Arginylation was previously found to promote stabilization of heat shock protein 70.3 (Hsp70.3) mRNA and cell survival in mouse embryonic fibroblasts (MEFs) on exposure to heat stress (HS). In search of a factor responsible for these phenomena, the current study identified human antigen R (HuR) as a direct target of arginylation. HS induced arginylation of HuR affected its stability and RNA binding activity. Arginylated HuR failed to bind Hsp70.3 3′ UTR, allowing the recruitment of cleavage stimulating factor 64 (CstF64) in the proximal poly-A-site (PAS), generating transcripts with short 3′UTR. However, HuR from Ate1 knock out (KO) MEFs bound to proximal PAS region with higher affinity, thus excluded CstF64 recruitment. This inhibited the alternative polyadenylation (APA) of Hsp70.3 mRNA and generated the unstable transcripts with long 3′UTR. The inhibition of RNA binding activity of HuR was traced to arginylation-coupled phosphorylation of HuR, by check point kinase 2 (Chk2). Arginylation of HuR occurred at the residue D15 and the arginylation was needed for the phosphorylation. Accumulation of HuR also decreased cell viability upon HS. In conclusion, arginylation dependent modifications of HuR maintained its cellular homeostasis, and promoted APA of Hsp70.3 pre-mRNA, during early HS response.
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We thank Dr Anna Kashina (University of Pennsylvania, USA) for the WT and KO MEFs, ATE1 antibody. pMSCV-PIG was kindly provided by Dr David Bartel (MIT, USA). We thank Dr. A. N. Jha and Ms Sapna M. Borah, TU for their help with structure analysis; Dr. Gaurangi Maitra for careful editing of the text. SS is supported by SERB-India (EEQ/2016/000772 and EMR/2016/004001). KD is supported by scholarship from SERB-India (EEQ/2016/000772). We thank funding agencies for their support to MBBT, TU (UGC-SAP, DST FIST, DBT Strengthening, DBT-Hub, and DBT-BIF) and Department of Biotechnology, NIT, Durgapur (DST-FIST).
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Deka, K., Saha, S. Heat stress induced arginylation of HuR promotes alternative polyadenylation of Hsp70.3 by regulating HuR stability and RNA binding. Cell Death Differ 28, 730–747 (2021). https://doi.org/10.1038/s41418-020-00619-5