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.

先前发现精氨酸化可促进热休克蛋白 70.3 ( Hsp70.3) mRNA 的稳定和暴露于热应激 (HS) 的小鼠胚胎成纤维细胞 (MEF) 中的细胞存活。为了寻找导致这些现象的因素，目前的研究将人类抗原 R (HuR) 确定为精氨酸化的直接靶标。HS诱导的HuR精氨酸化影响其稳定性和RNA结合活性。精氨酸化的 HuR 未能结合Hsp70.3 3'UTR，从而允许在近端 poly-A 位点 (PAS) 中募集切割刺激因子 64 (CstF64)，从而产生具有短 3'UTR 的转录物。然而，来自Ate1的 HuR敲除 (KO) MEFs 以更高的亲和力与近端 PAS 区域结合，因此排除了 CstF64 招募。这抑制了Hsp70.3 mRNA 的选择性多聚腺苷酸化 (APA) 并产生了具有长 3'UTR 的不稳定转录本。通过检查点激酶 2 (Chk2)，HuR 对 RNA 结合活性的抑制可追溯到 HuR 的精氨酸化偶联磷酸化。HuR 的精氨酸化发生在残基 D15 处，并且磷酸化需要精氨酸化。HuR 的积累也降低了 HS 后的细胞活力。总之，在早期 HS 反应期间，HuR 的精氨酸化依赖性修饰维持其细胞稳态，并促进Hsp70.3前 mRNA 的 APA。