HuR binding to cytoplasmic mRNA is perturbed by heat shock

AU-rich elements (AREs) located in the 3' untranslated region target the mR NAs encoding many protooncoproteins, cytokines, and lymphokines for rapid d egradation. HuR, a ubiquitously expressed member of the embryonic lethal ab normal Vision (ELAV) family of RNA-binding proteins, binds ARE sequences an d selectively stabilizes ARE-containing reporter mRNAs when overexpressed i n transiently transfected cells. HuR appears predominantly nucleoplasmic bu t has been shown to shuttle between the nucleus and cytoplasm via a novel s huttling sequence HNS. We report generation of a mouse monoclonal antibody 3A2 that both immunoblots and immunoprecipitates HuR protein; it recognizes an epitope located in the first of HuR's three RNA recognition motifs. Thi s antibody was used to probe HuR interactions with mRNA before and after he at shock, a condition that has been reported to stabilize ARE-containing mR NAs. At 37 degrees C, approximately one-third of the cytoplasmic HuR appear s polysome associated, and in vivo UV crosslinking reveals that HuR interac tions with poly(A)(+) RNA are predominantly cytoplasmic rather than nuclear . This comprises evidence that HuR directly interacts with mRNA in vivo. Af ter heat shock, 12-15% of HuR accumulates in discrete foci in the cytoplasm , but surprisingly the majority of HuR crosslinks instead to nuclear poly(A )(+) RNA, whose levels are dramatically increased in the stressed cells. Th is behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. We inter pret these differences to mean that the temporal association of HuR with AR E-containing mRNAs is different from that of these other two proteins.