HuR binding to cytoplasmic mRNA is perturbed by heat shock

Imed Gallouzi, Christopher M. Brennan, Myrna G. Stenberg, Maurice S. Swanson, Ashley Eversole, Nancy Maizels, Joan A. Steitz

Research output: Contribution to journalArticle

256 Citations (Scopus)

Abstract

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°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.

Original languageEnglish
Pages (from-to)3073-3078
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number7
DOIs
Publication statusPublished - 28 Mar 2000
Externally publishedYes

Fingerprint

AU Rich Elements
Shock
Hot Temperature
Messenger RNA
RNA-Binding Proteins
Cytoplasm
Heterogeneous-Nuclear Ribonucleoproteins
Polyribosomes
Lymphokines
RNA Stability
3' Untranslated Regions
Epitopes
Carrier Proteins
Monoclonal Antibodies
Cytokines
Antibodies

ASJC Scopus subject areas

  • General

Cite this

HuR binding to cytoplasmic mRNA is perturbed by heat shock. / Gallouzi, Imed; Brennan, Christopher M.; Stenberg, Myrna G.; Swanson, Maurice S.; Eversole, Ashley; Maizels, Nancy; Steitz, Joan A.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 97, No. 7, 28.03.2000, p. 3073-3078.

Research output: Contribution to journalArticle

Gallouzi, Imed ; Brennan, Christopher M. ; Stenberg, Myrna G. ; Swanson, Maurice S. ; Eversole, Ashley ; Maizels, Nancy ; Steitz, Joan A. / HuR binding to cytoplasmic mRNA is perturbed by heat shock. In: Proceedings of the National Academy of Sciences of the United States of America. 2000 ; Vol. 97, No. 7. pp. 3073-3078.
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AU - Steitz, Joan A.

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