HuR Binding to Cytoplasmic mRNA Is Perturbed by Heat Shock

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 97; no. 7; pp. 3073 - 3078
• Main Authors: Gallouzi, Imed-Eddine, Brennan, Christopher M., Stenberg, Myrna G., Swanson, Maurice S., Eversole, Ashley, Maizels, Nancy, Steitz, Joan A.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 28-03-2000; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Subjects: Antibodies; Antibodies, Monoclonal - immunology; Antibody Specificity; Antigens, Surface; ARE-binding protein; Base Sequence; Biochemistry; Biological Sciences; Cytoplasm; Cytoplasm - metabolism; DNA Primers; ELAV Proteins; ELAV-Like Protein 1; Fluorescent antibody techniques; HeLa Cells; Heterogeneous nuclear ribonucleoproteins; hnRNP A1 protein; hnRNP D protein; Hot Temperature; Humans; HuR protein; Messenger RNA; Monoclonal antibodies; Polyribosomes; Protein Binding; Proteins; Ribonucleic acid; RNA; RNA, Messenger - metabolism; RNA-Binding Proteins - immunology; RNA-Binding Proteins - metabolism; Shock heating
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.97.7.3073

AU-rich elements (AREs) located in the 3′untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling 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. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that has been reported to stabilize ARE-containing mRNAs. At 37 degrees C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A)+RNA are predominantly cytoplasmic rather than nuclear. This comprises evidence that HuR directly interacts with mRNA in vivo. After 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. This 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 interpret these differences to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of these other two proteins.