HuR binding to cytoplasmic mRNA is perturbed by heat shock

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.

Apoptotic-induced cleavage shifts HuR from being a promoter of survival to an activator of caspase-mediated apoptosis

“…HuR shuttles between the nucleus and the cytoplasm, and while only a fraction of it (B10-20%) localizes to the cytoplasm, 36,37 it is in this compartment where HuR exercises its main biological effects. [15][16][17]30,[38][39][40] Our recent papers 18,24,25,41 strengthen this idea and show that the HuR-CPs, generated from B50% of cytoplasmic HuR, are both sufficient and necessary for HuR-mediated effects. 18 However, as not all cytoplasmic HuR is cleaved, wt HuR and both of its CPs are all present simultaneously, and thus may be affected by one another when exercising their effects.…”

“…To do this, an immunoprecipitation (IP) was carried out on HeLa cells using anti-HuR antibody (3A2), 30 and the mRNA associating with HuR were hybridized on an apoptosis-specific cDNA microarray. Numerous messages were identified (Supplementary Figure 1), but of these, caspase-9 stood out as an mRNA encoding for a component of the apoptotic response that acts downstream of Bax.…”

“…Cell extracts were prepared as described previously. 24 Western blotting was carried out as described previously 37 using antibodies against HuR, 30 Ras-GAP SH3 domain-binding Immunoprecipitation and RT-PCR/qPCR. Cells were collected and immunoprecipitation was performed as described previously 29 to isolate mRNA that was precipitated using antibodies against Sam68 (generous gift from S. Richard), HuR, GFP or an IgG control.…”

Post‐transcriptional regulation of gene expression by degradation of messenger RNAs

“…Alternatively, Hsp70 may also modulate the interaction of a given mRNA with the regulatory proteins that influence mRNA stability. In agreement with this hypothesis, Hsp70 has been identified in a cellular complex containing an ARE-binding protein as well as being detected in direct contact with RNA ARE (Laroia et al, 1999;Gallouzi et al, 2000). It is also noteworthy that heat shock is associated with a block in the degradation of ARE-mRNAs.…”

Evidence That Ternary Complex (eIF2-GTP-tRNAiMet)–Deficient Preinitiation Complexes Are Core Constituents of Mammalian Stress Granules

“…Although HSP27 has been reported to coaggregate with eIF4G and form insoluble cytosolic heat-shock granules, these were reported to exclude eIF4E, eIF3, and PABP (Cuesta et al, 2000) based on sedimentation, suggesting that heat-shock granules are distinct from SGs. The RNA-binding protein HuR was reported to form cytosolic aggregates in response to heat shock (Gallouzi et al, 2000), leading us to examine its recruitment to SGs. We find that HuR is recruited to HA-eIF2␣ (S51D)-induced TIA-1 ϩ SGs ( Figure 7I; red, yellow arrows) and is also recruited to arsenite-induced TIA-1 ϩ SGs ( Figure 3D; red).…”