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.