Hsp70's RNA-binding and mRNA-stabilizing activities are independent of its protein chaperone functions

Abstract

Hsp70 is a protein chaperone that prevents protein aggregation and aids protein folding by binding to hydrophobic peptide domains through a reversible mechanism directed by an ATPase cycle. However, Hsp70 also binds U-rich RNA including some AU-rich elements (AREs) that regulate the decay kinetics of select mRNAs and has recently been shown to bind and stabilize some ARE-containing transcripts in cells. Previous studies indicated that both the ATP- and peptide-binding domains of Hsp70 contributed to the stability of Hsp70-RNA complexes and that ATP might inhibit RNA recruitment. This suggested the possibility that RNA binding by Hsp70 might mimic features of its peptide-directed chaperone activities. Here, using purified, cofactor-free preparations of recombinant human Hsp70 and quantitative biochemical approaches, we found that high-affinity RNA binding requires at least 30 nucleotides of RNA sequence but is independent of Hsp70's nucleotide-bound status, ATPase activity, or peptide-binding roles. Furthermore, although both the ATP- and peptide-binding domains of Hsp70 could form complexes with an ARE sequence from mRNA, only the peptide-binding domain could recover cellular mRNA in ribonucleoprotein immunoprecipitations. Finally, Hsp70-directed stabilization of mRNA in cells was mediated exclusively by the protein's peptide-binding domain. Together, these findings indicate that the RNA-binding and mRNA-stabilizing functions of Hsp70 are independent of its protein chaperone cycle but also provide potential mechanical explanations for several well-established and recently discovered cytoprotective and RNA-based Hsp70 functions.

Expanding horizons: new roles for non-canonical RNA-binding proteins in cancer

“…Strikingly, in vitro experiments show that certain HSPs can bind AU-rich elements (ARE) in the 3′-UTR of their target mRNAs, mediating RNA decay [ 27 ]. RNA binding appears to be independent of chaperone and ATPase activities, at least for HSP70 [ 28 ]. HSPs play key roles in RNA metabolism, including miRNA loading in the RNA-induced silencing complex (RISC) [ 29 ] and folding of nascent polypeptides concomitantly to ribosome's activity [ 30 ].…”

Unconventional RNA‐binding proteins: an uncharted zone in RNA biology

“…As opposed to the previously reported regulation of HSP70 mRNA, the depletion of Hsp70 led to the destabilization of VEGF and COX‐2 mRNAs under heat shock conditions, suggesting a role of Hsp70 in the stabilization of selected ARE‐containing mRNAs . Furthermore, it was later shown that AREs consisting of at least 30 nucleotides are required for high‐affinity binding of human Hsp70 to RNA substrates, indicating high specificity of the Hsp70–RNA interaction . RIP studies further suggested that only the peptide‐binding domain but not the ATP‐binding domain of Hsp70 interacted and stabilized the mRNA in vivo .…”

“…The interaction with RNA was shown to be strongly inhibited by physiological concentrations of ATP (5 mM), which led to the suggestion that RNA binding could be established via an N-terminal ATPase domain [69][70][71]. Given that AREs in 3 0 UTRs often control mRNA stability [72], it was further proposed that Hsp70 and Hsp110 aid proper folding of their RNA substrates, possibly recruiting other factors that regulate the degradation and/or translation of the bound mRNA [69]. In agreement with these ideas, RBP immunoprecipitation (RIP) and gel-shift assay experiments with recombinant proteins showed that human Hsp70 binds directly to its own mRNA after heat shock [70].…”

“…Furthermore, it was later shown that AREs consisting of at least 30 nucleotides are required for high-affinity binding of human Hsp70 to RNA substrates, indicating high specificity of the Hsp70-RNA interaction [72]. RIP studies further suggested that only the peptide-binding domain but not the ATP-binding domain of Hsp70 interacted and stabilized the mRNA in vivo [72].…”

P23 Acts as Functional RBP in the Macrophage Inflammation Response

“…Specifically, HSPBP1 is implicated in stress-granule formation control and interacts with related proteins G3BP1, HUR and TIA-1/TIAR (Mahboubi et al, 2020). In addition, the chaperone HSP70 independent of its function, exhibits in vitro affinity for AU-rich elements (ARE) and stabilizes the ARE containing VEGFA mRNA in vivo (Kishor et al, 2017). Interestingly, a motor protein is encoded by Kif15 mRNA, which is differentially bound by P23 in untreated and LPSactivated macrophages.…”