Post-transcriptional control of gene expression by the major inducible heat shock protein Hsp70

Abstract

AU-rich elements (AREs) encoded within the 3'-untranslated regions (3'UTRs) of many mRNAs are targets for factors that control transcript longevity and translational efficiency. Hsp70, best known as a protein chaperone, also interacts with ARE-like RNA substrates in vitro. The findings here define cellular roles for the interaction between Hsp70 and ARE-containing transcripts as well as mechanistic aspects of binding. Suppression of Hsp70 in cells destabilizes a reporter mRNA containing a high-affinity Hsp70 binding site. Consistent with these data, Hsp70 interacts with and stabilizes endogenous ARE-containing mRNAs encoding VEGF and Cox-2 in HeLa cells. Hsp70 recognition and stabilization of VEGF mRNA is mediated by an ARE-like sequence in the proximal 3'UTR. Stabilization of VEGF mRNA coincides with accumulation of Hsp70 protein levels in HL60 promyelocytic leukemia cells recovering from acute thermal stress. The minimum length of RNA recognized by Hsp70 is probably on the order of 30 nucleotides, which must be single-stranded. Hsp70 has no canonical RNA-binding motifs, but both the ATPase and peptide-binding domains, the two major structural modules of the protein, recognize and bind AREs with high specificity and mid-nanomolar affinity. The ATPase domain, however, is unable to restore VEGF transcript stability in HeLa cells where endogenous Hsp70 has been suppressed. In contrast, the peptide-binding domain is able to partially restore the half-life of this transcript. The ARE-directed mRNA stabilization and protein chaperone roles of Hsp70 are likely to be independent functions. This conclusion is supported by observations that the nucleotide cofactor-bound status of the ATPase domain does not impact the binding affinity between Hsp70 and an ARE-containing substrate, suggesting that the protein conformational changes associated with the chaperone cycle are unrelated to the interaction between Hsp70 and RNA. Additionally, stabilization of an endogenous mRNA target involves a mechanism that is unaffected by an inhibitor of Hsp70 chaperone function. Since constitutively elevated Hsp70 levels are a negative prognostic indicator for several types of cancer, we propose that Hsp70-directed stabilization of ARE-containing mRNAs that encode regulators of tumor growth and metastasis may provide a novel mechanism linking this protein to the development of aggressive neoplastic phenotypes.