ARE-containing mRNAs, depending on physiological situations. By ectopically overexpressing HuR and its mutant derivatives in NIH 3T3 cells to mimic HuR upregulation of specific ARE-containing mRNAs in other systems, we have examined the in vivo ARE-binding specificity of HuR and dissected its functionally critical domains. We show that in NIH 3T3 cells, HuR stabilizes reporter messages containing only the c-fos ARE and not other AREs. Two distinct binding sites were identified within the c-fos ARE, the 5' AUUUA-containing domain and the 3' U-stretch-containing domain. These actions of HuR are markedly different from those of another ARE-binding protein, hnRNP D (also termed AUF1), which in vivo recognizes AUUUA repeats found in cytokine AREs and can exert both stabilizing and destabilizing effects. Further experiments showed that any combination of two of the three RRM domains of HuR is sufficient for strong binding to the c-fos ARE in vitro and to exert an RNA stabilization effect in vivo comparable to that of intact HuR and that the hinge region containing nucleocytoplasmic shuttling signals is dispensable for the stabilization effect of HuR. Our data suggest that the ARE-binding specificity of HuR in vivo is modulated to interact only with and thus regulate specific AREs in a cell type- and physiological state-dependent manner.
Chen
Chyi-Ying A
CY
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030, USA.
Xu
Nianhua
N
Shyu
Ann-Bin
AB
D016475:3T3 Cells; D000818:Animals; D000954:Antigens, Surface; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D020871:RNA Stability; D012333:RNA, Messenger; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D012261:Ribonucleoproteins / Q000378:metabolism
D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; C000630986:HNRNPD protein, human; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C000630987:Hnrpd protein, mouse; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins